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Sample records for tumefaciens ipt gene

  1. Senescence-induced ectopic expression of A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx and nitrate reductase activity but does not affect grain yield

    Czech Academy of Sciences Publication Activity Database

    Sýkorová, Blanka; Kurešová, G.; Daskalova, S.; Trčková, M.; Hoyerová, Klára; Raimanová, I.; Motyka, Václav; Trávníčková, Alena; Elliott, M. C.; Kamínek, Miroslav

    2008-01-01

    Roč. 59, č. 2 (2008), s. 377-387 ISSN 0022-0957 R&D Projects: GA ČR GA522/02/0530; GA MŠk 1M06030; GA AV ČR(CZ) IAA600380507 Keywords : Cytokinins * grain yield * ipt gene Subject RIV: EF - Botanics Impact factor: 4.001, year: 2008

  2. Construction of eukaryotic plant expression vector with the SAG12 promoter and the leaf senescence-related IPT gene and its genetic transformation in rice

    International Nuclear Information System (INIS)

    Cai, P.; Qian, Q.; Kang, Z.; Zhang, Z.

    2012-01-01

    A pair of primers was designed according to the sequences published by GenBank to amplify the isopentenyl transferase gene (IPT gene) of the plasmid in Agrobacterium tumefaciens (strain C58). The 5 '-untranslated sequence of the senescence-associated SAG12 gene of Arabidopsis was isolated by the polymerase chain reaction (PCR). In addition, we constructed the plant-expression vectors (pBI121-IPT and pCAMBIA1301-SAG12-IPT), which were regulated by the SAG12 promoter and the IPT gene. The plant-expression vector was used to transform the embryos of rice cultivar Zhonghua 16 with the help of the A. tumefaciens system. Plantlets were regenerated in vitro by selection on medium containing hygromycin. PCR amplification showed that the target gene was successfully integrated into the transformed plants. (author)

  3. Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P( SAG12 )-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene.

    Science.gov (United States)

    Zakizadeh, Hedayat; Lütken, Henrik; Sriskandarajah, Sridevy; Serek, Margrethe; Müller, Renate

    2013-02-01

    KEY MESSAGE : The P ( SAG12 ) -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions. Transgenic plants of Rosa hybrida 'Linda' were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P( SAG12 )-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P(35S)-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1-6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 μl l(-1)) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.

  4. Effects of PSAG12-IPT gene expression on development and senescence in transgenic Lettuce

    NARCIS (Netherlands)

    McCabe, M.S.; Garratt, L.C.; Schepers, F.; Jordi, W.J.R.M.; Stoopen, G.M.; Davelaar, E.; Rhijn, van J.H.A.; Power, J.B.; Davey, M.R.

    2001-01-01

    An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (PSAG12-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf

  5. Transformation of miniature potted rose (Rosa hybrida cv. Linda) with PSAG12-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene

    DEFF Research Database (Denmark)

    Zakizadeh, Hedayatollah; Lütken, Henrik Vlk; Sriskandarajah, Sridevy

    2013-01-01

    Transgenic plants of Rosa hybrida ‘Linda’ were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the PSAG12-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P35S-INTGUS gene) were used for transformation...... of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1–6 copies of the nptII gene...

  6. Identification and Expression Analysis of Medicago truncatula Isopentenyl Transferase Genes (IPTs Involved in Local and Systemic Control of Nodulation

    Directory of Open Access Journals (Sweden)

    Mahboobeh Azarakhsh

    2018-03-01

    Full Text Available Cytokinins are essential for legume plants to establish a nitrogen-fixing symbiosis with rhizobia. Recently, the expression level of cytokinin biosynthesis IPTs (ISOPENTENYLTRANSFERASES genes was shown to be increased in response to rhizobial inoculation in Lotus japonicus, Medicago truncatula and Pisum sativum. In addition to its well-established positive role in nodule primordium initiation in root cortex, cytokinin negatively regulates infection processes in the epidermis. Moreover, it was reported that shoot-derived cytokinin inhibits the subsequent nodule formation through AON (autoregulation of nodulation pathway. In L. japonicus, LjIPT3 gene was shown to be activated in the shoot phloem via the components of AON system, negatively affecting nodulation. However, in M. truncatula, the detailed analysis of MtIPTs expression, both in roots and shoots, in response to nodulation has not been performed yet, and the link between IPTs and AON has not been studied so far. In this study, we performed an extensive analysis of MtIPTs expression levels in different organs, focusing on the possible role of MtIPTs in nodule development. MtIPTs expression dynamics in inoculated roots suggest that besides its early established role in the nodule primordia development, cytokinin may be also important for later stages of nodulation. According to expression analysis, MtIPT3, MtIPT4, and MtIPT5 are activated in the shoots in response to inoculation. Among these genes, MtIPT3 is the only one the induction of which was not observed in leaves of the sunn-3 mutant defective in CLV1-like kinase, the key component of AON, suggesting that MtIPT3 is activated in the shoots in an AON-dependent manner. Taken together, our findings suggest that MtIPTs are involved in the nodule development at different stages, both locally in inoculated roots and systemically in shoots, where their expression can be activated in an AON-dependent manner.

  7. Overexpression of the bacterial ipt gene in developing Arabidopsis seedlins carrying auxin reporters

    Czech Academy of Sciences Publication Activity Database

    Kuderová, Alena; Brzobohatý, Břetislav

    2005-01-01

    Roč. 49, - (2005), S23 [2nd International Symposium Auxins and Cytokinins in Plant Development. 07.07.2005-12.07.2005, Prague] R&D Projects: GA AV ČR(CZ) IAA600380507 Institutional research plan: CEZ:AV0Z50040507 Keywords : ipt gene * auxin Subject RIV: BO - Biophysics

  8. Binary transformation systems based on 'shooter' mutants of Agrobacterium tumefaciens: a simple, efficient and universal gene transfer technology that permits marker gene elimination.

    Science.gov (United States)

    Mihálka, V; Balázs, E; Nagy, I

    2003-04-01

    A simple transformation procedure with a positive selection scheme using the expression of the isopentenyl transferase ( ipt) gene of transfer DNA (T-DNA) 'shooter' mutants of Agrobacterium tumefaciens was elaborated. After comparing several 'shooter' mutants we found that particular strains frequently produced phenotypically normal shoots after co-culturing with tobacco leaf explants. Shoots selected for normal phenotype showed apical dominance and could be rooted with the same efficiency as non-transformed shoots. When binary vectors were introduced into these strains, stably integrated binary vector T-DNA sequences were found in some regenerants, which were produced under non-selective conditions on growth-regulator-free medium. Such phenotypically normal transformants typically lacked a stably integrated ipt gene. Normal looking shoots could also be produced in tomato, muskmelon and sweet pepper.

  9. Water-deficit inducible expression of a cytokinin biosynthetic gene IPT improves drought tolerance in cotton.

    Directory of Open Access Journals (Sweden)

    Sundaram Kuppu

    Full Text Available Water-deficit stress is a major environmental factor that limits agricultural productivity worldwide. Recent episodes of extreme drought have severely affected cotton production in the Southwestern USA. There is a pressing need to develop cotton varieties with improved tolerance to water-deficit stress for sustainable production in water-limited regions. One approach to engineer drought tolerance is by delaying drought-induced senescence via up-regulation of cytokinin biosynthesis. The isopentenyltransferase gene (IPT that encodes a rate limiting enzyme in cytokinin biosynthesis, under the control of a water-deficit responsive and maturation specific promoter P(SARK was introduced into cotton and the performance of the P(SARK::IPT transgenic cotton plants was analyzed in the greenhouse and growth chamber conditions. The data indicate that P(SARK::IPT-transgenic cotton plants displayed delayed senescence under water deficit conditions in the greenhouse. These plants produced more root and shoot biomass, dropped fewer flowers, maintained higher chlorophyll content, and higher photosynthetic rates under reduced irrigation conditions in comparison to wild-type and segregated non-transgenic lines. Furthermore, P(SARK::IPT-transgenic cotton plants grown in growth chamber condition also displayed greater drought tolerance. These results indicate that water-deficit induced expression of an isopentenyltransferase gene in cotton could significantly improve drought tolerance.

  10. Identification and expression analysis of the IPT and CKX gene families during axillary bud outgrowth in apple (Malus domestica Borkh.).

    Science.gov (United States)

    Tan, Ming; Li, Guofang; Qi, Siyan; Liu, Xiaojie; Chen, Xilong; Ma, Juanjuan; Zhang, Dong; Han, Mingyu

    2018-04-20

    Cytokinins (CKs) play a crucial role in promoting axillary bud outgrowth and targeting the control of CK metabolism can be used to enhance branching in plants. CK levels are maintained mainly by CK biosynthesis (isopentenyl transferase, IPT) and degradation (dehydrogenase, CKX) genes in plants. A systematic study of the IPT and CKX gene families in apple, however, has not been conducted. In the present study, 12 MdIPTs and 12 MdCKXs were identified in the apple genome. Systematic phylogenetic, structural, and synteny analyses were performed. Expression analysis of these genes in different tissues was also assessed. MdIPT and MdCKX genes exhibit distinct expression patterns in different tissues. The response of MdIPT, MdCKX, and MdPIN1 genes to various treatments (6-BA, decapitation and Lovastatin, an inhibitor of CKs synthesis) that impact branching were also investigated. Results indicated that most of the MdIPT and MdCKX, and MdPIN1 genes were upregulated by 6-BA and decapitation treatment, but inhibited by Lovastatin, a compound that effectively suppresses axillary bud outgrowth induced by decapitation. These findings suggest that cytokinin biosynthesis is required for the activation of bud break and the export of auxin from buds in apple tree with intact primary shoot apex or decapitated apple tree. MdCKX8 and MdCKX10, however, exhibited little response to decapitation, but were significantly up-regulated by 6-BA and Lovastatin, a finding that warrants further investigation in order to understand their function in bud-outgrowth. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Mutants of Agrobacterium tumefaciens with elevated vir gene expression

    International Nuclear Information System (INIS)

    Pazour, G.J.; Ta, C.N.; Das, A.

    1991-01-01

    Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG

  12. Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression.

    OpenAIRE

    Gray, J; Wang, J; Gelvin, S B

    1992-01-01

    vir regulon expression in Agrobacterium tumefaciens involves both chromosome- and Ti-plasmid-encoded gene products. We have isolated and characterized a new chromosomal gene that when mutated results in a 2- to 10-fold reduction in the induced expression of vir genes by acetosyringone. This reduced expression occurs in AB minimal medium (pH 5.5) containing either sucrose or glucose and containing phosphate at high or low concentrations. The locus was cloned and used to complement A. tumefacie...

  13. Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene

    NARCIS (Netherlands)

    Heidekamp, F.; Dirkse, W.G.; Hille, J.; Ormondt, H. van

    1983-01-01

    The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined. The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant

  14. The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens.

    Science.gov (United States)

    Hwang, Hau-Hsuan; Yang, Fong-Jhih; Cheng, Tun-Fang; Chen, Yi-Chun; Lee, Ying-Ling; Tsai, Yun-Long; Lai, Erh-Min

    2013-09-01

    The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection.

  15. Nitrogen metabolism and gas exchange parameters associated with zinc stress in tobacco expressing an ipt gene for cytokinin synthesis.

    Science.gov (United States)

    Pavlíková, Daniela; Pavlík, Milan; Procházková, Dagmar; Zemanová, Veronika; Hnilička, František; Wilhelmová, Naďa

    2014-04-15

    Increased endogenous plant cytokinin (CK) content through transformation with an isopentyl transferase (ipt) gene has been associated with improved plant stress tolerance. The impact of zinc (tested levels Zn1=250, Zn2=500, Zn3=750mgkg(-1)soil) on gas exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration) and nitrogen utilization by plants resulted in changes of free amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, glycine, serine, cystein) and differed for transformed and non-transformed tobacco plants. For pot experiments, tobacco plants (Nicotiana tabacum L., cv. Wisconsin 38) transformed with a construct consisting of SAG12 promoter fused with the ipt gene for cytokinin synthesis (SAG plants) and its wild type (WT plants as a control) were used. Physiological analyses confirmed that SAG plants had improved zinc tolerance compared with the WT plants. The enhanced Zn tolerance of SAG plants was associated with the maintenance of accumulation of amino acids and with lower declines of photosynthetic and transpiration rates. In comparison to WT plants, SAG plants exposed to the highest Zn concentration accumulated lower concentrations of asparagine, which is a major metabolic product during senescence. Copyright © 2013 Elsevier GmbH. All rights reserved.

  16. [Genetic transformation of Pinellia ternata with Agrobacterium tumefaciens-mediated sHSP genes].

    Science.gov (United States)

    Guo, Zhao-Yang; Cui, Ting-Ting; Xue, Jian-Ping; Zhu, Yan-Fang; Zhang, Ai-Min; Sheng, Wei; Teng, Jing-Tong

    2012-12-01

    To establish an efficient genetic transformation system of Pinellia ternata. With petioles from test-tube seedlings of P. ternata as explants, Agrobacterium tumefaciens mediation method was adopted to explore the effect of phenolic substances, A. tumefaciens's concentration, infection time, pre-incubation time and co-cultivation time on genetic transformation efficiency of P. ternata. The genetic transformation efficiency could be effectively enhanced by infecting in A. tumefaciens culture containing AS 40 mg x L(-1) for 15 min for three days. The petioles were put into the differentiation medium containing 150 mg x L(-1) Kan and 350 mg x L(-1) Carb to screening and cultivation. After around 30 days, microtubers could be observed at both sides of the petioles. Gus staining and PCR verification on the regenerated plants showed that the exogenous gene sHSP had been integrated into genome of P. ternata.

  17. Virus-induced gene silencing and Agrobacterium tumefaciens-mediated transient expression in Nicotiana tabacum.

    Science.gov (United States)

    Zhang, Zhao; Thomma, Bart P H J

    2014-01-01

    Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the corresponding Verticillium effector Ave1 through Agrobacterium tumefaciens-mediated transient transformation (agroinfiltration), we established a rapid system for assessing the requirement of candidate plant genes for Ve1-mediated immune signaling.

  18. Theoretical prediction and experimental verification of protein-coding genes in plant pathogen genome Agrobacterium tumefaciens strain C58.

    Science.gov (United States)

    Wang, Qian; Lei, Yang; Xu, Xiwen; Wang, Gejiao; Chen, Ling-Ling

    2012-01-01

    Agrobacterium tumefaciens strain C58 is a Gram-negative soil bacterium capable of inducing tumors (crown galls) on many dicotyledonous plants. The genome of A. tumefaciens strain C58 was re-annotated based on the Z-curve method. First, all the 'hypothetical genes' were re-identified, and 29 originally annotated 'hypothetical genes' were recognized to be non-coding open reading frames (ORFs). Theoretical evidence obtained from principal component analysis, clusters of orthologous groups of proteins occupation, and average length distribution showed that these non-coding ORFs were highly unlikely to encode proteins. Results from the reverse transcription-polymerase chain reaction (RT-PCR) experiments on three different growth stages of A. tumefaciens C58 confirmed that 23 (79%) of the identified non-coding ORFs have no transcripts in these growth stages. In addition, using theoretical prediction, 19 potential protein-coding genes were predicted to be new protein-coding genes. Fifteen (79%) of these genes were verified with RT-PCR experiments. The RT-PCR experimental results confirmed the reliability of our theoretical prediction, indicating that false-positive prediction and missing genes always exist in the annotation of A. tumefaciens C58 genome. The improved annotation will serve as a valuable resource for the research of the lifestyle, metabolism, and pathogenicity of A. tumefaciens C58. The re-annotation of A. tumefaciens C58 can be obtained from http://211.69.128.148/Atum/.

  19. Theoretical prediction and experimental verification of protein-coding genes in plant pathogen genome Agrobacterium tumefaciens strain C58.

    Directory of Open Access Journals (Sweden)

    Qian Wang

    Full Text Available Agrobacterium tumefaciens strain C58 is a Gram-negative soil bacterium capable of inducing tumors (crown galls on many dicotyledonous plants. The genome of A. tumefaciens strain C58 was re-annotated based on the Z-curve method. First, all the 'hypothetical genes' were re-identified, and 29 originally annotated 'hypothetical genes' were recognized to be non-coding open reading frames (ORFs. Theoretical evidence obtained from principal component analysis, clusters of orthologous groups of proteins occupation, and average length distribution showed that these non-coding ORFs were highly unlikely to encode proteins. Results from the reverse transcription-polymerase chain reaction (RT-PCR experiments on three different growth stages of A. tumefaciens C58 confirmed that 23 (79% of the identified non-coding ORFs have no transcripts in these growth stages. In addition, using theoretical prediction, 19 potential protein-coding genes were predicted to be new protein-coding genes. Fifteen (79% of these genes were verified with RT-PCR experiments. The RT-PCR experimental results confirmed the reliability of our theoretical prediction, indicating that false-positive prediction and missing genes always exist in the annotation of A. tumefaciens C58 genome. The improved annotation will serve as a valuable resource for the research of the lifestyle, metabolism, and pathogenicity of A. tumefaciens C58. The re-annotation of A. tumefaciens C58 can be obtained from http://211.69.128.148/Atum/.

  20. Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei.

    Science.gov (United States)

    Xiao, Xing; Feng, Jiao; Li, Yu; Chen, Zhiwen; Shi, Minglan; Xi, Liyan; Mylonakis, Eleftherios; Zhang, Junmin

    2017-09-25

    Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 10 6 yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.

  1. Agrobacterium tumefaciens exoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer, and virulence gene expression.

    Science.gov (United States)

    Heckel, Brynn C; Tomlinson, Amelia D; Morton, Elise R; Choi, Jeong-Hyeon; Fuqua, Clay

    2014-09-01

    Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Avaliação de plantas transgênicas de Petunia x hybrida contendo o gene Psag12 - ipt cultivadas sob deficiência nutricional.

    Directory of Open Access Journals (Sweden)

    Francine Lorena Cuquel

    2001-05-01

    Full Text Available Deficiência nutricional em plantas pode causar clorose, necrose, desfolha, redução do crescimento e da produtividade e senescência prematura. Aplicações xógenas de citocinina podem aliviar tais sintomas. Este estudo foi efetuado para avaliar os efeitos da produção auto-regulada de citocinina na tolerância de plantas de petúnia (Petunia x hybrida à deficiência nutricional. Um gene quimérico contendo o promotor SAG12 unido ao gene ipt, o qual codifica a produção de isopentenyl transferase, uma enzima da rota metabólica da biossíntese de citocinina, foi introduzido em petúnia através de transformação mediada por Agrobacterium. Duas plantas contendo o gene ipt foram selecionadas e autopolinizadas para obtenção das linhas a serem submetidas à deficiência nutricional. Ambas as linhas PSAG12–ipt avaliadas foram mais tolerantes à deficiência nutricional que as plantas do tipo selvagem. Os resultados indicam que a produção endógena de citocinina pode aumentar a tolerância das plantas à deficiência nutricional.

  3. Regulated expression of a cytokinin biosynthesis gene IPT delays leaf senescence and improves yield under rainfed and irrigated conditions in canola (Brassica napus L..

    Directory of Open Access Journals (Sweden)

    Surya Kant

    Full Text Available Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.

  4. Regulated expression of a cytokinin biosynthesis gene IPT delays leaf senescence and improves yield under rainfed and irrigated conditions in canola (Brassica napus L.).

    Science.gov (United States)

    Kant, Surya; Burch, David; Badenhorst, Pieter; Palanisamy, Rajasekaran; Mason, John; Spangenberg, German

    2015-01-01

    Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.

  5. Effects of cor15a-IPT gene expression on leaf senescence in transgenic Petuniaxhybrida and Dendranthemaxgrandiflorum

    Czech Academy of Sciences Publication Activity Database

    Khodakovskaya, M.; Li, Y.; Li, J.; Vaňková, Radomíra; Malbeck, Jiří; McAvoy, R.

    2005-01-01

    Roč. 56, č. 414 (2005), s. 1165-1175 ISSN 0022-0957 R&D Projects: GA MŠk LN00A081 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cold-inducible promoter * cytokinins * ipt expression Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.336, year: 2005

  6. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    Science.gov (United States)

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. Copyright © 2015 Elsevier GmbH. All rights reserved.

  7. Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58

    Science.gov (United States)

    Yu, Jia-Feng; Sui, Tian-Xiang; Wang, Hong-Mei; Wang, Chun-Ling; Jing, Li; Wang, Ji-Hua

    2015-12-01

    Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants. Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as “hypothetical” were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58. Project supported by the National Natural Science Foundation of China (Grant Nos. 61302186 and 61271378) and the Funding from the State Key Laboratory of Bioelectronics of Southeast University.

  8. Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58

    International Nuclear Information System (INIS)

    Yu Jia-Feng; Sui Tian-Xiang; Wang Ji-Hua; Wang Hong-Mei; Wang Chun-Ling; Jing Li

    2015-01-01

    Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants. Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as “hypothetical” were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58. (special topic)

  9. Identification of genes associated with asexual reproduction in Phyllosticta citricarpa mutants obtained through Agrobacterium tumefaciens transformation.

    Science.gov (United States)

    Goulin, Eduardo Henrique; Savi, Daiani Cristina; Petters, Desirrê Alexia Lourenço; Kava, Vanessa; Galli-Terasawa, Lygia; Silva, Geraldo José; Glienke, Chirlei

    2016-11-01

    Phyllosticta citricarpa is the epidemiological agent of Citrus Black Spot (CBS) disease, which is responsible for large economic losses worldwide. CBS is characterized by the presence of spores (pycnidiospores) in dark lesions of fruit, which are also responsible for short distance dispersal of the disease. The identification of genes involved in asexual reproduction of P. citricarpa can be an alternative for directional disease control. We analyzed a library of mutants obtained through Agrobacterium tumefaciens transformation system, looking for alterations in growth and reproductive structure formation. Two mutant strains were found to have lost the ability to form pycnidia. The flanking T-DNA insertion regions were identified on P. citricarpa genome by using blast analysis and further gene prediction. The predicted genes containing the T-DNA insertions were identified as Spindle Poison Sensitivity Scp3, Ion Transport protein, and Cullin Binding proteins. The Ion Transport and Cullin Binding proteins are known to be correlated with sexual and asexual reproduction in fungi; however, the exact mechanism by which these proteins act on spore formation in P. citricarpa needs to be better characterized. The Scp3 proteins are suggested here for the first time as being associated with asexual reproduction in fungus. This protein is associated with microtubule formation, and as microtubules play an essential role as spindle machinery for chromosome segregation and cytokinesis, insertions in this gene can lead to abnormal formations, such as that observed here in P. citricarpa. We suggest these genes as new targets for fungicide development and CBS disease control, by iRNA. Copyright © 2016 Elsevier GmbH. All rights reserved.

  10. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells.

    Science.gov (United States)

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-10-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. © 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    Science.gov (United States)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  12. The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation

    International Nuclear Information System (INIS)

    Jin, S.; Roitsch, T.; Ankenbauer, R.G.; Gordon, M.P.; Nester, E.W.

    1990-01-01

    The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. The authors overproduced truncated VirA proteins in Escherichia coli be deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [γ- 32 P]ATP but not with [γ- 32 P]GTP or [α- 32 P]ATP, which suggests an ATP γ-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens

  13. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

    KAUST Repository

    Kumar, Nitish

    2010-07-01

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

  14. Agrobacterium tumefaciens-MEDIATED IN-PLANTA TRANSFORMATION OF INDONESIAN MAIZE USING pIG121Hm-Cs PLASMID CONTAINING nptII AND hpt GENES

    Directory of Open Access Journals (Sweden)

    Edy Listanto

    2017-05-01

    Full Text Available Maize (Zea mays L. productivity in Indonesia is challenged to be increased using genetic engineering. Recent advances in Agrobacterium tumefaciens-mediated in-planta transforma-tion makes it possible to transform maize with low cost, and simple method. This study aimed to confirm pIG121Hm-Cs plasmid in A. tumefaciens, and to estimate the efficiency level of  A. tumefaciens-mediated in-planta transformation of Indonesian maize by using pIG121Hm-Cs plasmid containing nptII and hpt genes. A series of studies were conducted including confirmation of gene construct of pIG121Hm-Cs plasmid in A. tumefaciens, transformation of four maize lines through A. tumefaciens-mediated in-planta technique, acclimatization of transformant plants and molecular analysis of selected plants using polymerase chain reaction (PCR. The pIG121Hm-Cs plasmid was confirmed via PCR analysis using specific primers of nptII and hpt genes and resulted 700 bp and 500 bp for fragments of nptII and hpt, respectively. After selection, acclimatization and molecular analysis steps, the efficiency levels of transformation of four maize lines were low, ranging from 3.8% to 12.8%. The level of transformation efficiency of ST-27 line was the highest accounting for 12.8% of 45 planted embryos on selection medium based on PCR analysis using specific primer for nptII gene. Overall, A. tumefaciens-mediated in planta transformation on maize floral pistil in this study proved to be successful and rapid. Therefore, this enhanced transformation method will be beneficial for Indonesian maize genetic engineering.

  15. Identification of pathogenicity-related genes in the vascular wilt fungus verticillium dahliae by agrobacterium tumefaciens-mediated t-DNA insertional mutagenesis.

    Science.gov (United States)

    Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that underpin pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transform...

  16. Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression.

    Science.gov (United States)

    Zhao, Jinlei; Binns, Andrew N

    2016-02-15

    Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens.

    Science.gov (United States)

    Liu, Jikai; Li, Hanjing; Miao, Min; Tang, Xiaofeng; Giovannoni, Jim; Xiao, Fangming; Liu, Yongsheng

    2012-02-01

    Plants defend themselves against potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs). However, the molecular mechanisms underlying this PAMP-triggered immunity (PTI) are largely unknown. In this study, we show that tomato HP1/DDB1, coding for a key component of the CUL4-based ubiquitin E3 ligase complex, is required for resistance to Agrobacterium tumefaciens. We found that the DDB1-deficient mutant (high pigment-1, hp1) is susceptible to nontumorigenic A. tumefaciens. The efficiency of callus generation from the hp1 cotyledons was extremely low as a result of the necrosis caused by Agrobacterium infection. On infiltration of nontumorigenic A. tumefaciens into leaves, the hp1 mutant moderately supported Agrobacterium growth and developed disease symptoms, but the expression of the pathogenesis-related gene SlPR1a1 and several PTI marker genes was compromised at different levels. Moreover, exogenous application of salicylic acid (SA) triggered SlPR1a1 gene expression and enhanced resistance to A. tumefaciens in wild-type tomato plants, whereas these SA-regulated defence responses were abolished in hp1 mutant plants. Thus, HP1/DDB1 may function through interaction with the SA-regulated PTI pathway in resistance against Agrobacterium infection. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  18. Enhancement of flowering and branching phenotype in chrysanthemum by expression of ipt under the control of a 0.821 kb fragment of the LEACO1 gene promoter

    Czech Academy of Sciences Publication Activity Database

    Khodakovskaya, M.; Vaňková, Radomíra; Malbeck, Jiří; Li, A.; Li, Y.; McAvoy, R.

    2009-01-01

    Roč. 28, č. 9 (2009), s. 1351-1362 ISSN 0721-7714 Institutional research plan: CEZ:AV0Z50380511 Keywords : ipt Gen e * Cytokinins * Transgenic chrysanthemum Subject RIV: EB - Gen etics ; Molecular Biology Impact factor: 2.301, year: 2009

  19. Biological activity of the tzs gene of nopaline Agrobacterium tumefaciens GV3101 in plant regeneration and genetic transformation.

    Science.gov (United States)

    Han, Zhao-Fen; Hunter, David M; Sibbald, Susan; Zhang, Ji-Shu; Tian, Lining

    2013-11-01

    Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.

  20. An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

    Directory of Open Access Journals (Sweden)

    Pileggi Marcos

    2001-01-01

    Full Text Available An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration. A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996, while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

  1. Agrobacterium tumefaciens

    Science.gov (United States)

    Cao, Shi-Liang; Masilamany, Pathmalojiny; Li, Wen-Bin; Pauls, K Peter

    2014-03-04

    An Agrobacterium tumefaciens -mediated corn transformation method based on multiple shoot tissue cultures was developed, which is effective with a variety of corn inbred lines and standard binary vectors. Six factors that affected the success of corn transformation were tested, including A. tumefaciens strain, corn genotype, tissue culture growth stage, medium composition, co-culture temperature and surfactant treatment. Agropine-type bacteria (EHA 101 and AGL 1) were eightfold more effective than octopine-type strain for corn multi-shoot tissues transformation. The average frequency of Glucuronidase (GUS)-positive explants obtained from 14 corn genotypes ranged from 36% to 76%. L-proline (0.7 g L -1 ) in the co-culture medium apparently improved the frequency of transformation. The newly initiated multi-shoot tissues were most responsive to Agrobacterium infection. A positive correlation was found between multi-shoot tissue susceptibility to Agrobacterium and the proportion of cells in G1 phase. Transformants were identified by reverse transcription Polymerase Chain Reaction (PCR) and by southern blot hybridization assays. The frequency of transformants was approximately 2% based on the number of multi-shoot explants co-cultivated with Agrobacterium .

  2. Expresión transitoria del gen GUS en caña de azúcar usando Agrobacterium tumefaciens Transient gene expression in sugarcane using Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Martha Liliana Bonilla Betancourt

    2008-10-01

    Full Text Available En el estudio se desarrolló una metodología de transformación genética mediante Agrobacterium tumefaciens en cultivares colombianos de caña de azúcar. La transformación se evaluó mediante la expresión del gen GUS. Callos embriogénicos y explantes meristemáticos de los genotipos CC85-92, CC84-75 y CC87-505 se transformaron usando tres cepas (AGL-1, LBA4404 y EHA105 con el plásmido pCambia 1305.2 y dos (EHA105 y LBA4404 con pCambia 2301. Se usó el medio de infiltración (IM con acetosiringona y se evaluó el tiempo de cocultivo y la densidad óptica de la bacteria al momento de la inducción. Los genotipos mostraron respuesta diferencial con las combinaciones cepa-plásmido: obtuvieron mayor expresión del gen GUS cuando el genotipo CC85-92 se transformó con la cepa AGL-1-pCambia 1305.2. CC84-75 y CC87-505 mostraron mayor expresión cuando se transformaron con la cepa EHA105-pCambia 1305.2. Mayor eficiencia en la expresión se obtuvo cuando la bacteria se indujo en IM después de siete días de cocultivo y cuando la densidad óptica de la bacteria fue de 0.2(600nm al momento de la inducción. Se demostró superioridad de los explantes en la eficiencia de transformación.The aim of the present study was to develop a transformation method mediated by Agrobacterium in Colombian cultivars of sugarcane. Transformation was evaluated in each step through transient GUS expression. Embryogenic calli and meristematic explants of CC85-92, CC84-75 y CC87-505 cultivars, were transformed using Agrobacterium tumefaciens AGL-1, LBA 4404 and EHA 105 strains, harboring pCambia 1305.2 plasmid. Furthermore, strains LBA 4404 and EHA 105 harboring pCambia 2301 were also tested. Bacterian activator medium, named infiltration media (IM with acetosyringone was used. Co-cultivation time and bacteria optical density before induction were tested. Sugarcane cultivars evaluated showed differential response to different strain-plasmid combinations, obtaining

  3. The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

    Science.gov (United States)

    Niu, Xiaolei; Zhou, Meiliang; Henkel, Christiaan V; van Heusden, G Paul H; Hooykaas, Paul J J

    2015-12-01

    During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  4. Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae.

    Science.gov (United States)

    Dobinson, Katherine F; Grant, Sandra J; Kang, Seogchan

    2004-02-01

    A gene encoding a trypsin protease was isolated from a tomato isolate of Verticillium dahliae. The gene, designated VTP1, contains two introns and is predicted to encode a protein of 256 amino acids. The gene is present in V. dahliae isolates from different host plants and in V. albo-atrum; weakly hybridizing sequences are present in V. tricorpus. VTP1 cDNA sequences were identified in a sequence tag analysis of genes expressed under growth conditions that promote microsclerotia development. Replacement of the gene, by Agrobacterium tumefaciens-mediated transformation (ATMT), with a mutant allele construct did not noticeably alter either pathogenicity or growth in culture. Searches of expressed sequence tag databases showed that, in addition to the VTP1 gene, V. dahliae contains two genes encoding subtilisin-like proteases similar to those produced by pathogenic Aspergillus spp. This is the first description of the application of ATMT to the molecular analysis of phytopathogenic Verticillium spp.

  5. Virus-induced gene silencing and Agrobacterium tumefaciens-mediated transient expression in Nicotiana tabacum

    NARCIS (Netherlands)

    Zhang, Z.; Thomma, B.P.H.J.

    2014-01-01

    Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the

  6. Mechanisms of Change in Interpersonal Therapy (IPT)

    OpenAIRE

    Lipsitz, Joshua D.; Markowitz, John C.

    2013-01-01

    Although interpersonal therapy (IPT) has demonstrated efficacy for mood and other disorders, little is known about how IPT works. We present interpersonal change mechanisms that we hypothesize account for symptom change in IPT. IPT’s interpersonal model integrates both relational theory, building on work by Sullivan, Bowlby, and others, and insights based on research findings regarding stress, social support, and illness to highlight contextual factors thought to precipitate and maintain psyc...

  7. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    Science.gov (United States)

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

  8. Construction of a gene bank and use of the chromosome walking technique for the detection of new putative agrocin genes in Agrobacterium tumefaciens strain D286

    International Nuclear Information System (INIS)

    Herrera, G.

    1987-02-01

    A gene bank of Agrobacterium tumefaciens D286 wt has been constructed by cloning D286 wt DNA partially digested with EcoRI in the cosmid vector pLAFRI. The library; composed of 1750 members with a 27.7 kb average insert size was probed with pCDTn5-3, a cosmid vector carrying a D286:: Tn5 insert from the strain D286:: Tn5 Ag-. One recombinant cosmid of the library, pCDO932, was detected. The insert DNA of pCDO932 has sequences homologous to the D286:: Tn5 insert of pCDTn5-3, therefore it carries putative wt agrocin D286 genes. The insert DNA of pCDO932 was isolated and used to probe the D286 wt gene library. Chromosome walking resulted in the detection of pCD2375. EcoRI restriction digestions and DNA homology studies of pCDO932 and pCD2375 showed that their D286 wt inserts are both composed of 4 EcoRI DNA sub-fragments totalling 21.8 and 24.8 kb respectively, with an overlapping sequence extending 3.5 kb. In order to overcome the failure to detect A. tumefaciens cells transformed with pCDO932. Vectors pSUP204-1 was constructed. Such vector has been derived from pSUP204 which were slightly altered by cloning into it a 700 bp λ DNA SalI fragmet. This resulted in insertion inactivation of the Tc r gene, allows the use of pSUP204-1 as a subcloning vector in conjugations and transformations involving pCDO932 or pCD2375 and strains D286:: Tn5 Ag- and C58 C1G. Two recombinant cosmids bearing D286 wt DNA inserts, at least one of which (pCDO932) contains DNA sequences putatively affecting agrocin D286 production, are now available for further genetic manipulations. pSUP204-1 should prove useful as a subcloning vector for transformations and conjugations involving recombinant cosmids from the D286 wt gene bank and Agrobacterium strains. Future work on the molecular biology of agrocin D286 production is discussed. The DNA probe used in this study was labelled with phosphorus 32

  9. The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated.

    Science.gov (United States)

    Cho, K; Winans, S C

    1996-12-01

    The Agrobacterium tumefaciens putA gene, which encodes proline dehydrogenase, is transcriptionally induced by exogenous proline. In contrast to the putA genes of enteric bacteria, the A. tumefaciens putA gene is not regulated by the PutA protein, as the putA promoter remained strongly proline inducible in strains lacking PutA. A putA null mutation increased the expression of the putA promoter under a variety of conditions. However, this mutation is predicted to increase the cytoplasmic concentration of proline, and this alone probably accounts for its effects on putA expression. The putA promoter was also strongly induced by valine, and the putA genotype did not affect expression by this gratuitous inducer. An open reading frame (ORF) encoding an Lrp-like protein was found transcribed divergently from putA. Disruption of this ORF, designated putR, abolished induction of the putA promoter by proline or valine. In addition to activating putA, PutR also repressed its own transcription, and this autorepression was only slightly affected by exogenous proline. The transcription start sites for the putA and putR genes are separated by 64 nucleotides, suggesting that PutR could regulate both promoters by binding to a single operator.

  10. Global analysis of differentially expressed genes and proteins in the wheat callus infected by Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Xiaohong Zhou

    Full Text Available Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs and differentially expressed proteins (DEPs were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq and two-dimensional electrophoresis (2-DE in conjunction with mass spectrometry (MS. A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.

  11. Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

    Science.gov (United States)

    Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo

    2013-01-01

    Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131

  12. [Opine biosynthesis and catabolism genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes].

    Science.gov (United States)

    Vladimirov, I A; Matveeva, T V; Lutova, L A

    2015-02-01

    Agrobacterium is a genus of soil bacteria with the ability to transform plant cells by a T-DNA-sequence located on the pTi/pRi- plasmid containing a set of genes expressed in plant cells. Expression of these genes leads to a proliferation of transformed cells, with the subsequent formation of tumors or growths of roots and the synthesis of opines--products of the condensation of amino acids with ketoacids or sugars used by Agrobacteria as a source of carbon and nitrogen. In this review, we systematized the information about most common opines in plant--Agrobacterium systems and their biosynthesis and catabolism genes, as well as the role of opines in the interaction of pathogenic Agrobacterium with plants and with other Agrobacterium strains, including the genetic consequences of such interactions.

  13. Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Frandsen, Mette; Giese, Nanna Henriette

    2012-01-01

    cloning without the need for subcloning. The cloning efficiency is not always as high as desired, but it still presents an efficient alternative to restriction enzyme and ligase-based cloning systems. The USER technology offers a higher four fragment cloning efficiency than In-Fusion, but depends...... on specific structures in the binary vector. The available fungal binary vectors adapted for the USER system are described and protocols are provided for vector design and construction. A general protocol for verification of the resulting gene replacement events in the recipient fungal cells is also given...

  14. Mechanisms of change in interpersonal therapy (IPT).

    Science.gov (United States)

    Lipsitz, Joshua D; Markowitz, John C

    2013-12-01

    Although interpersonal therapy (IPT) has demonstrated efficacy for mood and other disorders, little is known about how IPT works. We present interpersonal change mechanisms that we hypothesize account for symptom change in IPT. Integrating relational theory and insights based on research findings regarding stress, social support, and illness, IPT highlights contextual factors thought to precipitate and maintain psychiatric disorders. It frames therapy around a central interpersonal problem in the patient's life, a current crisis or relational predicament that is disrupting social support and increasing interpersonal stress. By mobilizing and working collaboratively with the patient to resolve this problem, IPT seeks to activate several interpersonal change mechanisms. These include: 1) enhancing social support, 2) decreasing interpersonal stress, 3) facilitating emotional processing, and 4) improving interpersonal skills. We hope that articulating these mechanisms will help therapists to formulate cases and better maintain focus within an IPT framework. Here we propose interpersonal mechanisms that might explain how IPT's interpersonal focus leads to symptom change. Future work needs to specify and test candidate mediators in clinical trials. We anticipate that pursuing this more systematic strategy will lead to important refinements and improvements in IPT and enhance its application in a range of clinical populations. © 2013.

  15. Mechanisms of Change in Interpersonal Therapy (IPT)

    Science.gov (United States)

    Lipsitz, Joshua D.; Markowitz, John C.

    2014-01-01

    Although interpersonal therapy (IPT) has demonstrated efficacy for mood and other disorders, little is known about how IPT works. We present interpersonal change mechanisms that we hypothesize account for symptom change in IPT. IPT’s interpersonal model integrates both relational theory, building on work by Sullivan, Bowlby, and others, and insights based on research findings regarding stress, social support, and illness to highlight contextual factors thought to precipitate and maintain psychiatric disorders. IPT frames therapy around a central interpersonal problem in the patient’s life, a current crisis or relational predicament that is disrupting social support and increasing interpersonal stress. By mobilizing and working collaboratively with the patient to resolve (better manage or negotiate) this problem, IPT seeks to activate several interpersonal change mechanisms. These include: 1) enhancing social support, 2) decreasing interpersonal stress, 3) facilitating emotional processing, and 4) improving interpersonal skills. We hope that articulating these mechanisms will help therapists to formulate cases and better maintain focus within an IPT framework. We propose interpersonal mechanisms that might explain how IPT’s interpersonal focus leads to symptom change. Future work needs to specify and test candidate mediators in clinical trials of IPT. We anticipate that pursuing this more systematic strategy will lead to important refinements and improvements in IPT and enhance its application in a range of clinical populations. PMID:24100081

  16. Cellulose Synthesis in Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Alan R. White; Ann G. Matthysse

    2004-07-31

    We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants including CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one

  17. Agrobacterium tumefaciens-mediated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a fungal pathogen Sclerotium rolfsii.

    Science.gov (United States)

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Moore, Paul H; Zhu, Yun J

    2008-05-01

    Taro (Colocasia esculenta) is one of the most important crops in the Pacific Islands, however, taro yields have been declining in Hawaii over the past 30 years partly due to diseases caused by oomycete and fungal pathogens. In this study, an efficient Agrobacterium tumefaciens-mediated transformation method for taro is first reported. In total, approximately 200 pieces (8 g) of embryogenic calluses were infected with the super-virulent A. tumefaciens strain EHA105 harboring the plant transformation plasmid pBI121/ricchi11 that contains the rice chitinase gene ricchi11. The presence and expression of the transgene ricchi11 in six independent transgenic lines was confirmed using polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Southern blot analysis of the six independent lines indicated that three out of six (50%) had integrated a single copy of the transgene, and the other three lines had two or three copies of the transgene. Compared to the particle bombardment transformation of taro method, which was used in the previous studies, the Agrobacterium-mediated transformation method obtained 43-fold higher transformation efficiency. In addition, these six transgenic lines via Agrobacterium may be more effective for transgene expression as a result of single-copy or low-copy insertion of the transgene than the single line with multiple copies of the transgene via particle bombardment. In a laboratory bioassay, all six transgenic lines exhibited increased tolerance to the fungal pathogen Sclerotium rolfsii, ranging from 42 to 63% reduction in lesion expansion.

  18. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

    Science.gov (United States)

    Ward, J E; Dale, E M; Christie, P J; Nester, E W; Binns, A N

    1990-01-01

    The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity. Images PMID:2203743

  19. An efficient gene disruption method using a positive-negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi.

    Science.gov (United States)

    Su, Yu; Wang, Zhongkang; Shao, Changwen; Luo, Yuanli; Wang, Li; Yin, Youping

    2018-01-16

    Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0-4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.

  20. An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

    Science.gov (United States)

    Wang, Sheng; Xing, Haiying; Hua, Chenlei; Guo, Hui-Shan; Zhang, Jie

    2016-06-01

    The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.

  1. Cysteine desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative resistance in Agrobacterium tumefaciens.

    Science.gov (United States)

    Bhubhanil, Sakkarin; Niamyim, Phettree; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2014-01-01

    The Agrobacterium tumefaciens genome contains a cluster of genes that are predicted to encode Fe-S cluster assembly proteins, and this cluster is known as the sufS2BCDS1XA operon. sufS2 is the first gene in the operon, and it was inactivated to determine its physiological function. The sufS2 mutant exhibited a small colony phenotype, grew slower than the wild-type strain and was more sensitive to various oxidants including peroxide, organic hydroperoxide and superoxide. The sufS2 gene was negatively regulated by iron response regulator (Irr) and rhizobial iron regulator (RirA) under low and high iron conditions, respectively, and was inducible in response to oxidative stress. The oxidant-induced expression of sufS2 was controlled by Irr, RirA and an additional but not yet identified mechanism. sufS2 was required for RirA activity in the repression of a sufS2 promoter-lacZ fusion. RirA may use Fe-S as its cofactor. sufS2 disruption may cause a defect in the Fe-S supply and could thereby affect the RirA activity. The three conserved cysteine residues (C91, C99 and C105) in RirA were predicted to coordinate with the Fe-S cluster and were shown to be essential for RirA repression of the sufS2-lacZ fusion. These results suggested that sufS2 is important for the survival of A. tumefaciens.

  2. Effects upon metabolic pathways and energy production by Sb(III) and As(III)/Sb(III)-oxidase gene aioA in Agrobacterium tumefaciens GW4.

    Science.gov (United States)

    Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Li, Mingshun; Wang, Gejiao

    2017-01-01

    Agrobacterium tumefaciens GW4 is a heterotrophic arsenite [As(III)]/antimonite [Sb(III)]-oxidizing strain. The As(III) oxidase AioAB is responsible for As(III) oxidation in the periplasm and it is also involved in Sb(III) oxidation in Agrobacterium tumefaciens 5A. In addition, Sb(III) oxidase AnoA and cellular H2O2 are also responsible for Sb(III) oxidation in strain GW4. However, the deletion of aioA increased the Sb(III) oxidation efficiency in strain GW4. In the present study, we found that the cell mobility to Sb(III), ATP and NADH contents and heat release were also increased by Sb(III) and more significantly in the aioA mutant. Proteomics and transcriptional analyses showed that proteins/genes involved in Sb(III) oxidation and resistance, stress responses, carbon metabolism, cell mobility, phosphonate and phosphinate metabolism, and amino acid and nucleotide metabolism were induced by Sb(III) and were more significantly induced in the aioA mutant. The results suggested that Sb(III) oxidation may produce energy. In addition, without periplasmic AioAB, more Sb(III) would enter bacterial cells, however, the cytoplasmic AnoA and the oxidative stress response proteins were significantly up-regulated, which may contribute to the increased Sb(III) oxidation efficiency. Moreover, the carbon metabolism was also activated to generate more energy against Sb(III) stress. The generated energy may be used in Sb transportation, DNA repair, amino acid synthesis, and cell mobility, and may be released in the form of heat.

  3. Effects upon metabolic pathways and energy production by Sb(III and As(III/Sb(III-oxidase gene aioA in Agrobacterium tumefaciens GW4.

    Directory of Open Access Journals (Sweden)

    Jingxin Li

    Full Text Available Agrobacterium tumefaciens GW4 is a heterotrophic arsenite [As(III]/antimonite [Sb(III]-oxidizing strain. The As(III oxidase AioAB is responsible for As(III oxidation in the periplasm and it is also involved in Sb(III oxidation in Agrobacterium tumefaciens 5A. In addition, Sb(III oxidase AnoA and cellular H2O2 are also responsible for Sb(III oxidation in strain GW4. However, the deletion of aioA increased the Sb(III oxidation efficiency in strain GW4. In the present study, we found that the cell mobility to Sb(III, ATP and NADH contents and heat release were also increased by Sb(III and more significantly in the aioA mutant. Proteomics and transcriptional analyses showed that proteins/genes involved in Sb(III oxidation and resistance, stress responses, carbon metabolism, cell mobility, phosphonate and phosphinate metabolism, and amino acid and nucleotide metabolism were induced by Sb(III and were more significantly induced in the aioA mutant. The results suggested that Sb(III oxidation may produce energy. In addition, without periplasmic AioAB, more Sb(III would enter bacterial cells, however, the cytoplasmic AnoA and the oxidative stress response proteins were significantly up-regulated, which may contribute to the increased Sb(III oxidation efficiency. Moreover, the carbon metabolism was also activated to generate more energy against Sb(III stress. The generated energy may be used in Sb transportation, DNA repair, amino acid synthesis, and cell mobility, and may be released in the form of heat.

  4. Transport of nonmetabolizable opines by Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Krishnan, M.; Burgner, J.W.; Chilton, W.S.; Gelvin, S.B.

    1991-01-01

    We have examined the uptake of [ 14 C]octopine and [ 14 C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [ 14 C]octopine and [ 14 C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid

  5. Relationship between expression level of hygromycin B-resistant gene and Agrobacterium tumefaciens-mediated transformation efficiency in Beauveria bassiana JEF-007.

    Science.gov (United States)

    Nai, Y S; Lee, M R; Kim, S; Lee, S J; Kim, J C; Yang, Y T; Kim, J S

    2017-09-01

    Agrobacterium tumefaciens-mediated transformation (AtMT) is an effective method for generation of entomopathogenic Beauveria bassiana transformants. However, some strains grow on the selective medium containing hygromycin B (HygB), which reduces the selection efficiency of the putative transformants. In this work, a relationship between HygB resistance gene promoter and AtMT efficiency was investigated to improve the transformant selection. Ten B. bassiana isolates were grown on 800 μg ml -1 HygB medium, but only JEF-006, -007 and -013 showed susceptibility to the antibiotics. Particularly, JEF-007 showed the most dose-dependent susceptibility. Two different Ti-Plasmids, pCeg (gpdA promoter based) and pCambia-egfp (CaMV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene (hph) at 100, 150 and 200 μg ml -1 HygB medium. Eight days after the transformation, wild type, AtMT/pCeg and AtMT/pCambia-egfp colonies were observed on 100 μg ml -1 HygB, but significantly larger numbers of colonies were counted on AtMT/pCeg plates. At higher HygB concentration (150 μg ml -1 ), only AtMT/pCeg colonies were further observed, but very few colonies were observed on the wild type and AtMT/pCambia-egfp plates. Putative transformants were subjected to PCR, RT-PCR and qRT-PCR to investigate the T-DNA insertion rate and gene expression level. Consequently, >80% of colonies showed successful AtMT transformation, and the hph expression level in AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. In the HygB-susceptible B. bassianaJEF-007, gpdA promoter works better than CaMV 35S promoter in the expression of HygB resistance gene at 150 μg ml -1 HygB, consequently improving the selection efficiency of putative transformants. These results provide useful information for determining AtMT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters. © 2017 The

  6. Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation.

    Science.gov (United States)

    Fuentes, Alejandro; Ramos, Pedro Luis; Ayra, Camilo; Rodríguez, Meilyn; Ramírez, Nadia; Pujol, Merardo

    2004-06-01

    A transient gene-expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules. The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way. Conditions such as pre-culture of tobacco cells and the co-cultivation period were identified as determinants to achieve high expression levels. Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared. A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western-blot analysis. A monoclonal antibody against anti-(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.

  7. Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Qi Jia

    2012-01-01

    Full Text Available In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

  8. Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

    Science.gov (United States)

    Izawati, Abang Masli Dayang; Masani, Mat Yunus Abdul; Ismanizan, Ismail; Parveez, Ghulam Kadir Ahmad

    2015-01-01

    DOGR1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l-1 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOGR1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOGR1 gene and 2-DOG for regenerating transgenic oil palm. PMID:26442041

  9. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

    Science.gov (United States)

    Czolkoss, Simon; Fritz, Christiane; Hölzl, Georg; Aktas, Meriyem

    2016-01-01

    Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.

  10. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Simon Czolkoss

    Full Text Available Cardiolipin (CL is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG by phospholipase D-type cardiolipin synthases (PLD-type Cls. In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1 and atu2486 (cls2, coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.

  11. PLANT TRANSFORMATION VECTORS AND THEIR STABILITY IN AGROBACTERIUM TUMEFACIENS

    Directory of Open Access Journals (Sweden)

    Zuzana Polóniová,Pavol Dubnický

    2013-02-01

    Full Text Available The stability of the plant transformation vectors pTS2, pEV2 and pEV8 was tested in two Agrobacterium tumefaciens strains LBA 4404 and C58C1 that differed in chromosomal background. The T-DNAs of all binary vectors contained the β-glucuronidase reporter and the neomycin phosphotransferase selectable marker genes. In addition, the plasmids pEV2 and pEV8 contained the Cre/loxP recombination system. The binary vectors were transformed into A. tumefaciens strains and their stability was evaluated by restriction analyses after retransformation of plasmids from A. tumefaciens into E. coli. Our results showed that plasmids pTS2 and pEV2 were stable in both bacterial strains while the pEV8 exhibited instability in A. tunmefaciens LBA 4404.

  12. Is there any crosstalk between the chemotaxis and virulence induction signaling in Agrobacterium tumefaciens?

    Science.gov (United States)

    Guo, Minliang; Huang, Zhiwei; Yang, Jing

    2017-07-01

    Agrobacterium tumefaciens, a soil-born phytopathogenic bacterium, is well known as a nature's engineer due to its ability to genetically transform the host by transferring a DNA fragment (called T-DNA) from its Ti plasmid to host-cell genome. To combat the harsh soil environment and seek the appropriate host, A. tumefaciens can sense and be attracted by a large number of chemical compounds released by wounded host. As a member of α-proteobacterium, A. tumefaciens has a chemotaxis system different from that found in Escherichia coli, since many chemoattractants for A. tumefaciens chemotaxis are virulence (vir) inducers. However, advances in the study of the chemotaxis paradigm, E. coli chemotaxis system, have provided enough information to analyze the A. tumefaciens chemotaxis. At low concentration, chemoattractants elicit A. tumefaciens chemotaxis and attract the species to the wound sites of the host. At high concentration, chemoattractants induce the expression of virulence genes and trigger T-DNA transfer. Recent studies on the VirA and ChvE of the vir-induction system provide some evidences to support the crosstalk between chemotaxis and vir-induction. This review compares the core components of chemotaxis signaling system of A. tumefaciens with those observed in other species, discusses the connection between chemotaxis and vir-induction in A. tumefaciens, and proposes a model depicting the signaling crosstalk between chemotaxis and vir-induction. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Interpersonal psychotherapy (IPT) in major depressive disorder.

    Science.gov (United States)

    Brakemeier, Eva-Lotta; Frase, Lukas

    2012-11-01

    In this article, we will introduce interpersonal psychotherapy as an effective short-term treatment strategy in major depression. In IPT, a reciprocal relationship between interpersonal problems and depressive symptoms is regarded as important in the onset and as a maintaining factor of depressive disorders. Therefore, interpersonal problems are the main therapeutic targets of this approach. Four interpersonal problem areas are defined, which include interpersonal role disputes, role transitions, complicated bereavement, and interpersonal deficits. Patients are helped to break the interactions between depressive symptoms and their individual interpersonal difficulties. The goals are to achieve a reduction in depressive symptoms and an improvement in interpersonal functioning through improved communication, expression of affect, and proactive engagement with the current interpersonal network. The efficacy of this focused and structured psychotherapy in the treatment of acute unipolar major depressive disorder is summarized. This article outlines the background of interpersonal psychotherapy, the process of therapy, efficacy, and the expansion of the evidence base to different subgroups of depressed patients.

  14. Efficient Construction of Large Genomic Deletion in Agrobacterium tumefaciens by Combination of Cre/loxP System and Triple Recombineering.

    Science.gov (United States)

    Liu, Zhengqiang; Xie, Yali; Zhang, Xu; Hu, Xiaofeng; Li, Yusheng; Ding, Xuezhi; Xia, Liqiu; Hu, Shengbiao

    2016-04-01

    In order to develop an efficient system for deleting genomic segment in Agrobacterium tumefaciens to analyze gene functions and construct marker gene-free recombinant strains, a Cre recombinase expression plasmid was constructed by placing its encoding gene under the control of Ptet promoter and cloning into the plasmid replicable in both A. tumefaciens and E. coli. Triple recombineering was applied to efficiently construct integrative vectors which were used to introduce loxP sites and selection markers into the chromosome of A. tumefaciens. Cre recombinase could be properly induced by anhydrotetracycline in A. tumefaciens, which was revealed by the fact that kanamycin resistance gene flanked by two parallel loxP sites was excised from the genome of A. tumefaciens with virtually 100% efficiency. And what is more, an A. tumefaciens mutant carrying large-deletion (~85 kb) in genome was successfully constructed by Cre/loxP system. Here, we described the application of combination of Cre/loxP system and triple recombineering to efficiently excise genomic segment in A. tumefaciens, which also would facilitate efficient construction of multiple gene disruptions in A. tumefaciens.

  15. (IS136) of Agrobacterium tumefaciens

    Indian Academy of Sciences (India)

    Agrobacterium strains harbour insertion sequences, which are known to transpose into genomes as well as into Ti plasmids. In this study we report the inactivation of a transgene due to transposition of the A. tumefaciens insertion sequence IS136. The transposition was discovered following transformation of plant tissues, ...

  16. Agrobacterium tumefaciens-Mediated Transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand

    2015-01-01

    The use of Agrobacterium tumefaciens-mediated transformation for achieving genetic transformation of fungi has steadily increased over the last decade, and has proven to be almost universally applicable technique once suitable selection markers have been developed. In recent years the major...

  17. Overview of the Integrated Pressurized Thermal-Shock (IPTS) study

    International Nuclear Information System (INIS)

    Cheverton, R.D.

    1990-01-01

    By the early 1980s, (PTS)-related, deterministic, vessel-integrity studies sponsored by the US Nuclear Regulatory Commission (NRC) indicated a potential for failure of some PWR vessels before design end of life, in the event of a postulated severe PTS transient. In response, the NRC established screening criteria, in the form of limiting values of the reference nil-ductility transition temperature (RT NDT ), and initiated the development of a probabilistic methodology for evaluating vessel integrity. This latter effort, referred to as the Integrated Pressurized Thermal-Shock (IPTS) Program, included development of techniques for postulating PTS transients, estimating their frequencies, and calculating the probability of vessel failure for a specific transient. Summing the products of frequency of transient and conditional probability of failure for each of the many postulated transients provide a calculated value of the frequency of failure. The IPTS Program also included the application of the IPTS methodology to three US PWR plants (Oconee-1, Calvert Cliffs-1, and HBRobinson-2) and the specification of a maximum permissible value of the calculated frequency of vessel failure. Another important purpose of the IPTS study was to determine, through application of the IPTS methodology, which design and operating features, parameters, and PTS transients were dominant in affecting the calculated frequency of failure. The scope of the IPTS Program included the development of a probabilistic fracture-mechanics capability, modification of the TRAC and RELAP5 thermal/hydraulic codes, and development of the methodology for estimating the uncertainty in the calculated frequency of vessel failure

  18. Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens.

    Science.gov (United States)

    Soares da Costa, Tatiana P; Patel, Madhvi; Desbois, Sebastien; Gupta, Ruchi; Faou, Pierre; Perugini, Matthew A

    2017-11-01

    Agrobacterium tumefaciens is a Gram-negative bacterium and causative agent of Crown Gall disease that infects a variety of economically important plants. The annotated A. tumefaciens genome contains 10 putative dapA genes, which code for dihydrodipicolinate synthase (DHDPS). However, we have recently demonstrated that only one of these genes (dapA7) encodes a functional DHDPS. The function of the other nine putative dapA genes is yet to be determined. Here, we demonstrate using bioinformatics that the product of the dapA5 gene (DapA5) possesses all the catalytic residues canonical to 2-keto-3-deoxygluconate (KDG) aldolase, which is a class I aldolase involved in glucose metabolism. We therefore expressed, purified, and characterized recombinant DapA5 using mass spectrometry, circular dichroism spectroscopy, analytical ultracentrifugation, and enzyme kinetics. The results show that DapA5 (1) adopts an α/β structure consistent with the TIM-barrel fold of KDG aldolases, (2) possesses KDG aldolase enzyme activity, and (3) exists as a tight dimer in solution. This study shows for the first time that dapA5 from A. tumefaciens encodes a functional dimeric KDG aldolase. © 2017 Wiley Periodicals, Inc.

  19. The plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.

    Science.gov (United States)

    Lang, Julien; Gonzalez-Mula, Almudena; Taconnat, Ludivine; Clement, Gilles; Faure, Denis

    2016-05-01

    In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified plants and bacteria, we evaluated the impact of plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type tumors relative to uninfected plant tissues. Moreover, comparisons of tumors induced on Col-0 and her1 plants showed that the increase in the plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in plant tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights plant GABA content as an important trait in the struggle against pathogenic bacteria. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  20. Succinic Semialdehyde Promotes Prosurvival Capability of Agrobacterium tumefaciens.

    Science.gov (United States)

    Wang, Chao; Tang, Desong; Gao, Yong-Gui; Zhang, Lian-Hui

    2016-01-11

    Succinic semialdehyde (SSA), an important metabolite of γ-aminobutyric acid (GABA), is a ligand of the repressor AttJ regulating the expression of the attJ-attKLM gene cluster in the plant pathogen Agrobacterium tumefaciens. While the response of A. tumefaciens to GABA and the function of attKLM have been extensively studied, genetic and physiological responses of A. tumefaciens to SSA remain unknown. In combination with microarray and genetic approaches, this study sets out to explore new roles of the SSA-AttJKLM regulatory mechanism during bacterial infection. The results showed that SSA plays a key role in regulation of several bacterial activities, including C4-dicarboxylate utilization, nitrate assimilation, and resistance to oxidative stress. Interestingly, while the SSA relies heavily on the functional AttKLM in mediating nitrate assimilation and oxidative stress resistance, the compound could regulate utilization of C4-dicarboxylates independent of AttJKLM. We further provide evidence that SSA controls C4-dicarboxylate utilization through induction of an SSA importer and that disruption of attKLM attenuates the tumorigenicity of A. tumefaciens. Taken together, these findings indicate that SSA could be a potent plant signal which, together with AttKLM, plays a vital role in promoting the bacterial prosurvival abilities during infection. Agrobacterium tumefaciens is a plant pathogen causing crown gall diseases and has been well known as a powerful tool for plant genetic engineering. During the long history of microbe-host interaction, A. tumefaciens has evolved the capabilities of recognition and response to plant-derived chemical metabolites. Succinic semialdehyde (SSA) is one such metabolite. Previous results have demonstrated that SSA functions to activate a quorum-quenching mechanism and thus to decrease the level of quorum-sensing signals, thereby avoiding the elicitation of a plant defense. Here, we studied the effect of SSA on gene expression at a

  1. Establishment of an efficient Agrobacterium tumefaciens -mediated ...

    African Journals Online (AJOL)

    Spine gourd (Momordica dioica Roxb. ex Willd) is a medicinally and economically important plant and also used as vegetable. In this study, we established an Agrobacterium tumefaciens-mediated transformation procedure for M. dioica. Leaf explants were incubated with A. tumefaciens strain LBA4404 containing a binary ...

  2. Quantification of Agrobacterium tumefaciens C58 attachment to Arabidopsis thaliana roots.

    Science.gov (United States)

    Petrovicheva, Anna; Joyner, Jessica; Muth, Theodore R

    2017-10-02

    Agrobacterium tumefaciens is the causal agent of crown gall disease and is a vector for DNA transfer in transgenic plants. The transformation process by A. tumefaciens has been widely studied, but the attachment stage has not been well characterized. Most measurements of attachment have used microscopy and colony counting, both of which are labor and time intensive. To reduce the time and effort required to analyze bacteria attaching to plant tissues, we developed a quantitative real-time PCR (qPCR) assay to quantify attached A. tumefaciens using the chvE gene as marker for the presence of the bacteria. The qPCR detection threshold of A. tumefaciens from pure culture was 104 cell equivalents/ml. The A. tumefaciens minimum threshold concentration from root-bound populations was determined to be 105 cell equivalents/ml inoculum to detect attachment above background. The qPCR assay can be used for measuring A. tumefaciens attachment in applications such as testing the effects of mutations on bacterial adhesion molecules or biofilm formation, comparing attachment across various plant species and ecotypes, and detecting mutations in putative attachment receptors expressed in plant roots. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Transformação genética de cereais via Agrobacterium tumefaciens Cereal genetic transformation via Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Cristine Luise Handel

    1997-06-01

    Full Text Available A transformação genética via Agrobacterium tumefaciens é um método que permite a inserção de uma ou poucas cópias do transgene no DNA da planta hospedeira. Esta pode ser uma ferramenta importante para os melhoristas, pois, além de aumentar a variabilidade genética existente, torna possível criar variabilidade não disponível via métodos de melhoramento convencional. No entanto, ainda existem algumas dificuldades a serem superadas para que os genes de interesse agronômico sejam incorporados no genoma dos cereais, como aidentificação de estirpes de bactérias que infectem monocotiledôneas e a adequação da técnica. O objetivo deste trabalho é de revisar as potencialidades e problemas do uso da A. tumefaciens para transformação de cereais no presentemomento e abordar suas perspectivas futuras. Trabalhos recentes com arroz e trigo indicam que estas culturas podem ser transformadas com A. tumefaciens, sendo que em arroz plantas transgênicas foram obtidas com este método. Esta tecnologia vem sendo aprimorada e a curto prazo possibilitará a transferência de genes para diversas espécies monocotiledôneas.The genetic transformation via Agrobacterium tumefaciens allows the insertion of one or few copies of a transgene into the host DNA. This can be an important tool to plant breeders because it expands the genetic variability in breeding programs, developing variability not readily available from traditional methods. However, there are some difficulties that have to be overcome before this technology may be used in cereals, as the identification of highly ineffective bacteria strains and the adjustments of the technique to various crops. The objective of this paper is to revise the potentialities and limitations of using A. tumefaciens to transform cereals and to indicate the future perspectives of this technology. Recent studies have indicated that it is possible to transform rice and wheat with A. tumefaciens, so that rice

  4. Reproductive fitness of outcrossed hybrids between transgenic broccoli (brassica oleracea) carrying the ipt transgene and conventional varieties of kale, broccoli and cauliflower

    International Nuclear Information System (INIS)

    Ting, P.; Tu, Y.; Lin, C.; Chang, H.; Chen, L.; Litfu, A

    2014-01-01

    Pollens are potential carriers for genetically modified crops to transfer genetic materials horizontally to other plants. For phanerogams, pollen viability and cross-compatibility are critical factors for successful outcross hybridization. To evaluate this possibility, this project investigated pollen viability and pod setting rate by comparing broccoli (Brassica oleracea L. var. italica Planck) and broccoli transformed with the isopentenyl transferase (ipt) gene. Both served as pollen donors and four other varieties as pollen receptors to determine outcross rates. For pollen viability, F1 progeny was higher (p?0.05) for the cross of transgenic ipt broccoli with Li Syue significantly by FDA (fluorescein diacetate) assay. Higher successful hybrids were observed for transgenic ipt broccoli with Fu Yue, Li Syue and Green King. As pollen properties, number and grain diameter were significantly larger (p?0.05) in hybrid combinations of transgenic ipt broccoli with Li Syue and Green King significantly (p?0.05). The pod setting rates were higher while transgenic ipt broccoli served as donor plant. These results analyzing pollen properties between transgenic crops with possible outcross candidates would serve as one of those critical strategies for evaluating environmental biosafety issues for transgenic crops. (author)

  5. Unexpected phytostimulatory behavior for Escherichia coli and Agrobacterium tumefaciens model strains.

    Science.gov (United States)

    Walker, Vincent; Bruto, Maxime; Bellvert, Floriant; Bally, René; Muller, Daniel; Prigent-Combaret, Claire; Moënne-Loccoz, Yvan; Comte, Gilles

    2013-05-01

    Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

  6. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens.

    Science.gov (United States)

    Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

    2014-01-01

    Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.

  7. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens

    Science.gov (United States)

    Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

    2014-01-01

    Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132

  8. The multifaceted roles of the interspecies signalling molecule indole in Agrobacterium tumefaciens.

    Science.gov (United States)

    Lee, Jin-Hyung; Kim, Yong-Guy; Baek, Kwang-Hyun; Cho, Moo Hwan; Lee, Jintae

    2015-04-01

    Bacteria utilize signal molecules to ensure their survival in environmental niches, and indole is an interspecies and interkingdom signalling molecule, which is widespread in the natural environment. In this study, we sought to identify novel roles of indole in soil-borne bacterium Agrobacterium tumefaciens. Agrobacterium tumefaciens was found not to synthesize indole and to degrade it rapidly. The addition of exogenous indole dose-dependently inhibited A. tumefaciens growth and decreased its motility. Surprisingly, indole markedly increased A. tumefaciens biofilm formation on polystyrene, glass and nylon membrane surfaces and enhanced its antibiotic tolerance. Transcriptional analysis showed that indole markedly up-regulated several biofilm-related (celA, cheA, exoR, phoB, flgE, fliR and motA), stress-related genes (clpB, dnaK, gsp, gyrB, marR and soxR) and efflux genes (emrA, norM, and Atu2551) in A. tumefaciens, which partially explained the increased biofilm formation and antibiotic tolerance. In contrast, the plant auxin indole-3-acetic acid did not affect biofilm formation, antibiotic tolerance or gene expression. Interestingly, indole was found to exhibit several similarities with antibiotics, as it inhibited the growth of non-indole-producing bacteria, whereas these bacteria countered its effects by rapidly degrading indole, and by enhancing biofilm formation and antibiotic tolerance. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  9. Light affects motility and infectivity of Agrobacterium tumefaciens.

    Science.gov (United States)

    Oberpichler, Inga; Rosen, Ran; Rasouly, Aviram; Vugman, Michal; Ron, Eliora Z; Lamparter, Tilman

    2008-08-01

    Response to changes in light conditions involves a variety of receptors that can modulate gene expression, enzyme activity and/or motility. For the study of light-regulated effects of Agrobacterium tumefaciens, we used a global analysis approach - proteomics - and compared the protein patterns of dark- and light-grown bacteria. These analyses revealed a significant reduction of FlaA and FlaB - proteins of the flagellum - when the cells were grown in light. The light effect was confirmed by SDS-PAGE with isolated flagella. Quantitative PCR experiments showed a 10-fold increase of the transcription level of flaA, flaB and flaC within 20 min after the transfer from light to darkness. Electron microscopy revealed that these molecular events result in a light-induced reduction of the number of flagella per cell. These changes have major physiological consequences regarding motility, which is considerably reduced with exposure to light. The inhibitory effect of light on the motility is not unique to A. tumefaciens and was also seen in other species of the Rhizobiaceae. Previous studies suggested that the flagella function is significant for bacteria-plant interactions and bacterial virulence. In our studies, light reduced the attachment of A. tumefaciens to tomato roots and the virulence of the bacteria in a cucumber infection assay.

  10. [Interpersonal therapy (IPT) in child psychiatry and adolescent].

    Science.gov (United States)

    Lavigne, B; Audebert-Mérilhou, E; Buisson, G; Kochman, F; Clément, J P; Olliac, B

    2016-12-01

    Depression disorder may become the first cause of morbidity by 2030, according to the World Health Organization. It is actually one of the main causes of disease and handicap in children aged from 10 to 19. The major risk is suicide, whose prevalence is estimated, in France, around 6.7 for 100,000, which is probably underestimated. At present, the discussions about prescription of antidepressants in an adolescent's depression remain intense which is why psychotherapy becomes the first choice of treatment. We propose here to present one of them, Interpersonal PsychoTherapy (IPT), which remains largely unknown in France, and its adaptations in the adolescent population. IPT is a brief psychotherapy, structured in twelve to sixteen sessions, which was created by Klerman and Weissman in the seventies inspired by the biopsychosocial model of Meyer, interpersonal theory of Sullivan, and attachment theory of Bowlby. It is divided into three parts: the initial phase, the intermediate phase, and the termination phase. IPT was adapted for adolescents by Mufson in 1993, but a few modifications must be considered. Parental implication is the first. Indeed, parents, rather than the adolescent, often ask for the consultation; but it is the latter who benefits from the therapy. Parents may be met at some point in the therapy, for example between each phase and at the end. The initial phase is very close for the adolescent as for the adult; but the therapist must be careful about employing the "sick role" which can be used by the adolescent to avoid school, and as a consequence, to exacerbate the interpersonal deficit. The intermediate phase focuses on one of the four interpersonal issues: complicated bereavement, role transition, interpersonal role disputes, and interpersonal deficit. Complicated bereavement may become problematic when prolonged or when the adolescent had complicated relations with the deceased. The therapist essentially works on emotion verbalization. The role

  11. A Case Study of Internet Protocol Telephony (IPT) Implementation at United States Coast Guard Headquarters

    National Research Council Canada - National Science Library

    Patton, Mark B

    2005-01-01

    .... This thesis investigates the successful implementation practices and security policies of commercial, educationa, and goverment organizations in order to create recommendations for IPT security...

  12. Improvement of soybean transformation via Agrobacterium tumefaciens methods involving α-aminooxyacetic acid and sonication treatments enlightened by gene expression profile analysis.

    Science.gov (United States)

    Zhang, Yan-Min; Liu, Zi-Hui; Yang, Rui-Juan; Li, Guo-Liang; Guo, Xiu-Lin; Zhang, Hua-Ning; Zhang, Hong-Mei; Di, Rui; Zhao, Qing-Song; Zhang, Meng-Chen

    2016-06-01

    Antagonists and sonication treatment relieved the structural barriers of Agrobacterium entering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility to Agrobacterium infection. Soybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3'H, HCT, β-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant-pathogen interaction; and genes coding for GPAT and α-L-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of 'invasion' signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation

  13. [Obtaining transgenic rice plants and their progenies using Agrobacterium tumefaciens].

    Science.gov (United States)

    Yin, Z C; Yang, F; Xu, Y; Li, B J

    1998-12-01

    Rice (Oriza sativa L.) suspension cells of Taipei 309 were co-cultivated with A. tumefaciens stran EHA101 harbouring binary vector pBYT2 for 3 days in the presence of vir inducer, 100 mumol/L acetosyringone (AS). After 2 months of continuous selection, 17 stable hygromycin-resistant, GUS-positive calli were recovered from 364 suspension cell clusters co-cultivated with A. tumefaciens. 10 putative transgenic R0 plants obtained from 8 tansformed calli and their progenies were analyzed for the integration and expression of foreign genes. Southern blot analysis of R0 and R1 generations indicated that foreign genes had been stably integrated in the genome of transgenic rice and sexually transmitted. One of the transgenic lines showed 5 copies of T-DNA integration, while the others had only one copy. Histochemical staining observation and fluorometric assay of GUS activity in transgenic rice cells and plants showed ubiquitin promoter from maize was highly effective in driving the expression of gus reporter gene in transgenic rice cells. GUS protein and its activity were also investigated through ndPAGE-X-Gluc staining assay, and it was found that the GUS protein in transgenic rice cells was smaller in size than the standard GUS protein (Sigma Co. G0786) but as large as that from E.coli HB101 (pBI121). This study suggested that Agrobacterium-mediated transformation of plant is an efficient and reliable method to introduce foreign genes into rice.

  14. Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains

    Science.gov (United States)

    Novel primers were developed to amplify a 243 bp fragment of an intergenic region between gene5 and tms2 on the T-DNA of Agrobacterium tumefaciens. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence and did not generate extraneous bands,...

  15. Transgenic carnation plants obtained by Agrobacterium tumefaciens mediated transformation of petal explants

    NARCIS (Netherlands)

    Altvorst, van A.C.; Koehorst, H.; Jong, de J.; Dons, M.M.

    1996-01-01

    Transgenic carnation plants were obtained after infection of petal explants with the supervirulent Agrobacterium tumefaciens strain AGLO. Southern blot techniques confirmed the transgenic nature of four transformed plants. The expression of the gus gene was verified in these plants by histochemical

  16. Interpersonal Psychotherapy for Depressed Adolescents (IPT-A): A Case Illustration

    Science.gov (United States)

    Hall, Elisabeth Baerg; Mufson, Laura

    2009-01-01

    This article describes the treatment of a depressed adolescent (15 years of age) boy using Interpersonal Psychotherapy for depressed adolescents (IPT-A). IPT-A is an empirically supported psychosocial intervention for adolescents suffering from a depressive episode. It is delivered as an individual psychotherapy with a minimum of parental…

  17. Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population.

    Science.gov (United States)

    Lang, Julien; Faure, Denis

    2016-05-03

    Accumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations.

  18. Polyhydroxybutyrate and polyhydroxydodecanoate produced by Burkholderia contaminans IPT553.

    Science.gov (United States)

    Matias, F; Brandt, C A; da Silva, E S; de Andrade Rodrigues, M F

    2017-04-06

    In this paper, we introduce a new Burkholderia contaminans capable of producing a newly characterized polymer. CG-MS and magnetic nuclear resonance 1 H and 13 C were used to determine the constitution of polymers produced in glucose, glucose with casein, sucrose and sucrose with casein. Three pairs of primers were used to find the polyhydroxyalkanoates (PHA) synthase class and sequence. The synthesized polymers were composed by short-chain length PHA (scl-PHA), especially polyhydroxybutyrate (PHB), and medium chain length PHA (mcl-PHA), especially polyhydroxydodecanoate (PHDd), and their concentration, constitution and molecular weight depend on carbon source used. The bacterium showed only class I synthase which could not explain the mcl-PHA production. Burkholderia contaminans has a class I PHA synthase and produces PHB combined to PHDd when cultivated in sucrose or glucose, and PHDd concentration is affected when casein is used. PHA are natural polymers produced by a wide range of bacteria. The presence of PHDd monomers confers to the polymer elastomeric properties. Previously, PHDd was only obtained when bacteria were cultivated in related carbon source. In this work, B. contaminansIPT553 produced PHB with PHDd using simple and low-cost carbon sources that can make possible the cheaper production of a more flexible biopolymer with crystallinity and elasticity different from the more common PHAs. Journal of Applied Microbiology © 2017 The Society for Applied Microbiology.

  19. Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L. Kurz

    Directory of Open Access Journals (Sweden)

    Mallesham Bulle

    2015-12-01

    Full Text Available In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L. Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA containing intron as the reporter gene and hygromycin phosphotransferase (hpt as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM and Indole-3-acetic acid IAA (1.14 μM + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1 for 4 weeks (includes a single subculture onto the same medium at a 2 week interval. They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM and Indole-3-acetic acid IAA (1.14 μM + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2 medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

  20. Stimulation of Agrobacterium tumefaciens Growth by Azotobacter vinelandii Ferrisiderophores

    OpenAIRE

    Page, William J.; Dale, Phyllis L.

    1986-01-01

    Azotobacter vinelandii stimulated the growth of Agrobacterium tumefaciens H2, H23, H24, H27, and ATCC 15955 on media containing insoluble iron sources. The Azotobacter vinelandii siderophores appeared to promote Agrobacterium tumefaciens growth by solubilizing mineral iron, and the ferrisiderophores so formed then acted as iron sources for Agrobacterium tumefaciens. Agrobactin, the Agrobacterium siderophore, appeared to be inefficient in solubilizing mineral iron directly.

  1. Agrobacterium tumefaciens is a diazotrophic bacterium

    International Nuclear Information System (INIS)

    Kanvinde, L.; Sastry, G.R.K.

    1990-01-01

    This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate 15 N supplied as 15 N 2 . As with most other well-characterized diazotrophic bacteria, the presence of NH 4 + in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship

  2. Optimization of agrobacterium tumefaciens mediated transformation in populus deltoides

    International Nuclear Information System (INIS)

    John, E.; Maqbool, A.; Malik, K.A.

    2014-01-01

    The objective of the study was to develop an efficient protocol for Populus deltoides transformation through Agrobacterium tumefaciens LBA4404. Agrobacterium strain harboring binary plasmid pGA482 with Gus (uidA) gene under CamV35S promoter and Neomycin phosphotransferase (nptII) gene under Nos promoter was used for the transformation. Nodal, internodal and leaf explants from 4-5 months In vitro and fieldgrown plants were used for the transformation. Transformation was done under different conditions including, preculture time, optical density, acetosyringone concentration, infection time and co-cultivation time. Confirmation of transformation was done through GUS histochemical staining. Highest transformation efficiency was observed in one week precultured leaf explants from field grown source on preculture medium containing 200 meu M acetosyringone. Precultured explants from In vitro source also gave good results for transformation but the callus formation was found to be slow in leaf explant. Calli from the both sources did not show any transformation when infected with O.D A600nm range from 0.3-0.8. Node and internode though showed less transformation rate but the callogenesis was found to be highest in node and internode explants on CIM 1. Leaf explants from field source also gave high callus induction on CIM 5. A. tumefaciens O.D A600nm 0.3-0.5 was found to be effective. Infection time of 1-2 hour and co-cultivation time of 1day in dark were found to be optimum for the transformation. 200mg/l of timentin was found the best to control the overgrowth of Agrobacterium.100mg/l Kanamycin in growth medium was found to sufficient for selection for transformants. Selected transformants were confirmed through PCR for the presence of transgene. The present protocol for P. deltoides was found to be efficient for genetic transformation and can be used to introduce novel traits in the P. deltoides. (author)

  3. Genome sequence of the nicotine-degrading Agrobacterium tumefaciens S33.

    Science.gov (United States)

    Yu, Wenjun; Li, Huili; Xie, Kebo; Huang, Haiyan; Xie, Huijun; Wang, Shuning

    2016-06-20

    Agrobacterium tumefaciens S33 is capable of growing with nicotine as the sole source of carbon and nitrogen, and has the potential to dispose of tobacco wastes and transform nicotine into functionalized pyridines intermediates, which are important precursors for some valuable drugs and insecticides. Here we report the complete genome sequence of strain S33 and predict the gene cluster involved in nicotine catabolism according to the annotation. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

    NARCIS (Netherlands)

    Muniz, C.R.; Silva, da C.F.; Souza, M.T.; Freire, F.C.O.; Kema, G.H.J.; Guedes, M.I.F.

    2014-01-01

    Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp)

  5. [Meristematic characteristics of tumors initiated by Agrobacterium tumefaciens in pea plants].

    Science.gov (United States)

    Vinogradova, A P; Lebedeva, M A; Lutova, L A

    2015-01-01

    It is known that two key groups of plant hormones--auxins and cytokinins--play an important role in plant tumor development. The formation of Agrobacterium-induced tumors results from the horizontal transfer of bacterial oncogenes involved in the biosynthesis of these hormones in the plant genome. The role of transcriptional factors in plant tumor development is poorly investigated. It can be assumed that tumor development associated with abnormal cell proliferation can be controlled by the same set of transcription factors that control normal cell proliferation and, in particular, transcription factors that regulate meristem activity. In the present study, we analyzed the histological organization and distribution of proliferating cells in tumors induced by Agrobacterium tumefaciens on pea hypocotyls. In addition, the expression of a set of meristem-specific genes with Agrobacterium tumefaciens-induced tumor development was analyzed. In general, our results indicate that meristematic structures are present in A. tumefaciens-induced tumors and that the development of such tumors is associated with increased expression of a key gene regulating the root apical meristem--the WOX5 gene.

  6. Interpersonal psychotherapy (IPT for late-life depression in general practice: uptake and satisfaction by patients, therapists and physicians

    Directory of Open Access Journals (Sweden)

    de Haan Marten

    2007-09-01

    Full Text Available Abstract Background Interpersonal Psychotherapy (IPT is recommended in most depression treatment guidelines and proved to be a suitable treatment for elderly depressed patients. Despite the favorable results of IPT in research populations, the dissemination to general practice is surprisingly limited. Little is known about uptake and satisfaction when this therapy is introduced into real-life general practice. Methods Motivation and evaluation of patients, GPs and therapists were recorded and organizational barriers described alongside a randomized controlled trial. IPT, given by mental health workers, was compared with usual general practitioner (GP care. Included were patients (≥55 years who met the DSM-IV criteria for major depressive disorder. Results Patients were motivated for the psychotherapy intervention: of the 205 eligible patients, 143 (70% entered the study, and of the 69 patients who were offered IPT, 77% complied with the treatment. IPT proved to be an attractive therapy for patients as well as for therapists from mental health organizations. General practitioners evaluated the intervention positively afterwards, mainly because of the time-limited and structured approach. Organizational barriers: no IPT therapists were available; an IPT trainer and supervisor had to be trained and training materials had to be developed and translated. Additionally, there was a lack of office space in some general practices; for therapists from private practices it was not feasible to participate because of financial reasons. IPT was superior to usual care in patients with moderate to severe depression. Conclusion As we succeeded in delivering IPT in primary care practice, and as IPT was superior to usual care, there are grounds to support the implementation of IPT for depressed elderly patients within general practice, as long as the practices have room for the therapists and financial barriers can be overcome. Consolidation may be achieved by

  7. Impact of biological amendments on Agrobacterium tumefaciens soil survival

    Science.gov (United States)

    Paradox, the primary walnut rootstock used in California, is susceptible to Agrobacterium tumefaciens, which causes crown gall. While A. tumefaciens is susceptible to commonly used fumigants such as methyl bromide (MeBr) and Telone-C35 (1,3-dichloropropene and chloropicrin), these fumigants also sig...

  8. Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

    Science.gov (United States)

    2010-01-01

    Background White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi. Results This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene

  9. Agrobacterium tumefaciens responses to plant-derived signaling molecules

    Directory of Open Access Journals (Sweden)

    Sujatha eSubramoni

    2014-07-01

    Full Text Available As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium-plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its T-DNA (Transferred DNA from its Tumour-inducing (Ti plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA, cytokinin (CK and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including -amino butyric acid (GABA and salicylic acid (SA to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene (ET to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium-plant interactions.

  10. Agrobacterium tumefaciens responses to plant-derived signaling molecules

    Science.gov (United States)

    Subramoni, Sujatha; Nathoo, Naeem; Klimov, Eugene; Yuan, Ze-Chun

    2014-01-01

    As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium–plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including γ-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium–plant interactions. PMID:25071805

  11. Transformation of medicinal plants using Agrobacterium tumefaciens.

    Science.gov (United States)

    Bandurska, Katarzyna; Berdowska, Agnieszka; Król, Małgorzata

    2016-12-20

    For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall). This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

  12. Transformation of medicinal plants using Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Katarzyna Bandurska

    2016-12-01

    Full Text Available For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall. This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

  13. Hfq influences multiple transport systems and virulence in the plant pathogen Agrobacterium tumefaciens.

    Science.gov (United States)

    Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min; Narberhaus, Franz

    2012-10-01

    The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.

  14. Bacillus amyloliquefaciens strain 32a as a source of lipopeptides for biocontrol of Agrobacterium tumefaciens strains.

    Science.gov (United States)

    Ben Abdallah, D; Frikha-Gargouri, O; Tounsi, S

    2015-07-01

    A Bacillus amyloliquefaciens strain, designated 32a, was used to identify new compounds active against Agrobacterium tumefaciens and to evaluate their efficiency to control crown gall on carrot discs. Based on PCR-assays, four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A, bacillomycin D and fengycin. Mass spectrometry analysis of culture supernatant led to the identification of these secondary metabolites, except bacillomycin, with heterogeneous mixture of homologues. Antimicrobial assays using lipopeptides-enriched extract showed a strong inhibitory activity against several bacterial and fungal strains, including Ag. tumefaciens. Biological control assays on carrot discs using both 32a spores and extract resulted in significant protection against crown gall disease, similar to that provided by the reference antagonistic strain Agrobacterium rhizogenes K1026. In contrast to all active compounds against Ag. tumefaciens that are of proteinaceous nature, this work enables for the first time to correlate the strong protective effect of B. amyloliquefaciens strain 32a towards crown gall disease with the production of a mixture of lipopeptides. The findings could be useful for growers and nursery men who are particularly interested in the biocontrol of the crown gall disease. © 2015 The Society for Applied Microbiology.

  15. Cloning and characterization of uronate dehydrogenases from two pseudomonads and Agrobacterium tumefaciens strain C58.

    Science.gov (United States)

    Yoon, Sang-Hwal; Moon, Tae Seok; Iranpour, Pooya; Lanza, Amanda M; Prather, Kristala Jones

    2009-03-01

    Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k(cat)) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K(m)) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k(cat) = 1.9 x 10(2) s(-1) on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

  16. Optimization of agrobacterium tumefaciens mediated transformation in eucalyptus camaldulensis

    International Nuclear Information System (INIS)

    Ahad, A.; Maqbool, A.; Malik, K.A.

    2014-01-01

    This study was conducted to optimize Agrobacterium tumefaciens mediated transformation for Eucalyptus camaldulensis. Transformation was done by using LBA4404 containing binary plasmid pGA482 with uidA (Gus) gene under CamV35S promoter and nptII gene under nos promoter. For optimization, different explants (Cotyledonary leaves, plantlet leaves and hypocotyls of young In vitro plants and calli) with and without preculture were infected with a range of optical densities (O.D.600nm=0.3-0.6). Effect of different concentrations of Acetosyringone, infection time and co-cultivation time on transformation efficiency was evaluated. Confirmation of transformation was done through GUS histochemical staining and through PCR. Callogenesis and regeneration was found fast on MS medium containing 0.5 mg/L NAA and 1.5 mg/L BAP. Highest transformation efficiency was obtained with bacterial suspension of O.D.600nm = 0.5 for non-precultured explants and O.D.600nm=0.3 for precultured explants. (author)

  17. Plant responses to Agrobacterium tumefaciens and crown gall development

    Science.gov (United States)

    Gohlke, Jochen; Deeken, Rosalia

    2014-01-01

    Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (“omic”) approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant. PMID:24795740

  18. Do depression treatments reduce suicidal ideation? The effects of CBT, IPT, pharmacotherapy, and placebo on suicidality.

    Science.gov (United States)

    Weitz, Erica; Hollon, Steven D; Kerkhof, Ad; Cuijpers, Pim

    2014-01-01

    Many well-researched treatments for depression exist. However, there is not yet enough evidence on whether these therapies, designed for the treatment of depression, are also effective for reducing suicidal ideation. This research provides valuable information for researchers, clinicians, and suicide prevention policy makers. Analysis was conducted on the Treatment for Depression Research Collaborative (TDCRP) sample, which included CBT, IPT, medication, and placebo treatment groups. Participants were included in the analysis if they reported suicidal ideation on the HRSD or BDI (score of ≥1). Multivariate linear regression indicated that both IPT (b=.41, pdepression these treatment effects were no longer significant. Moderate Cohen׳s d effect sizes from baseline to post-test differences in suicide score by treatment group are reported. These analyses were completed on a single suicide item from each of the measures. Moreover, the TDCRP excluded participants with moderate to severe suicidal ideation. This study demonstrates the specific effectiveness of IPT and medications in reducing suicidal ideation (relative to placebo), albeit largely as a consequence of their more general effects on depression. This adds to the growing body of evidence that depression treatments, specifically IPT and medication, can also reduce suicidal ideation and serves to further our understanding of the complex relationship between depression and suicide. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth

    Czech Academy of Sciences Publication Activity Database

    Schnablová, Renáta; Synková, Helena; Vičánková, Anna; Burketová, Lenka; Eder, Josef; Cvikrová, Milena

    2006-01-01

    Roč. 44, - (2006), s. 526-534 ISSN 0981-9428 R&D Projects: GA ČR GA206/03/0310 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinins * ipt * transgenic tobacco * in vitro cultivation Subject RIV: EF - Botanics Impact factor: 1.847, year: 2006

  20. Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Kim, J.B.; Raemakers, C.J.J.M.; Jacobsen, E.; Visser, R.G.F.

    2007-01-01

    A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone

  1. Dopamine transporter density of the basal ganglia assessed with I-123 IPT SPECT in methamphetamine abusers

    International Nuclear Information System (INIS)

    Lee, Joo Ryung; Ahn, Byeong Cheol; Kewm, Do Hun

    2005-01-01

    Functional imaging of dopamine transporter (DAT) defines integrity of the dopaminergic system, and DAT is the target site of drugs of abuse such as cocaine and methamphetamine. Functional imaging the DAT may be a sensitive and selective indicator of neurotoxic change by the drug. The aim of the present study is to evaluate the clinical implications of qualitative/quantitative analyses of dopamine transporter imaging in methamphetamine abusers. Six detoxified methamphetamine abusers (abuser group) and 4 volunteers (control group) were enrolled in this study. Brain MRI was performed in all of abuser group. Abuser group underwent psychiatric and depression assessment using brief psychiatric rating scale (BPRS) and Hamilton depression rating scale (HAMD), respectively. All of the subjects underwent I-123 IPT SPECT (IPT SPECT). IPT SPECT image was analysed with visual qualitative method and quantitative method using basal ganglia dopamine transporter (DAT) specific/non-specific binding ratio (SBR). Comparison of DAT SBR between abuser and control groups was performed. We also performed correlation tests between psychiatric and depression assessment results and DAT SBR in abuser group. All of abuser group showed normal MRI finding, but had residual psychiatric and depressive symptoms, and psychiatric and depressive symptom scores were exactly correlated (r=1.0, ρ =0.005) each other. Five of them showed abnormal finding on qualitative visual I-123 IPT SPECT. Abuser group had lower basal ganglia DAT SBR than that of control (2.38 ± 0.20 vs 3.04 ± 0.27, ρ =0.000). Psychiatric and depressive symptoms were negatively well correlated with basal ganglia DAT SBR (r=-0.908, ρ =0.012, r=-0.924, ρ =0.009) This results suggest that dopamine transporter imaging using I-123 IPT SPECT may be used to evaluate dopaminergic system of the basal ganglia and the clinical status in methamphetamine abusers

  2. Dopamine transporter density of the basal ganglia assessed with I-123 IPT SPECT in methamphetamine abusers

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joo Ryung; Ahn, Byeong Cheol [Kyungpook National University Medical School, Daegu (Korea, Republic of); Kewm, Do Hun [National Bugok Mental Hospital, Changryung (Korea, Republic of)] (and others)

    2005-10-15

    Functional imaging of dopamine transporter (DAT) defines integrity of the dopaminergic system, and DAT is the target site of drugs of abuse such as cocaine and methamphetamine. Functional imaging the DAT may be a sensitive and selective indicator of neurotoxic change by the drug. The aim of the present study is to evaluate the clinical implications of qualitative/quantitative analyses of dopamine transporter imaging in methamphetamine abusers. Six detoxified methamphetamine abusers (abuser group) and 4 volunteers (control group) were enrolled in this study. Brain MRI was performed in all of abuser group. Abuser group underwent psychiatric and depression assessment using brief psychiatric rating scale (BPRS) and Hamilton depression rating scale (HAMD), respectively. All of the subjects underwent I-123 IPT SPECT (IPT SPECT). IPT SPECT image was analysed with visual qualitative method and quantitative method using basal ganglia dopamine transporter (DAT) specific/non-specific binding ratio (SBR). Comparison of DAT SBR between abuser and control groups was performed. We also performed correlation tests between psychiatric and depression assessment results and DAT SBR in abuser group. All of abuser group showed normal MRI finding, but had residual psychiatric and depressive symptoms, and psychiatric and depressive symptom scores were exactly correlated (r=1.0, {rho} =0.005) each other. Five of them showed abnormal finding on qualitative visual I-123 IPT SPECT. Abuser group had lower basal ganglia DAT SBR than that of control (2.38 {+-} 0.20 vs 3.04 {+-} 0.27, {rho} =0.000). Psychiatric and depressive symptoms were negatively well correlated with basal ganglia DAT SBR (r=-0.908, {rho} =0.012, r=-0.924, {rho} =0.009) This results suggest that dopamine transporter imaging using I-123 IPT SPECT may be used to evaluate dopaminergic system of the basal ganglia and the clinical status in methamphetamine abusers.

  3. Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae-induced hypersensitive response and non-host resistance in Nicotiana benthamiana.

    Science.gov (United States)

    Li, Wen; Cao, Jia-Yi; Xu, You-Ping; Cai, Xin-Zhong

    2017-05-01

    Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non-host resistance in its non-host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo-induced HR in N. benthamiana when pre-infiltrated or co-infiltrated, but not when post-infiltrated at 4 h after Xoo inoculation. This suppression by A. tumefaciens is local and highly efficient to Xoo. The HR-inhibiting efficiency of A. tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo-induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR-inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild-type A. tumefaciens strain C58 and a set of C58-derived strains reveals that this Xoo antibiosis activity of A. tumefaciens is negatively, but not solely, regulated by the transferred-DNA (T-DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo-induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A. tumefaciens on Xoo in N. benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A. tumefaciens and the non-host resistance induced by Xoo. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  4. Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens.

    Science.gov (United States)

    Wang, Yi; Kim, Sok Ho; Natarajan, Ramya; Heindl, Jason E; Bruger, Eric L; Waters, Christopher M; Michael, Anthony J; Fuqua, Clay

    2016-10-01

    In bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene in Agrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants for odc grew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite in A. tumefaciens and is synthesized from putrescine in A. tumefaciens via the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to the odc mutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for the odc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. The odc mutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue. Polyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several

  5. Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division.

    Science.gov (United States)

    Anderson-Furgeson, James C; Zupan, John R; Grangeon, Romain; Zambryski, Patricia C

    2016-07-01

    alphaproteobacteria, including Agrobacterium tumefaciens Essential components for unipolar growth are largely uncharacterized, and the mechanism constraining growth to one pole of a wild-type cell is unknown. Here, we report that the deletion of a polar development gene, podJAt, results in cells exhibiting ectopic polar growth, including multiple growth poles and aberrant localization of cell division and polar growth-associated proteins. These data suggest that PodJAt is a critical factor in normal polar growth and impacts cell division in A. tumefaciens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. A simplified and efficient Agrobacterium tumefaciens electroporation method.

    Science.gov (United States)

    Kámán-Tóth, Evelin; Pogány, Miklós; Dankó, Tamás; Szatmári, Ágnes; Bozsó, Zoltán

    2018-03-01

    Agrobacterium tumefaciens is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent Agrobacterium cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g., Agrobacterium strains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly into Agrobacterium omitting the E. coli transformation step and accelerating the cloning procedure further.

  7. Characterization of four ribosomal RNA operons in the genome of Agrobacterium tumefaciens MAFF301001.

    Science.gov (United States)

    Bautista-Zapanta, J-ney; Suzuki, Katsunori; Yoshida, Kazuo

    2002-01-01

    The four ribosomal RNA (rrn) operons rrnA, rrnB, rrnC and were identified, sequenced completely and characterized individually rrnD in the whole genome of A tumefaciens MAFF301001. These rrn operons were located in the two topologically different chromosomal DNAs; rrnA and rrnD in the linear chromosome and rrnB and rrnC in the circular chromosome. Each operon coded for three ribosomal RNA subunit genes; 16S, 23S and 5S rRNA. The 16S-23S internal transcribed spacers (ITS) of the four rrn operons contain genes for tRNA-Ile and tRNA-Ala and tRNA-Met downstream of 5S rDNA gene while the intergenic spacer between 23S rDNA and 5S rDNA lacked tRNA genes. Sequence alignment of 23S rRNAs of A. tumefaciens MAFF301001 and C58 strains showed unrelated sequences near the 5' end region suggesting the presence of intervening sequence (IVS). Primer extension analyses revealed that the primary transcription products for 16S and 23S rRNAs are 1497 and 2877 bases long, respectively.

  8. T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus Transferencia de T-DNA de Agrobacterium tumefaciens al hongo ectomicorrícico Pisolithus microcarpus

    Directory of Open Access Journals (Sweden)

    A.G. Pardo

    2005-06-01

    Full Text Available The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.

  9. An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants

    OpenAIRE

    Nonaka, Satoko; Someya, Tatsuhiko; Zhou, Sha; Takayama, Mariko; Nakamura, Kouji; Ezura, Hiroshi

    2017-01-01

    Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DN...

  10. Sikap Dan Motivasi Pelajar IPT terhadap Modul Pengajaran Bahasa Arab Pelancongan

    Directory of Open Access Journals (Sweden)

    M. Najib, J.

    2014-12-01

    Full Text Available Kajian ini dilakukan untuk menganalisis sikap dan motivasi pelajar IPT di Malaysia terhadap modul pengajaran Bahasa Arab Pelancongan. Sampel kajian terdiri daripada 126 pelajar di tiga IPT iaitu USIM, UiTM dan KUIS yang menawarkan subjek Bahasa Arab Pelancongan. Mereka terdiri daripada 30 pelajar lelaki dan 96 pelajar perempuan. Kajian ini mendapati peratusan tahap sikap dan motivasi terhadap modul pengajaran Bahasa Arab Pelancongan adalah tinggi dengan peratusan sebanyak hampir 90%. Responden juga mempunyai persetujuan yang agak baik terhadap keberkesanan modul pengajaran Bahasa Arab Pelancongan dengan skor min = 3.63 dan sp. = 0.65. Lebih 85% responden bersetuju bahawa modul pengajaran Bahasa Arab Pelancongan dapat diaplikasi dalam bidang kerjaya di sektor pelancongan. Kebolehpercayaan instrumen pengukuran adalah tinggi dengan Cronbach Alpha bagi sikap dan motivasi terhadap modul pengajaran Bahasa Arab Pelancongan ialah 0.96.

  11. Effects of Conditional IPT-Dependent Cytokinin Overproduction on Root Architecture of Arabidopsis Seedlings

    Czech Academy of Sciences Publication Activity Database

    Kuderová, A.; Urbánková, I.; Válková, M.; Malbeck, Jiří; Brzobohatý, Břetislav; Némethová, D.; Hejátko, J.

    2008-01-01

    Roč. 49, č. 4 (2008), s. 570-582 ISSN 0032-0781 R&D Projects: GA MŠk(CZ) LC06034; GA AV ČR(CZ) IAA600380507 Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50040507 Keywords : Arabidopsis thaliana * Auxin–cytokinin cross-talk * CaMV 35S>GR>ipt transactivation Subject RIV: ED - Physiology Impact factor: 3.542, year: 2008

  12. Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens.

    Science.gov (United States)

    Ahmad, Tauqeer; Venkataraman, Srividhya; Hefferon, Kathleen; AbouHaidar, Mounir G

    2014-09-12

    The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5' leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5' UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5' UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5' leader region upstream of the gene of interest. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Genetic transformation of the Brazilian BR 451 maize variety by the Agrobacterium tumefaciens method

    Directory of Open Access Journals (Sweden)

    Marilia Rodrigues de Silva

    2017-10-01

    Full Text Available ABSTRACT: The asexually gene introduction by genetic engineering has brought enormous possibilities to innovate plant breeding. However, principally because of the low in vitro response, genetic transformation has been restricted to only certain genotypes of agronomically significant species. With the objective of establishing a protocol for genetically transforming the Brazilian BR 451 maize variety through Agrobacterium tumefaciens, it was studied the capacity of plant regeneration in vitro from embryogenic calli cultivated in three regeneration media, each having different growth regulators. It was also evaluated the temperature stress effect on the transformation of the immature embryos with A. tumefaciens EHA 101 containing the plasmid pTF102 with uidA and bar genes. The BR 451 variety embryos and those of the Hi-II hybrid (control were exposed to three treatments applied as they were being infected with the agrobacteria (a infection at 25°C; (b infection at 40°C; (c pretreatment at 40°C for 5 seconds followed by infection at 25°C. Transformation was determined by uidA gene expression and through the callus resistant to the herbicide Bialaphos® formation. Embryos infected at 40°C showed a higher degree of genetic transformation in the Hi-II, although the same was not noted in BR 451. When growth regulators were added to the culture medium the number of regenerated BR 451 plants showed no increase.

  14. Universality of interpersonal psychotherapy (IPT problem areas in Thai depressed patients

    Directory of Open Access Journals (Sweden)

    Thavichachart Nuntika

    2010-10-01

    Full Text Available Abstract Background Many studies have shown the efficacy of interpersonal psychotherapy (IPT on depression; however, there are limited studies concerning the universality of the IPT problem areas in different countries. This study identifies whether the interpersonal problem areas defined in the IPT manual are endorsed by Thai depressed patients. Methods The Thai Hamilton Rating Scale for Depression (Thai HRSD and Thai Interpersonal Questionnaire were used to assess 90 depressed and 90 non-depressed subjects in King Chulalongkorn Memorial Hospital, during July 2007 - January 2008. The association between interpersonal problem areas/sociodemographic variables and depressive disorder were analyzed by chi-square test. A multivariable analysis was performed by using logistic regression to identify the remaining factors associated with depressive disorder. Results Most of the subjects were young to middle-aged females living in Bangkok and the Central Provinces. All four interpersonal problem areas (grief, interpersonal role disputes, role transitions, and interpersonal deficits were increased in the depressed subjects as compared to the non-depressed subjects, as were the sociodemographic variables (low education, unemployment, low income, and having a physical illness. Logistic regression showed that all interpersonal problem areas still remained problems associated with depression (grief: adjusted OR = 6.01, 95%CI = 1.93 - 18.69, p Conclusion All four interpersonal problem areas were applicable to Thai depressed patients.

  15. Family-Based Interpersonal Psychotherapy (FB-IPT) for Depressed Preadolescents: Examining Efficacy and Potential Treatment Mechanisms

    Science.gov (United States)

    Dietz, Laura J.; Weinberg, Rebecca J.; Brent, David A.; Mufson, Laura

    2014-01-01

    Objective To conduct a randomized controlled trial to evaluate the preliminary efficacy of family-based interpersonal psychotherapy (FB-IPT) for treating depression in preadolescents (ages 7–12) as compared to child-centered therapy (CCT), a supportive and nondirective treatment that closely approximates the standard of care for pediatric depression in community mental health. Method Preadolescents with depression (N=42) were randomly assigned FB-IPT or CCT. Pre- and posttreatment assessments included clinician-administered measures of depression, parent- and child-reported depression and anxiety symptoms, and parent-child conflict and interpersonal impairment with peers. Results Preadolescents receiving FB-IPT had higher rates of remission (66.0% vs. 31%), a greater decrease in depressive symptoms from pre- to posttreatment, and lower depressive symptoms at posttreatment (R2=0.35, Δ R2 = 0.22; B= -8.15, SE= 2.61, t(37)= -3.13, p=0.002, F2=0.28) than did preadolescents with depression receiving CCT. Furthermore, preadolescents in the FB-IPT condition reported significant reductions in anxiety and interpersonal impairment than did preadolescents in the CCT condition. Changes in social and peer impairment from pre- to posttreatment were associated with preadolescents’ posttreatment depressive symptoms. There was a significant indirect effect for decreased social impairment accounting for the association between the FB-IPT and preadolescents’ posttreatment depressive symptoms. Conclusion Findings indicate FB-IPT is an effective treatment for preadolescent depression and support further investigation of interpersonal mechanisms by which FB-IPT may reduce preadolescent depression. Clinical trial registration information Phase II Study of Family Based Interpersonal Psychotherapy (FB-IPT) for Depressed Preadolescents; http://clinicaltrials.gov/show/NCT02054312; NCT02054312. PMID:25721184

  16. Functions and regulation of quorum-sensing in Agrobacterium tumefaciens

    Science.gov (United States)

    Lang, Julien; Faure, Denis

    2014-01-01

    In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids. PMID:24550924

  17. Functions and regulation of quorum-sensing in Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Denis eFaure

    2014-01-01

    Full Text Available In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS. The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last twenty years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids.

  18. Identification and Characterization of a Second Quorum-Sensing System in Agrobacterium tumefaciens A6

    Science.gov (United States)

    Wang, Chao; Yan, Chunlan; Fuqua, Clay

    2014-01-01

    Quorum sensing (QS) is a widespread mechanism of bacterial communication in which individual cells produce and respond to small chemical signals. In Agrobacterium tumefaciens, an acylhomoserine lactone-dependent QS mechanism is known to regulate the replication and conjugation of the tumor-inducing (Ti) plasmid. Most of the QS regulatory proteins are encoded within the Ti plasmid. Among them, TraI is the LuxI-type enzyme synthesizing the QS signal N-3-oxooctanoyl-l-homoserine lactone (3OC8HSL), TraR is the LuxR-type transcriptional factor that recognizes 3OC8HSL, and TraM is an antiactivator that antagonizes TraR. Recently, we identified a TraM homolog encoded by the traM2 gene in the chromosomal background of A. tumefaciens A6. In this study, we further identified additional homologs (TraI2 and TraR2) of TraI and TraR in this strain. We showed that similar to TraI, TraI2 could predominantly synthesize the QS signal 3OC8HSL. We also showed that TraR2 could recognize 3OC8HSL and activate the tra box-containing promoters as efficiently as TraR. Further analysis showed that traM2, traI2, and traR2 are physically linked on a mobile genetic element that is not related to the Ti plasmid. These findings indicate that A. tumefaciens A6 carries a second QS system that may play a redundant role in the regulation of the replication and conjugation of the Ti plasmid. PMID:24464459

  19. Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens

    Science.gov (United States)

    Voigt, Boris; Menzel, Diedrik; Baluška, František; Villanueva, Marco A.

    2015-01-01

    Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium. PMID:26167858

  20. Growth kinetics and scale-up of Agrobacterium tumefaciens.

    Science.gov (United States)

    Leth, Ingrid K; McDonald, Karen A

    2017-06-01

    Production of recombinant proteins in plants through Agrobacterium-mediated transient expression is a promising method of producing human therapeutic proteins, vaccines, and commercial enzymes. This process has been shown to be viable at a large scale and involves growing large quantities of wild-type plants and infiltrating the leaf tissue with a suspension of Agrobacterium tumefaciens bearing the genes of interest. This study examined one of the steps in this process that had not yet been optimized: the scale-up of Agrobacterium production to sufficient volumes for large-scale plant infiltration. Production of Agrobacterium strain C58C1 pTFS40 was scaled up from shake flasks (50-100 mL) to benchtop (5 L) scale with three types of media: Lysogeny broth (LB), yeast extract peptone (YEP) media, and a sucrose-based defined media. The maximum specific growth rate (μ max ) of the strain in the three types of media was 0.46 ± 0.04 h -1 in LB media, 0.43 ± 0.03 h -1 in YEP media, and 0.27 ± 0.01 h -1 in defined media. The maximum biomass concentration reached at this scale was 2.0 ± 0.1, 2.8 ± 0.1, and 2.6 ± 0.1 g dry cell weight (DCW)/L for the three media types. Production was successfully scaled up to a 100-L working volume reactor with YEP media, using k L a as the scale-up parameter.

  1. Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

    Directory of Open Access Journals (Sweden)

    Wang Quan

    2012-06-01

    Full Text Available Abstract Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus produces many terpenoid indole alkaloids (TIAs, such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS gene and a selectable marker neomycin phosphotransferase II gene (NTPII. The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC

  2. Transgene expression in tick cells using agrobacterium tumefaciens

    Science.gov (United States)

    Ticks transmit infectious diseases to humans and other animals. Genetic manipulation of these arthropods would allow the development of alternative disease control strategies. Interestingly, Agrobacterium tumefaciens (At) mediated T-DNA transfer has been recently shown to promote the genetic modific...

  3. Attachment study of Agrobacterium tumefaciens to tef [ Eragrostis tef ...

    African Journals Online (AJOL)

    Agrobacterium tumefaciens attachment as a factor in transformation was investigated in tef (Eragrostis tef) zygotic embryos, seeds, seedlings, leaf bases and embryogenic callus; leaf discs of yam species (Dioscorea bulbifera, Dioscorea caynensis and Dioscorea alata); and leaf discs of tobacco (Nicotina tabaccum).

  4. Genetic Analysis of Agrobacterium tumefaciens Unipolar Polysaccharide Production Reveals Complex Integrated Control of the Motile-to-Sessile Switch

    Science.gov (United States)

    Xu, Jing; Kim, Jinwoo; Koestler, Benjamin J.; Choi, Jeong-Hyeon; Waters, Christopher M.; Fuqua, Clay

    2013-01-01

    Summary Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive cyclic diguanosine monophosphate phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment. PMID:23829710

  5. An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants

    Science.gov (United States)

    Nonaka, Satoko; Someya, Tatsuhiko; Zhou, Sha; Takayama, Mariko; Nakamura, Kouji; Ezura, Hiroshi

    2017-01-01

    Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DNA transfer. In this study, we examined the effect of GABA on T-DNA transfer using a tomato line with a low GABA content. Compared with the control, the T-DNA transfer frequency was increased in the low-GABA tomato line, indicating that GABA inhibits T-DNA transfer. Therefore, we bred a new A. tumefaciens strain with GABA transaminase activity and the ability to degrade GABA. The A. tumefaciens strain exhibited increased T-DNA transfer in two tomato cultivars and Erianthus arundinacues and an increased frequency of stable transformation in tomato. PMID:28220841

  6. An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants.

    Science.gov (United States)

    Nonaka, Satoko; Someya, Tatsuhiko; Zhou, Sha; Takayama, Mariko; Nakamura, Kouji; Ezura, Hiroshi

    2017-02-21

    Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DNA transfer. In this study, we examined the effect of GABA on T-DNA transfer using a tomato line with a low GABA content. Compared with the control, the T-DNA transfer frequency was increased in the low-GABA tomato line, indicating that GABA inhibits T-DNA transfer. Therefore, we bred a new A. tumefaciens strain with GABA transaminase activity and the ability to degrade GABA. The A. tumefaciens strain exhibited increased T-DNA transfer in two tomato cultivars and Erianthus arundinacues and an increased frequency of stable transformation in tomato.

  7. Small high-yielding binary Ti vectors pLSU with co-directional replicons for Agrobacterium tumefaciens-mediated transformation of higher plants.

    Science.gov (United States)

    Lee, Seokhyun; Su, Guiying; Lasserre, Eric; Aghazadeh, Monty Arta; Murai, Norimoto

    2012-05-01

    Small high-yielding binary Ti vectors of Agrobacterium tumefaciens were constructed to increase the cloning efficiency and plasmid yield in Escherichia coli and A. tumefaciens for transformation of higher plants. We reduced the size of the binary vector backbone to 4566bp with ColE1 replicon (715bp) for E. coli and VS1 replicon (2654bp) for A. tumefaciens, a bacterial kanamycin resistance gene (999bp), and the T-DNA region (152bp). The binary Ti vectors with the truncated VS1 replicon were stably maintained with more than 98% efficiency in A. tumefaciens without antibiotic selection for 4 days of successive transfers. The transcriptional direction of VS1 replicon can be the same as that of ColE1 replicon (co-directional transcription), or opposite (head-on transcription) as in the case of widely used vectors (pPZP or pCambia). New binary vectors with co-directional transcription yielded in E. coli up to four-fold higher transformation frequency than those with the head-on transcription. In A. tumefaciens the effect of co-directional transcription is still positive in up to 1.8-fold higher transformation frequency than that of head-on transcription. Transformation frequencies of new vectors are over six-fold higher than those of pCambia vector in A. tumefaciens. DNA yields of new vectors were three to five-fold greater than pCambia in E. coli. The proper functions of the new T-DNA borders and new plant selection marker genes were confirmed after A. tumefaciens-mediated transformation of tobacco leaf discs, resulting in virtually all treated leaf discs transformed and induced calli. Genetic analysis of kanamycin resistance trait among the progeny showed that the kanamycin resistance and sensitivity traits were segregated into the 3:1 ratio, indicating that the kanamycin resistance genes were integrated stably into a locus or closely linked loci of the nuclear chromosomal DNA of the primary transgenic tobacco plants and inherited to the second generation. © 2012

  8. Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens

    Science.gov (United States)

    Paradox (Juglans hindsii x J. regia), the dominant rootstock used in the California walnut industry, is susceptible to crown gall, caused by Agrobacterium tumefaciens. In practice, soil fumigation has been a common preplant management strategy for crown gall, but even an industry standard, methyl b...

  9. A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Traore Sy

    2011-12-01

    Full Text Available Abstract Background Cloning genes into plasmid vectors is one of the key steps for studying gene function. Recently, Invitrogen™ developed a convenient Gateway® cloning system based on the site-specific DNA recombination properties of bacteriophage lambda and the cytotoxic protein ccdB, which is lethal to most E. coli strains. The ccdB protein, however, is not toxic to Agrobacterium tumefaciens, an important player often used for studying gene function in planta. This limits the direct application of the Gateway® cloning system in plant transformation-mediated research. Results In this study, we constructed a novel Gateway®-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker. Conclusion Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway® cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.

  10. A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens.

    Science.gov (United States)

    Traore, Sy Mamadou; Zhao, Bingyu

    2011-12-07

    Cloning genes into plasmid vectors is one of the key steps for studying gene function. Recently, Invitrogen™ developed a convenient Gateway® cloning system based on the site-specific DNA recombination properties of bacteriophage lambda and the cytotoxic protein ccdB, which is lethal to most E. coli strains. The ccdB protein, however, is not toxic to Agrobacterium tumefaciens, an important player often used for studying gene function in planta. This limits the direct application of the Gateway® cloning system in plant transformation-mediated research. In this study, we constructed a novel Gateway®-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker. Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway® cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.

  11. Genome Sequence and Mutational Analysis of Plant-Growth-Promoting Bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a Zinc-Lead Mine Tailing

    Science.gov (United States)

    Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J.

    2012-01-01

    The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006

  12. Construction of an engineering strain producing high yields of α-transglucosidase via Agrobacterium tumefaciens-mediated transformation of Asperillus niger.

    Science.gov (United States)

    Li, Ming; Zhou, Liying; Liu, Meng; Huang, Yunyan; Sun, Xin; Lu, Fuping

    2013-01-01

    In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was used in breeding industrial strains for the purpose of improving α-transglucosidase production. Firstly, an efficient ATMT system for Asperillus niger was established by optimization of several influencing factors, in which transformation efficiency was improved up to 14-fold compared with the initial conditions. Furthermore, binary vector pBI-Glu containing an α-transglucosidase expression cassette was constructed and transferred into Agrobacterium tumefaciens LBA4404 in order to infect A. niger. By the efficient ATMT method, the gene for α-transglucosidase, driven by strong promoter PglaA (the glucoamylase gene promoter), had a high expression level in A. niger A-8 (25.02 U/mL). The optimized ATMT system was found to be effective and suitable for A. niger, and should be a useful tool for studying the function of A. niger genes and for industrial breeding of this strain.

  13. Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

    Science.gov (United States)

    Hoffmann, Marie-Christine; Ali, Koral; Sonnenschein, Marleen; Robrahn, Laura; Strauss, Daria; Narberhaus, Franz; Masepohl, Bernd

    2016-09-01

    Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators. © 2016 John Wiley & Sons Ltd.

  14. Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions.

    Science.gov (United States)

    Simmons, Christopher W; VanderGheynst, Jean S; Nitin, N

    2014-01-01

    Transient expression of recombinant proteins in plant tissues following Agrobacterium-mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub-optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10(8) , 10(9) , and 10(10) CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break-Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10(8) , 10(9) , and 10(10) CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue. © 2014 American Institute of Chemical Engineers.

  15. DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

    Directory of Open Access Journals (Sweden)

    Jochen Gohlke

    Full Text Available Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes

  16. Discrete Responses to Limitation for Iron and Manganese in Agrobacterium tumefaciens: Influence on Attachment and Biofilm Formation.

    Science.gov (United States)

    Heindl, Jason E; Hibbing, Michael E; Xu, Jing; Natarajan, Ramya; Buechlein, Aaron M; Fuqua, Clay

    2015-12-28

    Transition metals such as iron and manganese are crucial trace nutrients for the growth of most bacteria, functioning as catalytic cofactors for many essential enzymes. Dedicated uptake and regulatory systems have evolved to ensure their acquisition for growth, while preventing toxicity. Transcriptomic analysis of the iron- and manganese-responsive regulons of Agrobacterium tumefaciens revealed that there are discrete regulatory networks that respond to changes in iron and manganese levels. Complementing earlier studies, the iron-responsive gene network is quite large and includes many aspects of iron-dependent metabolism and the iron-sparing response. In contrast, the manganese-responsive network is restricted to a limited number of genes, many of which can be linked to transport and utilization of the transition metal. Several of the target genes predicted to drive manganese uptake are required for growth under manganese-limited conditions, and an A. tumefaciens mutant with a manganese transport deficiency is attenuated for plant virulence. Iron and manganese limitation independently inhibit biofilm formation by A. tumefaciens, and several candidate genes that could impact biofilm formation were identified in each regulon. The biofilm-inhibitory effects of iron and manganese do not rely on recognized metal-responsive transcriptional regulators, suggesting alternate mechanisms influencing biofilm formation. However, under low-manganese conditions the dcpA operon is upregulated, encoding a system that controls levels of the cyclic di-GMP second messenger. Mutation of this regulatory pathway dampens the effect of manganese limitation. Responses to changes in transition metal levels, such as those of manganese and iron, are important for normal metabolism and growth in bacteria. Our study used global gene expression profiling to understand the response of the plant pathogen Agrobacterium tumefaciens to changes of transition metal availability. Among the properties

  17. Movement disorders associated with focal midbrain lesion: correlation with clinical and I-123 IPT SPECT findings

    International Nuclear Information System (INIS)

    Kang, Ji Hoon; Im, Joo Hyuk; Kim, Jae Seung; Lee, Myoung Chong

    2001-01-01

    Midbrain lesion may produce a variety of movement disorders including tremor, dystonia, and parkinsonism. The anatomical and functional basis of the movement disorder associated with the midbrain lesion is still unclear. The purpose of this study was to correlate focal midbrain lesions with clinical and I-123 IPT SPECT findings. Five patients (aged 25 to 69 years, 3 men and 2 women) who presented with movement disorder associated with discrete focal midbrain lesion on the brain MRI were included. We reviewed the clinical characteristics of movement disorders and the brain MRI findings in all patients. I-123 IPT SPECT was performed in all patients and 9 normal controls to evaluate the integrity of the nigrostriatal dopaminergic system and specific binding ratios were also calculated. Patients consisted of 2 with parkinsonism, 1 with midbrain tremor, 1 with hemidystonia, and 1 with micrographia as the only manifestation. In all patients, movement disorders were confined to the limbs contralateral to the focal midbrain lesions. The causes of midbrain lesion were trauma (n=2), rupture of AVM (n=1), cerebral infarction (n=1), and encephalitis (n=1). The latency between the midbrain injury and the onset of movement disorder varied from 1.5 months to 2 years (mean 6.7 months). Specific binding ratios of ipsilateral striatum (1.6±1.4) were significantly lower than that of contralateral side (3.3±0.99) and normal control (3.5±0.5)(p<0.05). All of six patients had lesions involving substantia nigra on MRI and two of these with resting tremor had also lesions involving the red nucleus. Bradykinesia and rigidity were mild or absent in these two patients, despite severely decreased specific binding ratios (mean 0.55) of ipsilateral striatum. Movement disorders associated with focal midbrain lesion were partially related to the damage in the nigrostriatal dopaminergic system. However, the severity and nature of movement disorder were variable and not directly related to the

  18. Potential impact of intermittent preventive treatment (IPT on spread of drug-resistant malaria.

    Directory of Open Access Journals (Sweden)

    Wendy Prudhomme O'Meara

    2006-05-01

    Full Text Available Treatment of asymptomatic individuals, regardless of their malaria infection status, with regularly spaced therapeutic doses of antimalarial drugs has been proposed as a method for reducing malaria morbidity and mortality. This strategy, called intermittent preventive treatment (IPT, is currently employed for pregnant women and is being studied for infants (IPTi as well. As with any drug-based intervention strategy, it is important to understand how implementation may affect the spread of drug-resistant parasites. This is a difficult issue to address experimentally because of the limited size and duration of IPTi trials as well as the intractability of distinguishing the spread of resistance due to conventional treatment of malaria episodes versus that due to IPTi when the same drug is used in both contexts.Using a mathematical model, we evaluated the possible impact of treating individuals with antimalarial drugs at regular intervals regardless of their infection status. We translated individual treatment strategies and drug pharmacokinetics into parasite population dynamic effects and show that immunity, treatment rate, drug decay kinetics, and presumptive treatment rate are important factors in the spread of drug-resistant parasites. Our model predicts that partially resistant parasites are more likely to spread in low-transmission areas, but fully resistant parasites are more likely to spread under conditions of high transmission, which is consistent with some epidemiological observations. We were also able to distinguish between spread of resistance due to treatment of symptomatic infections and that due to IPTi. We showed that IPTi could accelerate the spread of resistant parasites, but this effect was only likely to be significant in areas of low or unstable transmission.The results presented here demonstrate the importance of considering both the half-life of a drug and the existing level of resistance when choosing a drug for IPTi

  19. Crystal Structure of AGR_C_4470p from Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Vorobiev,S.; Neely, H.; Seetharaman, J.; Ma, L.; Xiao, R.; Acton, T.; Montelione, G.; Tong, L.

    2007-01-01

    We report here the crystal structure at 2.0 {angstrom} resolution of the AGR{_}C{_}4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR{_}C{_}4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR{_}C{_}4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR{_}C{_}4470p in E. coli, in addition to the ChuS protein.

  20. Effectiveness Research: Transporting Interpersonal Psychotherapy for Depressed Adolescents (IPT-A) From the Lab to School-Based Health Clinics

    Science.gov (United States)

    Mufson, Laura H.; Dorta, Kristen Pollack; Olfson, Mark; Weissman, Myrna M.; Hoagwood, Kimberly

    2004-01-01

    This paper describes the process of modifying and transporting an evidence-based treatment, Interpersonal Psychotherapy for Depressed Adolescents (IPT-A), from a university setting to school-based health clinics. It addresses conceptual issues involved in the shift from efficacy to effectiveness research as well as operational issues specific to…

  1. Recovery of nonpathogenic mutant bacteria from tumors caused by several Agrobacterium tumefaciens strains: a frequent event?

    Science.gov (United States)

    Llop, Pablo; Murillo, Jesús; Lastra, Beatriz; López, María M

    2009-10-01

    We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.

  2. Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system

    DEFF Research Database (Denmark)

    Danhorn, T.; Hentzer, Morten; Givskov, Michael Christian

    2004-01-01

    The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions...

  3. Mini-Tn7 Insertion in an Artificial attTn7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens.

    Science.gov (United States)

    Figueroa-Cuilan, Wanda; Daniel, Jeremy J; Howell, Matthew; Sulaiman, Aliyah; Brown, Pamela J B

    2016-08-15

    Mechanistic studies of many processes in Agrobacterium tumefaciens have been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmid-based systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes in A. tumefaciens Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable. Agrobacterium tumefaciens is a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Polar Organizing Protein PopZ Is Required for Chromosome Segregation in Agrobacterium tumefaciens.

    Science.gov (United States)

    Ehrle, Haley M; Guidry, Jacob T; Iacovetto, Rebecca; Salisbury, Anne K; Sandidge, D J; Bowman, Grant R

    2017-09-01

    Despite being perceived as relatively simple organisms, many bacteria exhibit an impressive degree of subcellular organization. In Caulobacter crescentus , the evolutionarily conserved polar organizing protein PopZ facilitates cytoplasmic organization by recruiting chromosome centromeres and regulatory proteins to the cell poles. Here, we characterize the localization and function of PopZ in Agrobacterium tumefaciens , a genetically related species with distinct anatomy. In this species, we find that PopZ molecules are relocated from the old pole to the new pole in the minutes following cell division. PopZ is not required for the localization of the histidine kinases DivJ and PdhS1, which become localized to the old pole after PopZ relocation is complete. The histidine kinase PdhS2 is temporally and spatially related to PopZ in that it localizes to transitional poles just before they begin to shed PopZ and disappears from the old pole after PopZ relocalization. At the new pole, PopZ is required for tethering the centromere of at least one of multiple replicons (chromosome I), and the loss of popZ results in a severe chromosome segregation defect, aberrant cell division, and cell mortality. After cell division, the daughter that inherits polar PopZ is shorter in length and delayed in chromosome I segregation compared to its sibling. In this cell type, PopZ completes polar relocation well before the onset of chromosome segregation. While A. tumefaciens PopZ resembles its C. crescentus homolog in chromosome tethering activity, other aspects of its localization and function indicate distinct properties related to differences in cell organization. IMPORTANCE Members of the Alphaproteobacteria exhibit a wide range of phenotypic diversity despite sharing many conserved genes. In recent years, the extent to which this diversity is reflected at the level of subcellular organization has become increasingly apparent. However, which factors control such organization and how

  5. Stable Agrobacterium tumefaciens-mediated transformation related ...

    African Journals Online (AJOL)

    laddiya

    2012-07-17

    Jul 17, 2012 ... (2009) transformed the wheat late embryogenesis abundant (LEA) gene using ..... Effect of infection time on callus growth rate and transformation frequency according to various infection times (60, 30, 25, 20, 15 and 10 min) using 0.8 ..... This work was supported by Nutraceutical Bio Brain. Korea 21 Project ...

  6. Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops.

    Science.gov (United States)

    Singh, Roshan Kumar; Prasad, Manoj

    2016-05-01

    Steady increase in global population poses several challenges to plant science research, including demand for increased crop productivity, grain yield, nutritional quality and improved tolerance to different environmental factors. Transgene-based approaches are promising to address these challenges by transferring potential candidate genes to host organisms through different strategies. Agrobacterium-mediated gene transfer is one such strategy which is well known for enabling efficient gene transfer in both monocot and dicots. Due to its versatility, this technique underwent several advancements including development of improved in vitro plant regeneration system, co-cultivation and selection methods, and use of hyper-virulent strains of Agrobacterium tumefaciens harbouring super-binary vectors. The efficiency of this method has also been enhanced by the use of acetosyringone to induce the activity of vir genes, silver nitrate to reduce the Agrobacterium-induced necrosis and cysteine to avoid callus browning during co-cultivation. In the last two decades, extensive efforts have been invested towards achieving efficient Agrobacterium-mediated transformation in cereals. Though high-efficiency transformation systems have been developed for rice and maize, comparatively lesser progress has been reported in other graminaceous crops. In this context, the present review discusses the progress made in Agrobacterium-mediated transformation system in rice, maize, wheat, barley, sorghum, sugarcane, Brachypodium, millets, bioenergy and forage and turf grasses. In addition, it also provides an overview of the genes that have been recently transferred to these graminaceous crops using Agrobacterium, bottlenecks in this technique and future possibilities for crop improvement.

  7. Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis

    Science.gov (United States)

    Aktas, Meriyem; Danne, Linna; Möller, Philip; Narberhaus, Franz

    2014-01-01

    Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930

  8. Membrane lipids in Agrobacterium tumefaciens: Biosynthetic pathways and importance for pathogenesis

    Directory of Open Access Journals (Sweden)

    Meriyem eAktas

    2014-03-01

    Full Text Available Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs phosphatidylethanolamine (PE, phosphatidylglycerol (PG, phosphatidylcholine (PC and cardiolipin, and ornithine lipids (OLs. Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defence response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22 % of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation.

  9. Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

    OpenAIRE

    Penalver, R.; Serra, M. T.; Duran-Vila, N.; Lopez, M. M.

    1996-01-01

    Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips.

  10. Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)

    2012-09-05

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

  11. Transcriptome analysis revealed that a quorum sensing system regulates the transfer of the pAt megaplasmid in Agrobacterium tumefaciens.

    Science.gov (United States)

    Mhedbi-Hajri, Nadia; Yahiaoui, Noura; Mondy, Samuel; Hue, Nathalie; Pélissier, Franck; Faure, Denis; Dessaux, Yves

    2016-08-20

    Agrobacterium tumefaciens strain P4 is atypical, as the strain is not pathogenic and produces a for this species unusual quorum sensing signal, identified as N-(3-hydroxy-octanoyl)-homoserine lactone (3OH,C8-HSL). By sequence analysis and cloning, a functional luxI-like gene, named cinI, has been identified on the At plasmid of A. tumefaciens strain P4. Insertion mutagenesis in the cinI gene and transcriptome analyses permitted the identification of 32 cinI-regulated genes in this strain, most of them encoding proteins responsible for the conjugative transfer of pAtP4. Among these genes were the avhB genes that encode a type 4 secretion system (T4SS) involved in the formation of the conjugation apparatus, the tra genes that encode the DNA transfer and replication (Dtr) machinery and cinI and two luxR orthologs. These last two genes, cinR and cinX, exhibit an unusual organization, with the cinI gene surrounded by the two luxR orthologs. Conjugation experiments confirmed that the conjugative transfer of pAtP4 is regulated by 3OH,C8-HSL. Root colonization experiments indicated that the quorum sensing regulation of the conjugation of the pAtP4 does not confer a gain or a loss of fitness to the bacterial host in the tomato plant rhizosphere. This work is the first identification of the occurrence of a quorum sensing regulation of the pAt conjugation phenomenon in Agrobacterium.

  12. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens

    Science.gov (United States)

    Wilms, Ina; Overlöper, Aaron; Nowrousian, Minou; Sharma, Cynthia M.; Narberhaus, Franz

    2012-01-01

    Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium. PMID:22336765

  13. Membrane and core periplasmic Agrobacterium tumefaciens virulence Type IV secretion system components localize to multiple sites around the bacterial perimeter during lateral attachment to plant cells.

    Science.gov (United States)

    Aguilar, Julieta; Cameron, Todd A; Zupan, John; Zambryski, Patricia

    2011-01-01

    Type IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain of Agrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in the A. tumefaciens octopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression following vir induction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles. vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiple vir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates. Transfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of the Agrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model of A. tumefaciens attachment to a plant cell, where A. tumefaciens takes advantage of the multiple

  14. Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens.

    Science.gov (United States)

    Attai, Hedieh; Rimbey, Jeanette; Smith, George P; Brown, Pamela J B

    2017-12-01

    To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative p hage p eptidoglycan h ydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N -acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens , may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The

  15. Effects of biotic stress caused by Potato virus Y on photosynthesis in ipt transgenic and control Nicotiana tabacum L

    Czech Academy of Sciences Publication Activity Database

    Synková, Helena; Semorádová, Šárka; Schnablová, Renáta; Muller, K.; Pospíšilová, Jana; Ryšlavá, H.; Malbeck, Jiří; Čeřovská, Noemi

    2006-01-01

    Roč. 171, - (2006), s. 607-616 ISSN 0168-9452 R&D Projects: GA ČR GA206/03/0310 Grant - others:Grantová agentura University Karlovy GAUK428/2004/B-Ch/PrF Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinins * ipt * transgenic tobacco * photosynthesis * Potato virus Y Subject RIV: EF - Botanics Impact factor: 1.631, year: 2006

  16. Predictors of response to CBT and IPT for depression; the contribution of therapy process.

    Science.gov (United States)

    Carter, Janet D; Crowe, Marie T; Jordan, Jennifer; McIntosh, Virginia V W; Frampton, Christopher; Joyce, Peter R

    2015-11-01

    Little is known about the factors that contribute to a positive psychotherapy outcome. There is still considerable debate as to whether specific factors (e.g. severity of symptoms, comorbidity) or nonspecific factors (e.g. alliance, therapy process) are most important in influencing outcome. This study examined the additional contribution that therapeutic process and alliance made to previously identified specific predictors of response to CBT and IPT for depression over the course of therapy. The previously identified specific factors were belief that childhood reasons caused the depression, recurrent depression, perceptions about how logical therapy was and comorbid personality disorder symptoms. One hundred and sixty five adult outpatients with major depression were treated for depression in a randomised clinical trial examining predictors of response to Cognitive Behavior Therapy and Interpersonal Psychotherapy. All therapy sessions were audiorecorded to enable objective ratings of therapeutic process and alliance. Process factors - patient psychic distress, patient participation and patient alliance had the strongest associations with outcome. The early and middle stage of therapy process did not account for any additional variance other than that previously identified by the patient predictors, however, at the end stage of therapy process contributed a further 14%. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Gene and enhancer trap tagging of vascular-expressed genes in poplar trees

    Science.gov (United States)

    Andrew Groover; Joseph R. Fontana; Gayle Dupper; Caiping Ma; Robert Martienssen; Steven Strauss; Richard Meilan

    2004-01-01

    We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the β-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS...

  18. Relationship between clinical features of Parkinson`s disease and presynaptic dopamine transporter binding assessed with [{sup 123}I]IPT and single-photon emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Tatsch, K. [Department of Nuclear Medicine, University of Munich (Germany); Schwarz, J. [Department of Neurology, University of Munich (Germany); Mozley, P.D. [Department of Radiology, University of Pennsylvania (United States)]|[Department of Psychiatry, University of Pennsylvania (United States); Linke, R. [Department of Nuclear Medicine, University of Munich (Germany); Pogarell, O. [Department of Neurology, University of Munich (Germany); Oertel, W.H. [Department of Neurology, University of Munich (Germany); Fieber, R.S. [Department of Nuclear Medicine, University of Munich (Germany); Hahn, K. [Department of Nuclear Medicine, University of Munich (Germany); Kung, H.F. [Department of Radiology, University of Pennsylvania (United States)

    1997-04-01

    IPT [N-(3-iodopropen-2-yl)-2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl) tropane] is a new cocain analogue which allows the presynaptic dopamine transporters to be imaged with single-photon emission tomography (SPET) as early as 1-2 h post injection. In the present study [{sup 123}I]IPT SPET was performed in patients with Parkinson`s disease (PD) to analyse the relationship between specific dopamine tansporter binding and clinical features of the disease. Twenty-six PD patients (Hoehn and Yahr stages I-IV, age range 40-79 years) and eight age-matched controls were studied. SPET imaging was performed 90-120 min after injection of 160-185 MBq [{sup 123}I]IPT using a triple-head camera. For semiquantitative evaluation of specific [{sup 123}I]IPT binding, ratios between caudate, putamen and background regions were calculated. Specific [{sup 123}I]IPT uptake was significantly reduced in PD patients compared to controls. Most patients showed a marked asymmetry with a more pronounced decrease in [{sup 123}I]IPT binding on the side contralateral to the predominant clinical findings. The putamen was always more affected than the caudate. [{sup 123}I]IPT binding was significantly correlated with disease duration (r=-0.7, P<0.0001) but not with the age of PD patients (r=-0.10, P=0.61). Specific [{sup 123}I]IPT uptake in the caudate and putamen, and putamen to caudate ratios, decreased with increasing Hoehn and Yahr stage. (orig./AJ). With 2 figs., 2 tabs.

  19. A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

    Science.gov (United States)

    El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

    2015-08-01

    Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

  20. Effect of antibiotic ceftriaxone on elimination of ABI and GV3101 strains of Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Gorbatyuk I. R.

    2015-12-01

    Full Text Available Aim. To find out, at which concentration the antibiotic ceftriaxone of β-lactam group causes the elimination of ABI and GV3101 strains of Agrobacterium tumefaciens. Methods. The disc diffusion method. Results. Antibiotic ceftriaxone was used for the cell elimination of the Agrobacterium tumefaciens ABI strain for the first time. The same zones of inhibition were observed when using the 400 mg/l ceftriaxone and 500 mg/l cefotaxime solutions for both Agrobacterium strains (ABI and GV3101 studied. Conclusions. Ceftriaxone inhibits the Agrobacterium growth more effectively than cefotaxime. The ceftriaxone concentration for elimination of ABI and GV3101 Agrobacterium tumefaciens strains is 400 mg/l.

  1. Multilocus Sequence-Based Analysis Delineates a Clonal Population of Agrobacterium (Rhizobium) radiobacter (Agrobacterium tumefaciens) of Human Origin ▿

    Science.gov (United States)

    Aujoulat, Fabien; Jumas-Bilak, Estelle; Masnou, Agnès; Sallé, Fanny; Faure, Denis; Segonds, Christine; Marchandin, Hélène; Teyssier, Corinne

    2011-01-01

    The genus Agrobacterium includes plant-associated bacteria and opportunistic human pathogens. Taxonomy and nomenclature within the genus remain controversial. In particular, isolates of human origin were all affiliated with the species Agrobacterium (Rhizobium) radiobacter, while phytopathogenic strains were designated under the synonym denomination Agrobacterium tumefaciens. In order to study the relative distribution of Agrobacterium strains according to their origins, we performed a multilocus sequence-based analysis (MLSA) on a large collection of 89 clinical and environmental strains from various origins. We proposed an MLSA scheme based on the partial sequence of 7 housekeeping genes (atpD, zwf, trpE, groEL, dnaK, glnA, and rpoB) present on the circular chromosome of A. tumefaciens C58. Multilocus phylogeny revealed that 88% of the clinical strains belong to genovar A7, which formed a homogeneous population with linkage disequilibrium, suggesting a low rate of recombination. Comparison of genomic fingerprints obtained by pulsed-field gel electrophoresis (PFGE) showed that the strains of genovar A7 were epidemiologically unrelated. We present genetic evidence that genovar A7 may constitute a human-associated population distinct from the environmental population. Also, phenotypic characteristics, such as culture at 42°C, agree with this statement. This human-associated population might represent a potential novel species in the genus Agrobacterium. PMID:21398532

  2. Multilocus sequence-based analysis delineates a clonal population of Agrobacterium (Rhizobium) radiobacter (Agrobacterium tumefaciens) of human origin.

    Science.gov (United States)

    Aujoulat, Fabien; Jumas-Bilak, Estelle; Masnou, Agnès; Sallé, Fanny; Faure, Denis; Segonds, Christine; Marchandin, Hélène; Teyssier, Corinne

    2011-05-01

    The genus Agrobacterium includes plant-associated bacteria and opportunistic human pathogens. Taxonomy and nomenclature within the genus remain controversial. In particular, isolates of human origin were all affiliated with the species Agrobacterium (Rhizobium) radiobacter, while phytopathogenic strains were designated under the synonym denomination Agrobacterium tumefaciens. In order to study the relative distribution of Agrobacterium strains according to their origins, we performed a multilocus sequence-based analysis (MLSA) on a large collection of 89 clinical and environmental strains from various origins. We proposed an MLSA scheme based on the partial sequence of 7 housekeeping genes (atpD, zwf, trpE, groEL, dnaK, glnA, and rpoB) present on the circular chromosome of A. tumefaciens C58. Multilocus phylogeny revealed that 88% of the clinical strains belong to genovar A7, which formed a homogeneous population with linkage disequilibrium, suggesting a low rate of recombination. Comparison of genomic fingerprints obtained by pulsed-field gel electrophoresis (PFGE) showed that the strains of genovar A7 were epidemiologically unrelated. We present genetic evidence that genovar A7 may constitute a human-associated population distinct from the environmental population. Also, phenotypic characteristics, such as culture at 42°C, agree with this statement. This human-associated population might represent a potential novel species in the genus Agrobacterium.

  3. Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis

    Directory of Open Access Journals (Sweden)

    Yi-Han eLin

    2014-05-01

    Full Text Available Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction.

  4. A reliable in vitro fruiting system for Armillaria mellea for evaluation of Agrobacterium tumefaciens transformation vectors.

    Science.gov (United States)

    Ford, Kathryn L; Baumgartner, Kendra; Henricot, Béatrice; Bailey, Andy M; Foster, Gary D

    2015-10-01

    Armillaria mellea is a serious pathogen of horticultural and agricultural systems in Europe and North America. The lack of a reliable in vitro fruiting system for heterothallic A. mellea has hindered research and required dependence on intermittently available wild-collected basidiospores of endemic genotypes, necessitating the use of variable genetic material in transformation studies. Here we describe a reliable, reproducible in vitro fruiting method for heterothallic A. mellea from the western US. Isolates and growth conditions were evaluated to determine effective fruiting conditions. Following medium colonisation for 4 weeks, cultures were incubated under warm/bright conditions for 4-6 weeks before incubation in dim/cool conditions. Primordia emerged within 3-4 weeks following a temperature decrease and this was most efficient when coupled with a light reduction. Basidiocarps matured within 3-4 weeks and produced viable basidiospores. Agrobacterium tumefaciens and vectors were evaluated by transformation of in vitro-produced basidiospores and a versatile transformation vector was constructed to simplify promoter and marker gene exchange using homologous recombination in yeast. Fruiting bodies and viable basidiospores of A. mellea have been reliably produced in vitro which, coupled with the enhanced knowledge of suitable A. tumefaciens strains and vectors for transformation, will assist future genetic research into this important pathogen. Copyright © 2015 The British Mycological Society. All rights reserved.

  5. Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB, attD, and attR Mutants

    Science.gov (United States)

    Matthysse, Ann G.; McMahan, Susan

    1998-01-01

    Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 103 bacteria/cm of root length at the time of inoculation to more than 107 bacteria/cm after 10 days. The numbers of cellulose-minus and nonattaching attB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thaliana ecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel and att mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis. PMID:9647796

  6. Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

    Science.gov (United States)

    Liu, Mao-Sen; Ko, Miau-Hwa; Li, Hui-Chun; Tsai, Shwu-Jene; Lai, Ying-Mi; Chang, You-Ming; Wu, Min-Tze; Chen, Long-Fang O

    2014-08-28

    Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, "Green King". Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

  7. Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens.

    Science.gov (United States)

    Ali, Shawkat; Bakkeren, Guus

    2015-01-01

    The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

  8. Conversion of BAC Clones into Binary BAC (BIBAC) Vectors and Their Delivery into Basidiomycete Fungal Cells Using Agrobacterium tumefaciens

    KAUST Repository

    Ali, Shawkat

    2014-09-19

    The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

  9. attG and attC mutations of Agrobacterium tumefaciens are dominant negative mutations that block attachment and virulence.

    Science.gov (United States)

    Matthysse, Ann G; Jaeckel, Peter; Jeter, Cecelia

    2008-04-01

    The cryptic plasmid (pAT) of Agrobacterium tumefaciens was not required for virulence or attachment to plant surfaces. However, mutations in the attC and attG genes located on pAT caused the bacteria to become avirulent and non-attaching on tomato, carrot, and Bryophyllum daigremontiana. This was the case whether the mutation was in the copy of the genes located on pAT or whether it was carried in a second copy of the attA-G operon located on a plasmid in cells that contained a wild-type copy of pAT. Thus attC and attG mutations are dominant negative mutations. The mechanism by which these mutations block attachment and virulence is unknown.

  10. Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana.

    Science.gov (United States)

    Lee, Chil-Woo; Efetova, Marina; Engelmann, Julia C; Kramell, Robert; Wasternack, Claus; Ludwig-Müller, Jutta; Hedrich, Rainer; Deeken, Rosalia

    2009-09-01

    Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis-Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.

  11. The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.

    Science.gov (United States)

    Matthysse, Ann G; Marry, Mazz; Krall, Leonard; Kaye, Mitchell; Ramey, Bronwyn E; Fuqua, Clay; White, Alan R

    2005-09-01

    Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

  12. Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Cupp-Vickery, Jill R., E-mail: jvickery@uci.edu; Igarashi, Robert Y.; Meyer, Christopher R.

    2005-03-01

    Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å.

  13. Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Cupp-Vickery, Jill R.; Igarashi, Robert Y.; Meyer, Christopher R.

    2005-01-01

    Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å

  14. Agrobacterium tumefaciens-mediated transformation of corn (Zea mays L.) multiple shoots

    OpenAIRE

    Cao, Shi-liang; Masilamany, Pathmalojiny; Li, Wen-bin; Pauls, K. Peter

    2014-01-01

    An Agrobacterium tumefaciens-mediated corn transformation method based on multiple shoot tissue cultures was developed, which is effective with a variety of corn inbred lines and standard binary vectors. Six factors that affected the success of corn transformation were tested, including A. tumefaciens strain, corn genotype, tissue culture growth stage, medium composition, co-culture temperature and surfactant treatment. Agropine-type bacteria (EHA 101 and AGL 1) were eightfold more effective ...

  15. The Ctp type IVb pilus locus of Agrobacterium tumefaciens directs formation of the common pili and contributes to reversible surface attachment.

    Science.gov (United States)

    Wang, Yi; Haitjema, Charles H; Fuqua, Clay

    2014-08-15

    Agrobacterium tumefaciens can adhere to plant tissues and abiotic surfaces and forms biofilms. Cell surface appendages called pili play an important role in adhesion and biofilm formation in diverse bacterial systems. The A. tumefaciens C58 genome sequence revealed the presence of the ctpABCDEFGHI genes (cluster of type IV pili; Atu0216 to Atu0224), homologous to tad-type pilus systems from several bacteria, including Aggregatibacter actinomycetemcomitans and Caulobacter crescentus. These systems fall into the type IVb pilus group, which can function in bacterial adhesion. Transmission electron microscopy of A. tumefaciens revealed the presence of filaments, significantly thinner than flagella and often bundled, associated with cell surfaces and shed into the external milieu. In-frame deletion mutations of all of the ctp genes, with the exception of ctpF, resulted in nonpiliated derivatives. Mutations in ctpA (a pilin homologue), ctpB, and ctpG decreased early attachment and biofilm formation. The adherence of the ctpA mutant could be restored by ectopic expression of the paralogous pilA gene. The ΔctpA ΔpilA double pilin mutant displayed a diminished biovolume and lower biofilm height than the wild type under flowing conditions. Surprisingly, however, the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants formed normal biofilms and showed enhanced reversible attachment. In-frame deletion of the ctpA pilin gene in the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants caused the same attachment-deficient phenotype as the ctpA single mutant. Collectively, these findings indicate that the ctp locus is involved in pilus assembly and that nonpiliated mutants, which retain the CtpA pilin, are proficient in attachment and adherence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Dopamine transporter density assessed with [123]IPT SPECT before and after risperidone treatment in children with tourette's disorder

    International Nuclear Information System (INIS)

    Ryu, Young Hoon; Kim, Tae Hoon; Ryu, Won Gee

    2004-01-01

    Tourette's disorder (TD), which is characterized by multiple waxing and waning motor tics and one or more vocal tics, is known to be associated with abnormalities in the dopaminergic system. To testify our hypothesis that risperidone would improve tic symptoms of TD patients through the change of the dopaminergic system, we measured the dopamine transporter (DAT) densities between drug-naive children with TD and normal children, and investigated the DAT density before and after treatment with risperidone in drug-naive children with TD, using iodine-123 labelled N-(3-iodopropen-2-yl)-2β-carbomethoxy-3beta-(4-chlorophenyl)tropane ([ 123 I]IPT) single photon emission computed tomography (SPECT). [ 123I ]IPT SPECT imaging and Yale Global Tic Severity Scale-Korean version (YGTSS-K) for assessing the tic symptom severity were carried out before and after treatment with risperidone for 8 weeks in nine drug-naive children with TD. Eleven normal children also underwent SPECT imaging 2 hours after an intravenous administration of [ 123 I]IPT. Drug-naive children with TD had a significantly greater increase in the specific/nonspecific DAT binding ratio of both basal ganglia compared with the normal children. However, no significant difference in the specific/nonspecific DAT binding ratio of the basal ganglia before and after treatment with risperidone in children with TD was found, although tic symptoms were significantly improved with risperidone. These findings suggest that DAT densities are directly associated with the pathophysiology of TD, however, that the effect of risperidone on tic symptoms in children with TD is not attributed to the change of dopaminergic system

  17. Dopamine transporter density of the basal ganglia in children with attention deficit hyperactivity disorder assessed with I-123 IPT SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Won Gee; Kim, Tae Hoon; Ryu, Young Hoon; Yun, Mi Jin; Lee, Jong Doo; Cheon, Keun Ah [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of); Chi, Dae Yoon [College of Medicine, Inha Univ., Incheon (Korea, Republic of); Kim, Jong Ho; Choi, Tae Hyun [School of Medicine, Gachon Univ., Gachon (Korea, Republic of)

    2003-08-01

    Attention deficit hyperactivity disorder (ADHD) has been known as psychiatric disorder in childhood associated with dopamine dysregulation. In present study, we investigated changes in dopamine transporter (DAT) density of the basal ganglias using I-123 N-(3-iodopropen-2-yl) -2-carbomethoxy-3beta-(4-chlorphenyl) tropane (I-123 IPT) SPECT in children with ADHD before and after methylphenidate treatment. Nine drug-naive children with ADHD and seven normal children were included in the study. We performed brain SPECT two hours after the intravenous administration of I-123 IPT and made both quantitative and qualitative analyses using the obtained SPECT data, which were reconstructed for the assessment of specific/nonspecific DAT binding ratios in the basal ganglia. All children with ADHD reperformed (123I)IPT SPECT after treatment with methylphenidate (0.7mg/kg/d) during about 8 weeks. SPECT data reconstructed for the assessment of specific/nonspecific DAT binding ratio of the basal ganglia were compared between before and after treatment methyphenidate. We investigated correlation between the change of ADHD symptom severity assessed with ADHD rating scale-IV and specific/nonspecific DAT binding ratio of basal ganglia. Children with ADHD had a significantly greater specific/nonspecific DAT binding ratio of the basal ganglia comparing to normal children (Right : z = 2.057, p = 0.041 ; Left : z = 2.096, p = 0.032). Under treatment with methylphenidate in all children with ADHD, specific/nonspecific DAT binding ratio of both ganglia decreased significantly greater than before treatment with methylphenidate (Right : t = 3.239, p = 0.018 ; Left : t = 3.133, p 0.020). However, no significant correlation between the change of ADHD symptom severity scores and specific/nonspecific DAT binding ratio of the basal ganglia were found. These findings support the complex dysregulation of the dopaminergic neurotransmitter system in children with ADHD.

  18. Dopamine transporter density of the basal ganglia in children with attention deficit hyperactivity disorder assessed with I-123 IPT SPECT

    International Nuclear Information System (INIS)

    Ryu, Won Gee; Kim, Tae Hoon; Ryu, Young Hoon; Yun, Mi Jin; Lee, Jong Doo; Cheon, Keun Ah; Chi, Dae Yoon; Kim, Jong Ho; Choi, Tae Hyun

    2003-01-01

    Attention deficit hyperactivity disorder (ADHD) has been known as psychiatric disorder in childhood associated with dopamine dysregulation. In present study, we investigated changes in dopamine transporter (DAT) density of the basal ganglias using I-123 N-(3-iodopropen-2-yl) -2-carbomethoxy-3beta-(4-chlorphenyl) tropane (I-123 IPT) SPECT in children with ADHD before and after methylphenidate treatment. Nine drug-naive children with ADHD and seven normal children were included in the study. We performed brain SPECT two hours after the intravenous administration of I-123 IPT and made both quantitative and qualitative analyses using the obtained SPECT data, which were reconstructed for the assessment of specific/nonspecific DAT binding ratios in the basal ganglia. All children with ADHD reperformed (123I)IPT SPECT after treatment with methylphenidate (0.7mg/kg/d) during about 8 weeks. SPECT data reconstructed for the assessment of specific/nonspecific DAT binding ratio of the basal ganglia were compared between before and after treatment methyphenidate. We investigated correlation between the change of ADHD symptom severity assessed with ADHD rating scale-IV and specific/nonspecific DAT binding ratio of basal ganglia. Children with ADHD had a significantly greater specific/nonspecific DAT binding ratio of the basal ganglia comparing to normal children (Right : z = 2.057, p = 0.041 ; Left : z = 2.096, p = 0.032). Under treatment with methylphenidate in all children with ADHD, specific/nonspecific DAT binding ratio of both ganglia decreased significantly greater than before treatment with methylphenidate (Right : t = 3.239, p = 0.018 ; Left : t = 3.133, p 0.020). However, no significant correlation between the change of ADHD symptom severity scores and specific/nonspecific DAT binding ratio of the basal ganglia were found. These findings support the complex dysregulation of the dopaminergic neurotransmitter system in children with ADHD

  19. OPTIMIZATION OF FACTORS AFFECTING THE Agrobacterium tumefaciens- MEDIATED TRANSFORMATION OF Eucalyptus saligna

    Directory of Open Access Journals (Sweden)

    Yohana de Oliveira-Cauduro

    2018-02-01

    Full Text Available ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1, citric acid (25mg.L-1 and PVP-40 (1g.L-1. Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1 combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached.

  20. [ I - 123 ] IPT SPECT Dopamine Reuptake Site Imaging : Differences in Normal Controls and Parkinson's Patients by Semiquantitat

    International Nuclear Information System (INIS)

    Kim, Hee Joung; Yang, Seoung Oh; Ryu, Jin Sook; Choi, Yun Young; Lee, Hee Kyung; Im, Joo Hyuck; Lee, Myung Chong

    1996-01-01

    Dopamine transporter concentrations have been known to decrease in Parkinson's disease (PD) or increase in Tourette's disorder. The purpose of this study was to evaluate the effectiveness of [I-123]N-(3-iodopropene-2-yl)-2β-carbomethoxy-3β-(4-chlorophenyl) tropane (IPT) as an imaging agent for measuring changes in transporter concentrations with PD. IPT labelled with 6.69+/-0.64 mCi (247.53+/-23.68 MBq) of I-123 was intravenously injected into ten patients(age: 55+/-11) with PD, and six normal controls(NC)(age: 46+/-14) as a bolus. Dynamic SPECT scans of the brain were then performed for 5 minutes each over 120 minutes on a triple headed camera. Time activity curves were generated for the left basal ganglia(LBG), right basal ganglia(RBG), and occipital cortex(OCC). The statistical parameters included the time to peak activity, the contrast ratio of LEG and RBG to OCC at several time points, and the accumulated specific binding counts/mCi/pixel (ASBC) from 0 to 115 minutes. The uptake of IPT in the brains of PD and NC peaked within 10 minutes of injection in all subjects. The maximum target to background ratio in the basal ganglia of PD and NC occurred at 85+/-20 min and 110-+/-6 min of injection, respectively. The BG/OCC ratios at 115 minutes for PD and NC were 2.15+/-0.54 and 4.26+/-0.73, respectively. The ASBC at 115 minutes for PD and NC were 152.91+/-50.09 and 289.51+/-49.00, respectively. The ratio of BG/OCC for the NC was significantly higher than the ratio for PD. SPECT data matched with clinical diagnosis for PDs. The ratio between BG and OCC and the ASBC for PD were clearly separated from NC and may be useful outcome measures for clinical diagnosis. The findings suggest that IPT may be a very useful tracer for early diagnosis of PD and study of dopamine reuptake site.

  1. Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A.

    Science.gov (United States)

    Kang, Yoon-Suk; Brame, Keenan; Jetter, Jonathan; Bothner, Brian B; Wang, Gejiao; Thiyagarajan, Saravanamuthu; McDermott, Timothy R

    2016-06-15

    ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode

  2. Analysis of simulataneous I-123-IPT/Tc-99m-HMPAO dual isotope brain SPECT in Parkinson's disease and normal volunteers using SPM

    International Nuclear Information System (INIS)

    Chung, Y. A.; Juh, R. H.; Kim, S. H.; Park, Y. H.; Lee, S. Y.; Sohn, H. S.; Chung, S. K.

    2002-01-01

    The basal ganglia are usually poorly delineated in Parkinson's diseases on IPT images. We have studied simultaneous dual isotope brain SPECTs using I-123-IPT and Tc-99m-HMPAO, in order to overcome this limitation of IPT imaging. 17 patients (M: 7, F: 10) with Parkinson's disease (Idiopathic parkison's disease: 12, Multiple system atrophy: 5) and 4 normal volunteers (N) underwent the dual isotope brain SPECT following simultaneously injection of 370 MBq Tc-99m-HMPAO (energy window: 130-146 keV) and 111 MBq I-123-IPT (energy window: 152-168 keV). We first obtained parameters of spatial normalization during spatial normalization of Tc-99m-HMPAO brain SPECT using SPECT template. Using these parameters, we could spatially normalized I-123-IPT brain PSECT to standard space, because these images were obtained simultaneously. The difference between each groups(N vs IPD, N vs MSA, IPD vs MSA) were compared with t-test (p<0.01). We demonstrated decreased perfusion in the head and body caudate and globus pallidus on MSA compared with IPD. No significant hypo- and hyperperfusion area was observed in the other analysis. The method proposed in this study can effectively evaluate the dopamine function, and is easily applicable to conventional gamma camera system with any dual energy window acquisition modes

  3. Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens

    Science.gov (United States)

    Shaw, Gwo-Chyuan; Lai, Erh-Min

    2012-01-01

    The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens. PMID:23028331

  4. Agrobacterium tumefaciens-Mediated Transformation of Valsa mali: An Efficient Tool for Random Insertion Mutagenesis

    Directory of Open Access Journals (Sweden)

    Caixia Wang

    2013-01-01

    Full Text Available Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT, with the optimal transformation conditions as follows: 106/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200 μM acetosyringone (AS in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 106 recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels.

  5. Proteomics and Genetics for Identification of a Bacterial Antimonite Oxidase in Agrobacterium tumefaciens.

    Science.gov (United States)

    Li, Jingxin; Wang, Qian; Li, Mingshun; Yang, Birong; Shi, Manman; Guo, Wei; McDermott, Timothy R; Rensing, Christopher; Wang, Gejiao

    2015-05-19

    Antimony (Sb) and its compounds are listed by the United States Environmental Protection Agency (USEPA, 1979) and the European Union (CEC, 1976) as a priority pollutant. Microbial redox transformations are presumed to be an important part of antimony cycling in nature; however, regulation of these processes and the enzymology involved are unknown. In this study, comparative proteomics and reverse transcriptase-PCR analysis of Sb(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 revealed an oxidoreductase (anoA) is widely distributed in microorganisms, including at least some documented to be able to oxidize Sb(III). Deletion of the anoA gene reduced Sb(III) resistance and decreased Sb(III) oxidation by ∼27%, whereas the anoA complemented strain was similar to the wild type GW4 and a GW4 anoA overexpressing strain increased Sb(III) oxidation by ∼34%. Addition of Sb(III) up-regulated anoA expression and cloning anoA to Escherichia coli demonstrated direct transferability of this activity. A His-tag purified AnoA was found to require NADP(+) as cofactor, and exhibited a K(m) for Sb(III) of 64 ± 10 μM and a V(max) of 150 ± 7 nmol min(-1) mg(-1). This study contributes important initial steps toward a mechanistic understanding of microbe-antimony interactions and enhances our understanding of how microorganisms participate in antimony biogeochemical cycling in nature.

  6. Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications.

    Science.gov (United States)

    Krenek, Pavel; Samajova, Olga; Luptovciak, Ivan; Doskocilova, Anna; Komis, George; Samaj, Jozef

    2015-11-01

    Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Agrobacterium tumefaciens-mediated genetic transformation of embryogenesis cell suspensions of banana cultivar Grande naine (AAA

    Directory of Open Access Journals (Sweden)

    Idalmis Bermúdez-Caraballoso

    2004-01-01

    Full Text Available The black Sigatoka (Mycosphaerella fijiensis Morelet has become in the last years, the most destructive disease that affects the production of banana and plantains world-wide. The present work was made with the objective to obtain transgenic plants of banana cultivar Grand naine (AAA resistant to this disease with the use of genetic transformation. Embryogenenic cell suspensions obtained from somatic embryos formed from immature male flowers, were used for the transformation by Agrobacterium tumefaciens. The bacterial strain EHA-105 was used with the binary plasmids pHCA-58, pHCG-59 and pHGA-91, which contain different combinations of genes that encode for the antifungal chitinase, glucanase enzymes and the AP-24 osmotin. The commercial herbicide BASTA® was used as selective agent. One hundred ten putative transformed lines of the three constructions were obtained, after three selection months in the culture medium. The transgenic events were verified by means of Polymerase Chain Reaction analysis. Key words: AP-24, chitinase, glucanase, Musa, Mycosphaerella fijiensis

  8. A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand

    2011-01-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient transform......Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient...

  9. The thuEFGKAB operon of rhizobia and agrobacterium tumefaciens codes for transport of trehalose, maltitol, and isomers of sucrose and their assimilation through the formation of their 3-keto derivatives.

    Science.gov (United States)

    Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Jensen, John Beck; Bhuvaneswari, T V

    2013-09-01

    The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism.

  10. Absence of Substrate Channeling between Active Sites in the Agrobacterium tumefaciens IspDF and IspE Enzymes of the Methyl Erythritol Phosphate Pathway†

    OpenAIRE

    Lherbet, Christian; Pojer, Florence; Richard, Stéphane B.; Noel, Joseph P.; Poulter, C. D.

    2006-01-01

    The conversion of 2C-methyl-d-erythritol 4-phosphate (MEP) to 2C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2C-methyl-d-erythritol 2,4-c...

  11. Abiotic and biotic factors responsible for antimonite oxidation in Agrobacterium tumefaciens GW4

    Science.gov (United States)

    Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Wang, Qian; Wang, Gejiao

    2017-03-01

    Antimonite [Sb(III)]-oxidizing bacteria can transform the toxic Sb(III) into the less toxic antimonate [Sb(V)]. Recently, the cytoplasmic Sb(III)-oxidase AnoA and the periplasmic arsenite [As(III)] oxidase AioAB were shown to responsible for bacterial Sb(III) oxidation, however, disruption of each gene only partially decreased Sb(III) oxidation efficiency. This study showed that in Agrobacterium tumefaciens GW4, Sb(III) induced cellular H2O2 content and H2O2 degradation gene katA. Gene knock-out/complementation of katA, anoA, aioA and anoA/aioA and Sb(III) oxidation and growth experiments showed that katA, anoA and aioA were essential for Sb(III) oxidation and resistance and katA was also essential for H2O2 resistance. Furthermore, linear correlations were observed between cellular H2O2 and Sb(V) content in vivo and chemical H2O2 and Sb(V) content in vitro (R2 = 0.93 and 0.94, respectively). These results indicate that besides the biotic factors, the cellular H2O2 induced by Sb(III) also catalyzes bacterial Sb(III) oxidation as an abiotic oxidant. The data reveal a novel mechanism that bacterial Sb(III) oxidation is associated with abiotic (cellular H2O2) and biotic (AnoA and AioAB) factors and Sb(III) oxidation process consumes cellular H2O2 which contributes to microbial detoxification of both Sb(III) and cellular H2O2.

  12. Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33.

    Science.gov (United States)

    Li, Huili; Xie, Kebo; Yu, Wenjun; Hu, Liejie; Huang, Haiyan; Xie, Huijun; Wang, Shuning

    2016-01-04

    Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Ethanol inducible isopentenyl transferase as a high efficiency ...

    African Journals Online (AJOL)

    hope&shola

    2010-11-29

    Nov 29, 2010 ... The isopentenyl transforase gene from Agrobacterium tumefaciens is one positive selectable marker genes for plant transformation. In this research, the ipt gene was placed under the control of ethanol-inducible system and was introduced into Nicotiana tabacum cv. Xanthi via. Agrobacterium-mediated ...

  14. Ethanol inducible isopentenyl transferase as a high efficiency ...

    African Journals Online (AJOL)

    The isopentenyl transforase gene from Agrobacterium tumefaciens is one positive selectable marker genes for plant transformation. In this research, the ipt gene was placed under the control of ethanol-inducible system and was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation.

  15. Dopamine transporter density in the basal ganglia assessed with 123I-IPT SPECT in children with Tourette's deosoder

    International Nuclear Information System (INIS)

    Yoo, Y. H.; Cheon, K. A.; Yoon, M. J.; Kim, C. H.; Lee, J. D.; Kim, H. H.; Choi, T. H.

    2002-01-01

    Previous studies in patients with Tourette's disorder (TD) provided evidences of presynaptic dopaminergic dysfunction, demonstrating increased dopamine transporter densities. We investigated dopamine transporter densities using 123 I-IPT SPECT in drug-naive children with TD and postulated that dopamine transporter density reflected dopamine concentrations. 9 drug-naive children with ADHD and 8 normal children were included in the study. We performed brain SPECT 2 hours after administration of 123 I-IPT and made both quantitative and qualitative analyses for assessment of specific/nonspecific DAT binding ratio in the BG. We then investigated correlation between the severity of tics in children with TD assessed with the YGTSS and specific/nonspecific binding ratio on BG. Drug-naive children with TD showed a significantly increased specific/nonspecific DAT binding ratio in the BG compared with normal children. We found no significant correlation between severity of tics assessed with YGTSS in children with TD and specific/nonspecific DAT binding ratio in BG. These findings support the hypothesis of dopamine dysregulation in presynaptic dopamine function of BG being involved in pathophysiology of TD

  16. Community Norms and Human Rights: Supervising Haitian Colleagues on Interpersonal Psychotherapy (IPT) With a Depressed and Abused Pregnant Woman.

    Science.gov (United States)

    Verdeli, Helen; Therosme, Tatiana; Eustache, Eddy; Hilaire, Olissaint St; Joseph, Benissois; Sönmez, Cemile Ceren; Raviola, Giuseppe

    2016-08-01

    After the 2010 Haiti earthquake, Zanmi Lasante, a local health care organization, implemented a collaborative stepped-care model to address depression in community and primary care settings in rural Haiti. Specialized community health workers, the ajans santé, collaborate with local psychologists and primary care doctors to offer home-based evaluation, support, and follow-up. The services include brief interpersonal psychotherapy (IPT) and/or medication to persons who met locally defined criteria for depression. A cross-national (Haiti-United States) expert mental health team has been overseeing the program. The present IPT supervision case of a severely depressed, physically abused, and pregnant young woman illustrates the U.S.-based supervisor's internal struggle to reconcile awareness of and respect for local norms while maintaining a human rights-based framework. It also highlights the critical role of community health workers in addressing the mental health treatment gap in regions plagued by extreme poverty and adversity. © 2016 Wiley Periodicals, Inc.

  17. Coordination of division and development influences complex multicellular behavior in Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Jinwoo Kim

    Full Text Available The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJhomologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.

  18. Coordination of division and development influences complex multicellular behavior in Agrobacterium tumefaciens.

    Science.gov (United States)

    Kim, Jinwoo; Heindl, Jason E; Fuqua, Clay

    2013-01-01

    The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJhomologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.

  19. Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity

    Science.gov (United States)

    Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

    To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed

  20. Metabolic response of Agrobacterium tumefaciens 5A to arsenite.

    Science.gov (United States)

    Tokmina-Lukaszewska, Monika; Shi, Zunji; Tripet, Brian; McDermott, Timothy R; Copié, Valérie; Bothner, Brian; Wang, Gejiao

    2017-02-01

    Wide-spread abundance in soil and water, coupled with high toxicity have put arsenic at the top of the list of environmental contaminants. Early studies demonstrated that both concentration and the valence state of inorganic arsenic (arsenite, As(III) vs. arsenate As(V)) can be modulated by microbes. Using genetics, transcriptomic and proteomic techniques, microbe-arsenic detoxification, respiratory As(V) reduction and As(III) oxidation have since been examined. The effect of arsenic exposure on whole-cell intracellular microbial metabolism, however, has not been extensively studied. We combined LC-MS and 1 H NMR to quantify metabolic changes in Agrobacterium tumefaciens (strain 5A) upon exposure to sub-lethal concentrations of As(III). Metabolomics analysis reveals global differences in metabolite concentrations between control and As(III) exposure groups, with significant perturbations to intermediates shuttling into and cycling within the TCA cycle. These data are most consistent with the disruption of two key TCA cycle enzymes, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Glycolysis also appeared altered following As(III) stress, with carbon accumulating as complex saccharides. These observations suggest that an important consequence of As(III) contamination in nature will be to alter microbial carbon metabolism at the microbial community level and thus has the potential to foundationally impact all biogeochemical cycles in the environment. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Amikam, D.; Benziman, M.

    1989-01-01

    The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32 P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [ 14 C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes

  2. Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

    2011-01-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

  3. Evaluations and modifications of semi-selective media for improved isolation of Agrobacterium tumefaciens biovar 1 from cultivated walnut

    Science.gov (United States)

    Agrobacterium tumefaciens, the causal agent of crown gall of walnut, is an aerobic, Gram negative bacterium belonging to the family Rhizobiaceae. Like many in this group, A. tumefaciens is a common inhabitant of soil and plant host tissue. Isolation from these complex environments is difficult even ...

  4. Preparation and Imaging of [124I]IPT, [124I]β-CIT, [124I]FP-CIT

    International Nuclear Information System (INIS)

    Lee, Kyo Chul; Choi, Tae Hyun; Kim, Kyeong Min

    2006-01-01

    The dopamine transporter is a protein localized presynaptically on dopaminergic nerve terminals. The in vivo imaging of the dopamine transporter in the human brain with PET or single photon emission computer tomography (SPECT) may be useful for monitoring degenerative brain disorders such as Parkinson's disease. Within 10 years, several radioligands have been developed mainly for use in in vitro studies. For in vivo binding to the dopamine transporter, much attention has been given to the development of cocaine analogues based on the phenyltropane moiety, such as 2β- carbomethoxy-3β-(4-chlorophenyl)-8-([Z]-3-iodopropen- 1-yl)nortropane (IPT), 2β-carbomethoxy-3β-(4- iodophenyl)tropane (β-CIT), N-3-fluoropropyl-2β- carbomethoxy-3β-(4-iodophenyl)nortropane (FP-CIT) or the fluoro analogue β-CFT. The structure of β-CIT and FP-CIT allow labeling with 123 I in the aromatic ring and IPT allows labeling with 123 I in the olefin for SPECT studies or with 11C (t 1/2 20 min) in the Nmethyl group or with 18F (t 1/2 = 109.7 min) in the Nfluoroalkyl group for PET studies. An advantage with radiolabeled β-CIT and FP-CIT are that direct comparisons can be made between the results of the two imaging techniques, whereas IPT can only be used for SPECT. A problem with the quantity of β-CIT and FPCIT binding to the dopamine transporter is that the uptake of radioactivity in the dopamine-transporter rich striatum increases with time and does not reach equilibrium within the time of a PET examination 4. By following the kinetics of [ 123 I]β-CIT with SPECT, a binding equilibrium in the striatum was not reached for more than 18 h. There is a need for a radioligand for the dopamine transporter that gives an equilibrium within the time limit of a PET study (90 to 120 min) and that can be used for quantity of dopamine transporters in the human brain with the high-resolution PET cameras. Recently, new long half-life radioisotope developed for PET such as I-124 (t 1/2 = 4.18 days

  5. 6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.

    Science.gov (United States)

    Li, Huili; Xie, Kebo; Huang, Haiyan; Wang, Shuning

    2014-01-01

    Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8 ± 1.85 µmol min-1 mg protein-1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53 ± 0.03 mM 2,5-DHP was produced from 0.76 ± 0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP.

  6. Insertion Mutation in HMG-CoA Lyase Increases the Production Yield of MPA through Agrobacterium tumefaciens-Mediated Transformation.

    Science.gov (United States)

    Dong, Yuguo; Zhang, Jian; Xu, Rui; Lv, Xinxin; Wang, Lihua; Sun, Aiyou; Wei, Dongzhi

    2016-11-28

    Mycophenolic acid (MPA) is an antibiotic produced by Penicillium brevicompactum . MPA has antifungal, antineoplastic, and immunosuppressive functions, among others. β-Hydroxy-β-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the bypass metabolic pathway. The inhibitory activity of HMG-CoA lyase increases the MPA biosynthetic flux by reducing the generation of by-products. In this study, we cloned the P. brevicompactum HMG-CoA lyase gene using the thermal asymmetric interlaced polymerase chain reaction and gene walking technology. Agrobacterium tumefaciens -mediated transformation (ATMT) was used to insert a mutated HMG-CoA lyase gene into P. brevicompactum . Successful insertion of the HMG-CoA lyase gene was confirmed by hygromycin screening, PCR, Southern blot analysis, and enzyme content assay. The maximum MPA production by transformants was 2.94 g/l. This was 71% higher than wild-type ATCC 16024. Our results demonstrate that ATMT may be an alternative practical genetic tool for directional transformation of P. brevicompactum .

  7. A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

    Science.gov (United States)

    Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Do, Loc Thi Binh Xuan; Mai, Linh Thi Dam; Pham, Duc-Ngoc; Tran, Huyen Thi Thanh; Le, Diep Hong; Nguyen, Huy Quang; Tran, Van-Tuan

    2017-06-01

    Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 10 6 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

  8. Dopamine transporter density of basal ganglia assessed with [123I]IPT SPET in obsessive-compulsive disorder

    International Nuclear Information System (INIS)

    Kim, Chan-Hyung; Cheon, Keun-Ah; Lee, Hong-Shick; Koo, Min-Seong; Ryu, Young-Hoon; Lee, Jong-Doo

    2003-01-01

    It has been suggested that dopamine, as well as serotonin, is associated with the pathophysiology of obsessive-compulsive disorder (OCD). Thus, many studies have been performed on brain regions associated with dopamine in patients with OCD. In the present study, we investigated the DAT density of the basal ganglia using iodine-123 labelled N-(3-iodopropen-2-yl)-2β-carbomethoxy-3β-(4-chlorophenyl) tropane ([ 123 I]IPT) single-photon emission tomography (SPET) and evaluated the activity of the presynaptic dopamine function in patients with OCD. Fifteen patients with OCD and 19 normal control adults were included in the study. We performed brain SPET 2 h after the intravenous administration of [ 123 I]IPT and carried out both quantitative and qualitative analyses using the obtained SPET data, which were reconstructed for the assessment of the specific/non-specific dopamine transporter (DAT) binding ratio in the basal ganglia. We then investigated the correlation between the severity scores of OCD symptoms assessed with the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) and the specific/non-specific DAT binding ratio of the basal ganglia. Compared with normal control adults, patients with OCD showed a significantly increased specific/non-specific DAT binding ratio in the right basal ganglia and a tendency towards an increased specific/non-specific DAT binding ratio in the left basal ganglia. No significant correlation was found between the total scores on the Y-BOCS and the specific/non-specific DAT binding ratio of the basal ganglia. These findings suggest that the dopaminergic neurotransmitter system of the basal ganglia in patients with OCD could be involved in the pathophysiology of OCD. (orig.)

  9. Dopamine Transporter Density of the Basal Ganglia Assessed with I-123 IPT SPECT in Patients with Obsessive-Compulsive Disorder

    International Nuclear Information System (INIS)

    Ryu, W. K.; Ryu, Y. H.; Yoon, M. J.; Kim, C. H.; Chun, K. A.; Lee, J. D.; Jee, D. Y.; Choi, T. H.

    2003-01-01

    It has been suggested that dopamine as well as serotonin is associated with the pathophysiology of obsessive-compulsive disorder (OCD). Thus, many studies about brain regions associated with dopamine in OCD have been performed. In the present study, we investigated the DAT density of the basal ganglia using iodine-123 labelled N-(3-iodopropen-2-yl) - 2beta - carbomethoxy - 3beta - (4 - chloropheny1) tropane (I-123 IPT) single-photon emission tomography (SPECT) in patients with OCD and evaluated the activity of the presynaptic dopamine function in patients with OCD. Fifteen patients with OCD and nineteen normal control adults were included in the study. We performed brain SPET 2 hours after the intravenous administration of I-123 IPT and carried out both quantitative and qualitative analyses using the obtained SPET data, which were reconstructed for the assessment of the specific/non-specific DAT binding ratio in the basal ganglia. We then investigated the correlation between the severity scores of OCD symptoms assessed with the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) and the specific/non-specific DAT binding ratio of the basal ganglia. Patients with OCD showed a significantly increased specific/non-specific DAT binding ratio in right basal ganglia compared with normal control adults and an increased tendency in the specific/non-specific DAT binding ratio in left basal ganglia. No significant correlation was found between the total scores of the Y-BOCS and the specific/non-specific DAT binding ratio of the basal ganglia. Our findings suggest that the dopaminergic neurotransmitter system of the basal ganglia in patients with OCD plays an important role in fronto-subcortical circuit well-known as the pathophysiological mechanism of OCD

  10. Media development for large scale Agrobacterium tumefaciens culture.

    Science.gov (United States)

    Leth, Ingrid K; McDonald, Karen A

    2017-09-01

    A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH 4 ) 2 SO 4 ), magnesium sulfate heptahydrate (MgSO 4 *7H 2 O), calcium chloride dihydrate (CaCl 2 *2H 2 O), iron (II) sulfate heptahydrate (FeSO 4 *7H 2 O), manganese (II) sulfate monohydrate (MnSO 4 *H 2 O), zinc sulfate heptahydrate (ZnSO 4 *7H 2 O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na 2 HPO 4 /KH 2 PO 4 ). Calcium and zinc were found to have no detectable impact on biomass concentration or transient expression level, and concentrations of the other components that maximized final biomass concentration were determined. The maximum specific growth rate of Agrobacterium strain C58C1 pTFS40 in this media was 0.33 ± 0.01 h -1 and the final biomass concentration after 26 h of batch growth in shake flasks was 2.6 g dry cell weight/L. Transient expression levels of the reporter protein GUS following infiltration of a recombinant Agrobacterium strain C58C1 into N. benthamiana were comparable when the strain was grown in the defined media, Lysogeny Broth (LB) media, or yeast extract-peptone (YEP) media. In LB and YEP media, free amino acid concentration was measured at three points over the course of batch growth of Agrobacterium strain C58C1 pTFS40; results indicated that l-serine and l-asparagine were depleted from the media first, followed by l-alanine and l-glutamic acid. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1218-1225, 2017. © 2017 American Institute of Chemical Engineers.

  11. Is interpersonal counselling (IPC) sufficient treatment for depression in primary care patients? A pilot study comparing IPC and interpersonal psychotherapy (IPT).

    Science.gov (United States)

    Kontunen, Jarmo; Timonen, Markku; Muotka, Joona; Liukkonen, Timo

    2016-01-01

    Psychotherapeutic treatment is underused in primary care, where even short-term psychotherapy can be perceived as too lengthy and labour-intensive. We tested here for the first time the preliminary efficacy of seven sessions of interpersonal counselling (IPC) by comparison with sixteen sessions of interpersonal psychotherapy (IPT) in regular clinical settings. Patients seeking treatment for the first time who met the DSM-IV criteria for major depressive disorder (MDD, mild/moderate) were randomized to either IPC (n=20) or IPT (n=20). The efficacy of the treatments was assessed using the 34-item Clinical Outcomes in Routine Evaluation (CORE-OM) scale and the Beck Depression Inventory (BDI) scale. 90% of the patients completed all the treatment sessions. IPC delivered by psychiatric nurses in primary care proved equally as effective as IPT delivered by psychotherapists/psychologists in secondary care. The pre-treatment to 12-month follow-up within-group effect sizes were large: 1.52 (CORE-OM) and 1.41 (BDI) in the IPC group and 1.58 (CORE-OM) and 1.40 (BDI) in the IPT group. At the 12-month follow-up 59% of the patients in the IPC group and 63% in the IPT group were classified as recovered on the CORE-OM scale, with corresponding remission rates of 61% for both groups on the BDI scale. The small sample size limited the power to detect differences between the groups and the naturalistic settings may have confounded the results. This clinical trial suggests that IPC is an appropriate and even sufficient first-phase intervention for handling previously untreated mild to moderate depression in primary health care. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea.

    Science.gov (United States)

    Baumgartner, Kendra; Fujiyoshi, Phillip; Foster, Gary D; Bailey, Andy M

    2010-12-01

    Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.

  13. Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea▿

    Science.gov (United States)

    Baumgartner, Kendra; Fujiyoshi, Phillip; Foster, Gary D.; Bailey, Andy M.

    2010-01-01

    Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus. PMID:20952653

  14. Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions.

    Science.gov (United States)

    Chaoprasid, Paweena; Dokpikul, Thanittra; Johnrod, Jaruwan; Sirirakphaisarn, Sirin; Nookabkaew, Sumontha; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2016-06-15

    Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions. Both host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of host-pathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which ZinT functions in concert with

  15. Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum

    Directory of Open Access Journals (Sweden)

    Bipinchandra B. Kalbande

    2016-06-01

    Full Text Available A new method of transgenic development called “In-planta” transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.

  16. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    OpenAIRE

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-pro...

  17. Profound impact of Hfq on nutrient acquisition, metabolism and motility in the plant pathogen Agrobacterium tumefaciens.

    Science.gov (United States)

    Möller, Philip; Overlöper, Aaron; Förstner, Konrad U; Wen, Tuan-Nan; Sharma, Cynthia M; Lai, Erh-Min; Narberhaus, Franz

    2014-01-01

    As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.

  18. X-ray Structure of Imidazolonepropionase from Agrobacterium tumefaciens at 1.87 angstrom Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi,R.; Kumaran, D.; Burley, S.; Swaminathan, S.

    2007-01-01

    Histidine degradation in agrobacterium tumefaciens involves four enzymes, including histidase, urocanase, imidazolonepropionase, and N-formylglutamate amido hydrolase. The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-t-glutamate.

  19. Influence of volatile organic compounds emitted by Pseudomonas and Serratia strains on Agrobacterium tumefaciens biofilms.

    Science.gov (United States)

    Plyuta, Vladimir; Lipasova, Valentina; Popova, Alexandra; Koksharova, Olga; Kuznetsov, Alexander; Szegedi, Erno; Chernin, Leonid; Khmel, Inessa

    2016-07-01

    The ability to form biofilms plays an important role in bacteria-host interactions, including plant pathogenicity. In this work, we investigated the action of volatile organic compounds (VOCs) produced by rhizospheric strains of Pseudomonas chlororaphis 449, Pseudomonas fluorescens B-4117, Serratia plymuthica IC1270, as well as Serratia proteamaculans strain 94, isolated from spoiled meat, on biofilms formation by three strains of Agrobacterium tumefaciens which are causative agents of crown-gall disease in a wide range of plants. In dual culture assays, the pool of volatiles emitted by the tested Pseudomonas and Serratia strains suppressed the formation of biofilms of A. tumefaciens strains grown on polycarbonate membrane filters and killed Agrobacterium cells in mature biofilms. The individual VOCs produced by the tested Pseudomonas strains, that is, ketones (2-nonanone, 2-heptanone, 2-undecanone), and dimethyl disulfide (DMDS) produced by Serratia strains, were shown to kill A. tumefaciens cells in mature biofilms and suppress their formation. The data obtained in this study suggest an additional potential of some ketones and DMDS as protectors of plants against A. tumefaciens strains, whose virulence is associated with the formation of biofilms on the infected plants. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  20. DETECTION AND IMPLICATIONS OF EARLY AGROBACTERIUM TUMEFACIENS INFECTION OF PARADOX SEEDS AND SEEDLINGS

    Science.gov (United States)

    Paradox (Juglans hindsii x J. regia), the dominant rootstock used in California, USA walnut production, has many desirable horticultural characteristics, but is highly susceptible to crown gall. Crown gall, caused by the soil-borne bacterium Agrobacterium tumefaciens, can not be consistently control...

  1. Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans

    NARCIS (Netherlands)

    Vijn, I.; Govers, F.

    2003-01-01

    Agrobacterium tumefaciens is widely used for plant DNA transformation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late

  2. Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid

    NARCIS (Netherlands)

    Okker, Robert J.H.; Spaink, Herman; Hille, Jacques; Brussel, Ton A.N. van; Lugtenberg, Ben; Schilperoort, Rob A.

    1984-01-01

    Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for

  3. ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

    Czech Academy of Sciences Publication Activity Database

    Bleho, J.; Obert, B.; Takáč, T.; Petrovská, Beáta; Heym, C.; Menzel, D.; Šamaj, J.

    2012-01-01

    Roč. 249, č. 1 (2012), s. 53-63 ISSN 0033-183X Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : Agrobacterium rhizogenes * Agrobacterium tumefaciens * Endoplasmic reticulum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.855, year: 2012

  4. Susceptibility of Juglans Species andInterspecific Hybrids to Agrobacterium tumefaciens

    Science.gov (United States)

    James R. McKenna; Lynn Epstein

    2003-01-01

    Crown gall, caused by the common soil-borne bacterium Agrobacterium tumefaciens, can be an economic problem in walnut nurseries and production orchards in Caliiornia. The principal rootstocks used for commercial walnut production in California are the native Northern California black walnut, Juglans hindsii, and "Paradox,...

  5. VirD2 of Agrobacterium tumefaciens : functional domains and biotechnological applications

    NARCIS (Netherlands)

    Kregten, Maartje van

    2011-01-01

    Agrobacterium tumefaciens is a pathogenic bacterium, which can genetically transform plants and other organisms. It does so by translocating a part of its DNA, the T-strand, in complex with the relaxase protein VirD2. We have shown that VirD2 is the determinant of translocation of the VirD2-T-strand

  6. Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens.

    NARCIS (Netherlands)

    Mikosch, T.S.P.; Lavrijssen, B.; Griensven, van L.J.L.D.

    2001-01-01

    Agrobacterium tumefaciens is known to transfer parts of its tumour-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi. We have used this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete Agaricus bisporus.

  7. Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum)

    Energy Technology Data Exchange (ETDEWEB)

    VanderGheynst, J.S.; Guo, H.-Y.; Simmons, C.W. [Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616 (United States)

    2008-04-15

    Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) experiments were performed in harvested switchgrass (Panicum virgatum) leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta {beta}-glucuronidase expression and leaf decay. Expression was scored based on a consistent pattern of visual observations of histochemical staining over the leaf surface as might be observed in stable gene expression in switchgrass leaves. Assays on extracts from leaves were also performed to measure expression levels; however, these assays showed low expression levels, which may have been due to low recombinant protein recovery and decomposition in the leaf. Bead beating was successful for wounding the plant surface, but did not improve the consistency of expression based on histochemical staining observations. Surfactant was necessary for improving contact between the leaf surface and Agrobacterium suspension and consistently improved expression when vacuum application level was low (25 kPa). Increasing vacuum application from 25 to 5 kPa improved expression only when surfactant concentration was low. When a suspension of A. tumefaciens containing 1000 ppm Break-Thru surfactant was added to harvested leaves and 25 kPa vacuum applied, a fairly uniform expression was visualized across the leaf surface within 2-3 days of incubation, suggesting that agroinfiltration is a rapid tool for examining expression of transgenes in switchgrass leaves. (author)

  8. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

    2009-06-16

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer.

  9. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  10. Nitrogen metabolism and gas exchange parameters associated with zinc stress in tobacco expressing an ipt gene for cytokinin synthesis

    Czech Academy of Sciences Publication Activity Database

    Pavlíková, D.; Pavlík, Milan; Procházková, Dagmar; Zemanová, V.; Hnilička, F.; Wilhelmová, Naděžda

    2014-01-01

    Roč. 171, č. 7 (2014), s. 559-564 ISSN 0176-1617 R&D Projects: GA ČR(CZ) GAP501/11/1239 Institutional support: RVO:61389030 Keywords : Amino acid * Cytokinins * Photosynthetic rate Subject RIV: EF - Botanics Impact factor: 2.557, year: 2014

  11. Rapid, in situ detection of Agrobacterium tumefaciens attachment to leaf tissue.

    Science.gov (United States)

    Simmons, Christopher W; Nitin, N; Vandergheynst, Jean S

    2012-01-01

    Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging-based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum-infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post-infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  12. Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

    Science.gov (United States)

    Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

    2006-01-01

    The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%. PMID:16461638

  13. The feasibility of adapted group-based interpersonal therapy (IPT) for the treatment of depression by community health workers within the context of task shifting in South Africa.

    Science.gov (United States)

    Petersen, I; Bhana, A; Baillie, K

    2012-06-01

    Within the context of a large treatment gap for depression and a scarcity of specialist resources, there is a need for task shifting to scale up mental health services to address this gap in South Africa. This study assessed the feasibility of an adapted manualized version of grouped based Interpersonal Therapy (IPT) for use by supervised community health workers through a pilot study on 60 primary health care clinic users screened as having moderate to severe depression. Retention was good and participants in the group-based IPT intervention showed significant reduction in depressive symptoms on completion of the 12-week intervention as well as 24 weeks post baseline compared to the control group. Qualitative process evaluation suggests that improved social support, individual coping skills and improved personal agency assisted in the reduction of depressive symptoms.

  14. A pilot study of depressed adolescents' cortisol patterns during parent-adolescent conflict and response to interpersonal psychotherapy (IPT-A).

    Science.gov (United States)

    Gunlicks-Stoessel, Meredith; Mufson, Laura; Cullen, Kathryn R; Klimes-Dougan, Bonnie

    2013-09-25

    This study examined functioning of the Hypothalamic Pituitary Adrenal (HPA) axis, a neurobiological stress system centrally implicated in depression, as a predictor of treatment response to Interpersonal Psychotherapy for Depressed Adolescents (IPT-A; Mufson et al., 2004. Interpersonal psychotherapy for depressed adolescents. (second ed.). New York, Guilford Press). The sample consisted of fifteen depressed adolescents (age 12-17; mean age=15.2; 86.7% female) experiencing high levels of conflict with their parents who were recruited to participate in a pilot study of individual IPT-A and IPT-A delivered with greater and more structured involvement of parents. Adolescents came primarily from low-income Latino (93.3%) families. Prior to treatment, adolescents participated in a 15 min conflict negotiation task with their parents in which salivary cortisol was collected prior to and 10, 20, and 30 min post-conflict. Adolescents' depression symptoms were assessed pre-treatment and post-treatment (week 16) using the Children's Depression Rating Scale (CDRS-R). Higher levels of cortisol 30 min after the conflict task were associated with greater improvement in depression symptoms with IPT-A, controlling for baseline levels of depression. The study has a small sample size. This is the first study to examine HPA functioning as a predictor of depressed adolescents' response to a psychotherapeutic intervention. If replicated with larger samples, these results have important implications for advancing the use of neurobiological markers to personalize psychotherapeutic interventions for depressed adolescents. © 2013 Elsevier B.V. All rights reserved.

  15. Enhancing cytokinin synthesis by overexpressing ipt alleviated drought inhibition of root growth through activating ROS-scavenging systems in Agrostis stolonifera

    Science.gov (United States)

    Xu, Yi; Burgess, Patrick; Zhang, Xunzhong; Huang, Bingru

    2016-01-01

    Drought stress limits root growth and inhibits cytokinin (CK) production. Increases in CK production through overexpression of isopentenyltransferase (ipt) alleviate drought damages to promote root growth. The objective of this study was to investigate whether CK-regulated root growth was involved in the alteration of reactive oxygen species (ROS) production and ROS scavenging capacity under drought stress. Wild-type (WT) creeping bentgrass (Agrostis stolonifera L. ‘Penncross’) and a transgenic line (S41) overexpressing ipt ligated to a senescence-activated promoter (SAG12) were exposed to drought stress for 21 d in growth chambers. SAG12-ipt transgenic S41 developed a more extensive root system under drought stress compared to the WT. Root physiological analysis (electrolyte leakage and lipid peroxidation) showed that S41 roots exhibited less cellular damage compared to the WT under drought stress. Roots of SAG12-ipt transgenic S41 had significantly higher endogenous CK content than the WT roots under drought stress. ROS (hydrogen peroxide and superoxide) content was significantly lower and content of total and free ascorbate was significantly higher in S41 roots compared to the WT roots under drought stress. Enzymatic assays and transcript abundance analysis showed that superoxide dismutase, catalase, peroxidase, and dehydroascorbate reductase were significantly higher in S41 roots compared to the WT roots under drought stress. S41 roots also maintained significantly higher alternative respiration rates compared to the WT under drought stress. The improved root growth of transgenic creeping bentgrass may be facilitated by CK-enhanced ROS scavenging through antioxidant accumulation and activation of antioxidant enzymes, as well as higher alternative respiration rates when soil water is limited. PMID:26889010

  16. Regulation of the Cobalt/Nickel Efflux Operon dmeRF in Agrobacterium tumefaciens and a Link between the Iron-Sensing Regulator RirA and Cobalt/Nickel Resistance.

    Science.gov (United States)

    Dokpikul, Thanittra; Chaoprasid, Paweena; Saninjuk, Kritsakorn; Sirirakphaisarn, Sirin; Johnrod, Jaruwan; Nookabkaew, Sumontha; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2016-08-01

    The Agrobacterium tumefaciens C58 genome harbors an operon containing the dmeR (Atu0890) and dmeF (Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. The dmeRF operon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZ transcriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR represses dmeRF transcription through direct binding to the promoter region upstream of dmeR A strain lacking dmeF showed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting a Nicotiana benthamiana plant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated gene sodBII Furthermore, control of iron homeostasis via RirA is important for the ability of A. tumefaciens to cope with cobalt and nickel toxicity. The molecular mechanism of the regulation of dmeRF transcription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator site in vitro The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5'-ATATAGTATACCCCCCTATAGTATAT-3'). Cobalt and nickel cause DmeR to dissociate from the dmeRF promoter, which leads to expression of the metal efflux gene dmeF This work also revealed a connection between iron homeostasis and cobalt/nickel resistance in A. tumefaciens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Susceptibility of chrysanthemum (Dendranthema grandiflora Tzvelev cultivars to crown gall [Agrobacterium tumefaciens (Smith et Townsend Conn

    Directory of Open Access Journals (Sweden)

    Małgorzata Schollenberger

    2012-12-01

    Full Text Available The susceptibility of 29 cultivars of chrysanthemum, representing various types of growth, to crown gall was studied. Two isolates of Agrobacterium tumefaciens: chrysanthemum and apple were used for testing plants. The inoculation was made to the stem base of rooted cuttings and places of pinching off. The first method gave larger galls on most tested plants and only three cultivars were resistant to inoculation with both A. tumefaciens isolates. Weak symptoms were obtained after cutting of stem tips, thus plants of 9 and 5 cultivars (in subsequent tests showed no symptoms. Only three chrysanthemum cultivars were resistant to crown gall in all tests: Epidote Jaune, Epidote Orange and Epidote Rouge, all belonging to potted chrysanthemum. Plants of Maracas cultivar were resistant when bacteria were applied to the wound of stem tips. In all tests, the apple isolate was weak pathogenic, thus no gall were observed on many cultivars.

  18. Interaction between Meloidogyne incognita and Agrobacterium tumefaciens or Fusarium oxysporum f. sp. lycopersici on Tomato

    Science.gov (United States)

    El-Sherif, Ahmed G.; Elwakil, M. A.

    1991-01-01

    Agrobacterium tumefaciens stimulated and Fusarium oxysporum f. sp. lycopersici inhibited development and reproduction of Meloidogyne incognita when applied to the opposite split root of tomato, Lycopersicon esculentum cv. Tropic, plants. The lowest rate of nematode reproduction occurred after 2,000 juveniles were applied and the fungus was present in the opposite split root. The effects of all three pathogens alone on the growth of roots and shoots of tomato plants were evident, but M. incognita had a greater effect alone than did either of the other pathogens. The length of split roots was reduced by the infection of M. incognita and A. tumefaciens or F. oxysporum f. sp. lycopersici. The number of galls induced by nematodes on roots was higher where the bacterium was applied and lower where the fungus was applied to the opposite split root. PMID:19283119

  19. Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

    Science.gov (United States)

    Hwang, Hau-Hsuan; Liu, Yin-Tzu; Huang, Si-Chi; Tung, Chin-Yi; Huang, Fan-Chen; Tsai, Yun-Long; Cheng, Tun-Fang; Lai, Erh-Min

    2015-02-01

    Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.

  20. Colonization of Tomato Plants by Two Agrocin-Producing Strains of Agrobacterium tumefaciens

    OpenAIRE

    Macrae, Sharmane; Thomson, Jennifer A.; Van Staden, Johannes

    1988-01-01

    For a bacterium to be a successful biocontrol agent against crown gall disease, it must produce an effective agrocin specific for Agrobacterium tumefaciens and be able to colonize host plants efficiently. The colonization abilities of K84 and J73, successful and potential biocontrolling strains, respectively, were compared both in vivo and in vitro. Both strains produced fibrils attaching them to tomato root surfaces and had similar colonization efficiencies up to 14 days after inoculation. H...

  1. A signaling pathway involving the diguanylate cyclase CelR and the response regulator DivK controls cellulose synthesis in Agrobacterium tumefaciens.

    Science.gov (United States)

    Barnhart, D Michael; Su, Shengchang; Farrand, Stephen K

    2014-03-01

    The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK, the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR. These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK.

  2. Green route to synthesis of valuable chemical 6-hydroxynicotine from nicotine in tobacco wastes using genetically engineered Agrobacterium tumefaciens S33.

    Science.gov (United States)

    Yu, Wenjun; Wang, Rongshui; Li, Huili; Liang, Jiyu; Wang, Yuanyuan; Huang, Haiyan; Xie, Huijun; Wang, Shuning

    2017-01-01

    Tobacco is widely planted as an important nonfood economic crop throughout the world, and large amounts of tobacco wastes are generated during the tobacco manufacturing process. Tobacco and its wastes contain high nicotine content. This issue has become a major concern for health and environments due to its toxicity and complex physiological effects. The microbial transformation of nicotine into valuable functionalized pyridine compounds is a promising way to utilize tobacco and its wastes as a potential biomass resource. Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways, in which several intermediates, such as 6-hydroxynicotine, can be used as renewable precursors to synthesize drugs and insecticides. This provides an opportunity to produce valuable chemical 6-hydroxynicotine from nicotine via biocatalysis using strain S33. To accumulate the intermediate 6-hydroxynicotine, we firstly identified the key enzyme decomposing 6-hydroxynicotine, named 6-hydroxynicotine oxidase, and then disrupted its encoding gene in A. tumefaciens S33. With the whole cells of the mutant as a biocatalyst, we tested the possibility to produce 6-hydroxynicotine from the nicotine of tobacco and its wastes and optimized the reaction conditions. At 30 °C and pH 7.0, nicotine could be efficiently transformed into 6-hydroxynicotine by the whole cells cultivated with glucose/ammonium/6-hydroxy-3-succinoylpyridine medium. The molar conversion and the specific catalytic rate reached approximately 98% and 1.01 g 6-hydroxynicotine h -1  g -1 dry cells, respectively. The product could be purified easily by dichloromethane extraction with a recovery of 76.8%, and was further confirmed by UV spectroscopy, mass spectroscopy, and NMR analysis. We successfully developed a novel biocatalytic route to 6-hydroxynicotine from nicotine by blocking the nicotine catabolic pathway via gene disruption, which provides an alternative green

  3. Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

    Science.gov (United States)

    Dai, Ziyu; Deng, Shuang; Culley, David E; Bruno, Kenneth S; Magnuson, Jon K

    2017-08-01

    Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces

  4. Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Ziyu; Deng, Shuang; Culley, David E.; Bruno, Kenneth S.; Magnuson, Jon K.

    2017-06-19

    Background: Because of interest in the production of renewable bio-hydrocarbon fuels, various living organisms have been explored for their potential use in producing fuels and chemicals. The oil-producing (oleaginous) yeast Lipomyces starkeyi is the subject of active research regarding the production of lipids using a wide variety of carbon and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements using the tools of synthetic biology and metabolic engineering. However, using these tools for strain improvement requires the establishment of effective and reliable transformation methods with suitable selectable markers (antibiotic resistance or auxotrophic marker genes) and the necessary genetic elements (promoters and terminators) for expression of introduced genes. Chemical-based methods have been published, but suffer from low efficiency or the requirement for targeting to rRNA loci. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. Results: In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species and that the introduced DNA can be reliably integrated into the chromosomes of these species. The gene deletion of Ku70 and Pex10 was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial -glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1 promoter was also stably expressed in seven different Lipomyces species. Conclusion: The results from this study clearly demonstrate that Agrobacterium-mediated transformation is a reliable genetic tool for gene deletion and integration and expression of heterologous genes in L. starkeyi and other Lipomyces species.

  5. Dopamine transporter density assessed with [{sup 123}]IPT SPECT before and after risperidone treatment in children with tourette's disorder

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Young Hoon; Kim, Tae Hoon; Ryu, Won Gee [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of)] [and others

    2004-02-01

    Tourette's disorder (TD), which is characterized by multiple waxing and waning motor tics and one or more vocal tics, is known to be associated with abnormalities in the dopaminergic system. To testify our hypothesis that risperidone would improve tic symptoms of TD patients through the change of the dopaminergic system, we measured the dopamine transporter (DAT) densities between drug-naive children with TD and normal children, and investigated the DAT density before and after treatment with risperidone in drug-naive children with TD, using iodine-123 labelled N-(3-iodopropen-2-yl)-2{beta}-carbomethoxy-3beta-(4-chlorophenyl)tropane ([{sup 123}I]IPT) single photon emission computed tomography (SPECT). [{sup 123I}]IPT SPECT imaging and Yale Global Tic Severity Scale-Korean version (YGTSS-K) for assessing the tic symptom severity were carried out before and after treatment with risperidone for 8 weeks in nine drug-naive children with TD. Eleven normal children also underwent SPECT imaging 2 hours after an intravenous administration of [{sup 123}I]IPT. Drug-naive children with TD had a significantly greater increase in the specific/nonspecific DAT binding ratio of both basal ganglia compared with the normal children. However, no significant difference in the specific/nonspecific DAT binding ratio of the basal ganglia before and after treatment with risperidone in children with TD was found, although tic symptoms were significantly improved with risperidone. These findings suggest that DAT densities are directly associated with the pathophysiology of TD, however, that the effect of risperidone on tic symptoms in children with TD is not attributed to the change of dopaminergic system.

  6. Assessing the Genetic Diversity of Agrobacterium Tumefaciens in CA Walnut Growing Regions and Resistance to the Biocontrol Agent, A. Rhizogenes K84

    Science.gov (United States)

    Crown gall of walnut (Juglans sp.), caused by the bacterium Agrobacterium tumefaciens, greatly impacts the CA walnut industry. To determine the genetic diversity of A. tumefaciens throughout the Central Valley of CA, we collected isolates from ten walnut growing counties. A total of 340 A. tumefac...

  7. Incidence of Agrobacterium tumefaciens biovar 1 in and on ‘Paradox’ (Juglans hindsii x Juglans regia) walnut seed collected from commercial nurseries

    Science.gov (United States)

    The walnut rootstock Paradox (Juglans hindsii (Jeps) Rehder x J. regia L.) is susceptible to Agrobacterium tumefaciens (7) which often results in a high incidence of crown gall in nursery or walnut production orchards. Though A. tumefaciens is susceptible to the commonly used preplant soil fumigant...

  8. Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

    Science.gov (United States)

    Houdelet, Marcel; Galinski, Anna; Holland, Tanja; Wenzel, Kathrin; Schillberg, Stefan; Buyel, Johannes Felix

    2017-04-01

    Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD 600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco

    Czech Academy of Sciences Publication Activity Database

    Šámalová, Markéta; Brzobohatý, Břetislav; Moore, Ian

    2005-01-01

    Roč. 41, - (2005), s. 919-935 ISSN 0960-7412 R&D Projects: GA AV ČR(CZ) IAA5004001; GA AV ČR(CZ) KSK5052113 Institutional research plan: CEZ:AV0Z50040507 Keywords : chemically inducible gene expression * cytokinin-biosynthetic gene ipt * tobacco Subject RIV: BO - Biophysics Impact factor: 6.969, year: 2005

  10. Transient Expression of Viral Proteins in Plants Using Agrobacterium tumefaciens.

    Science.gov (United States)

    Hitzeroth, Inga I; van Zyl, Albertha R

    2016-01-01

    Transient expression of viral proteins in plants is a novel alternative to other expression platforms. The viral proteins can be used as potential vaccines or in diagnostics. Nicotiana benthamiana leaves or whole plants are infiltrated with recombinant Agrobacterium that harbor the gene of interest. Protein expression in the plants is rapid and results are obtained within 2-7 days. Here we describe how to make electrocompetent Agrobacterium, how to transform Agrobacterium, how to infiltrate leaves or plants with the recombinant Agrobacterium, and lastly how to extract the protein for analysis by gel electrophoresis.

  11. Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector

    Directory of Open Access Journals (Sweden)

    Nikolić Radomirka

    2007-01-01

    Full Text Available Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa­ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1 over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42% plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by β-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

  12. Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens.

    Science.gov (United States)

    Howell, Matthew; Aliashkevich, Alena; Salisbury, Anne K; Cava, Felipe; Bowman, Grant R; Brown, Pamela J B

    2017-09-01

    Agrobacterium tumefaciens is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The A. tumefaciens homolog of the Caulobacter crescentus polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of A. tumefaciens Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division. IMPORTANCE A. tumefaciens is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of A. tumefaciens growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in A. tumefaciens may be broadly applicable for devising antimicrobial strategies. Copyright © 2017 American Society for Microbiology.

  13. Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

    2009-05-14

    ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

  14. Antisense-induced suppression of taxoid 14β- hydroxylase gene ...

    African Journals Online (AJOL)

    Following the construction of an antisense RNA expression vector of 14OH from Taxus chinensis, the antisense 14OH cDNA (as14OH) was introduced into TM3 cells by Agrobacterium tumefaciens-mediated transformation. Southern blot analysis of hygromycin phosphotransferase gene (HYG) revealed that this selection ...

  15. Isolation of OmpA gene from Salmonella typhimurium and ...

    African Journals Online (AJOL)

    Isolation of OmpA gene from Salmonella typhimurium and transformation into alfalfa in order to develop an edible plant based vaccine. ... The recombinant OmpA was expressed in Escherichia coli TG1. The new construct was used to transform the Agrobacterium tumefaciens Strain LBA4404 before plant transformation.

  16. Isolation of OmpA gene from Salmonella typhimurium and ...

    African Journals Online (AJOL)

    user

    2011-01-31

    Jan 31, 2011 ... into a plant expression vector PBI121.The recombinant OmpA was expressed in Escherichia coli TG1. The new construct was used to transform the Agrobacterium tumefaciens Strain LBA4404 before plant transformation. Transgenic alfalfa plants were then developed by introducing OmpA gene in the plant.

  17. Dopamine transporter density in the basal ganglia assessed with {sup 123}I-IPT SPECT in children with Tourette's deosoder

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Y. H.; Cheon, K. A.; Yoon, M. J.; Kim, C. H.; Lee, J. D. [Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, H. H.; Choi, T. H. [Gachon Medical School, Incheon (Korea, Republic of)

    2002-07-01

    Previous studies in patients with Tourette's disorder (TD) provided evidences of presynaptic dopaminergic dysfunction, demonstrating increased dopamine transporter densities. We investigated dopamine transporter densities using {sup 123}I-IPT SPECT in drug-naive children with TD and postulated that dopamine transporter density reflected dopamine concentrations. 9 drug-naive children with ADHD and 8 normal children were included in the study. We performed brain SPECT 2 hours after administration of {sup 123}I-IPT and made both quantitative and qualitative analyses for assessment of specific/nonspecific DAT binding ratio in the BG. We then investigated correlation between the severity of tics in children with TD assessed with the YGTSS and specific/nonspecific binding ratio on BG. Drug-naive children with TD showed a significantly increased specific/nonspecific DAT binding ratio in the BG compared with normal children. We found no significant correlation between severity of tics assessed with YGTSS in children with TD and specific/nonspecific DAT binding ratio in BG. These findings support the hypothesis of dopamine dysregulation in presynaptic dopamine function of BG being involved in pathophysiology of TD.

  18. Dopamine transporter density in the basal ganglia assessed with [123I]IPT SPET in children with attention deficit hyperactivity disorder

    International Nuclear Information System (INIS)

    Cheon, Keun-Ah; Kim, Young-Kee; Namkoong, Kee; Kim, Chan-Hyung; Ryu, Young Hoon; Lee, Jong Doo

    2003-01-01

    Attention deficit hyperactivity disorder (ADHD) is a psychiatric disorder in childhood that is known to be associated with dopamine dysregulation. In this study, we investigated dopamine transporter (DAT) density in children with ADHD using iodine-123 labelled N-(3-iodopropen-2-yl)-2β-carbomethoxy-3β-(4-chlorophenyl) tropane ([ 123 I]IPT) single-photon emission tomography (SPET) and postulated that an alteration in DAT density in the basal ganglia is responsible for dopaminergic dysfunction in children with ADHD. Nine drug-naive children with ADHD and six normal children were included in the study. We performed brain SPET 2 h after the intravenous administration of [ 123 I]IPT and carried out both quantitative and qualitative analyses using the obtained SPET data, which were reconstructed for the assessment of the specific/non-specific DAT binding ratio in the basal ganglia. We then investigated the correlation between the severity scores of ADHD symptoms in children with ADHD assessed with ADHD rating scale-IV and the specific/non-specific DAT binding ratio in the basal ganglia. Drug-naive children with ADHD showed a significantly increased specific/non-specific DAT binding ratio in the basal ganglia compared with normal children. However, no significant correlation was found between the severity scores of ADHD symptoms in children with ADHD and the specific/non-specific DAT binding ratio in the basal ganglia. Our findings support the complex dysregulation of the dopaminergic neurotransmitter system in children with ADHD. (orig.)

  19. Dopamine transporter density in the basal ganglia assessed with [{sup 123}I]IPT SPET in children with attention deficit hyperactivity disorder

    Energy Technology Data Exchange (ETDEWEB)

    Cheon, Keun-Ah; Kim, Young-Kee; Namkoong, Kee; Kim, Chan-Hyung [Department of Psychiatry, College of Medicine, Yonsei University, Seoul (Korea); Ryu, Young Hoon; Lee, Jong Doo [Division of Nuclear Medicine, Department of Radiology, College of Medicine, Yonsei University, 146-92 Dogokdong, Gangnam-Gu, Seoul, 135-720 (Korea)

    2003-02-01

    Attention deficit hyperactivity disorder (ADHD) is a psychiatric disorder in childhood that is known to be associated with dopamine dysregulation. In this study, we investigated dopamine transporter (DAT) density in children with ADHD using iodine-123 labelled N-(3-iodopropen-2-yl)-2β-carbomethoxy-3β-(4-chlorophenyl) tropane ([{sup 123}I]IPT) single-photon emission tomography (SPET) and postulated that an alteration in DAT density in the basal ganglia is responsible for dopaminergic dysfunction in children with ADHD. Nine drug-naive children with ADHD and six normal children were included in the study. We performed brain SPET 2 h after the intravenous administration of [{sup 123}I]IPT and carried out both quantitative and qualitative analyses using the obtained SPET data, which were reconstructed for the assessment of the specific/non-specific DAT binding ratio in the basal ganglia. We then investigated the correlation between the severity scores of ADHD symptoms in children with ADHD assessed with ADHD rating scale-IV and the specific/non-specific DAT binding ratio in the basal ganglia. Drug-naive children with ADHD showed a significantly increased specific/non-specific DAT binding ratio in the basal ganglia compared with normal children. However, no significant correlation was found between the severity scores of ADHD symptoms in children with ADHD and the specific/non-specific DAT binding ratio in the basal ganglia. Our findings support the complex dysregulation of the dopaminergic neurotransmitter system in children with ADHD. (orig.)

  20. Dopamine transporter density in the basal ganglia assessed with {sup 123}I-IPT SPECT in children with attention deficit hyperactivity disorder

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Y. H.; Cheon, K. A.; Yoon, M. J.; Kim, C. H.; Lee, J. D. [Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, H. H.; Choi, T. H. [Gachon Medical School, Incheon (Korea, Republic of)

    2002-07-01

    Attention deficit hyperactivity disorder (ADHD) is known as a psychiatric disorder in childhood associated with dopamine dysregulation. We investigated dopamine transporter (DAT) density in children with ADHD in the present study using {sup 123}I-IPT SPECT and postulated that an alteration in DAT density in the basal ganglia (BG) is responsible for dopaminergic dysfunction in children with ADHD. 9 durg-naive children with ADHD and 6 normal children were included in the study. We performed brain SPECT 2 hours after administration of {sup 123}I-IPT and made both quantitative and qualitative analyses for assessment of specific/nonspecific DAT binding ratio in the BG. We investigated the correlation between the severity scores of ADHD symptoms in children with ADHD assessed with ADHD rating scale and specific/nonspecific DAT binding ratio in the BG. Drug-naive children with ADHD showed a significantly incresed specific/nonspecific DAT binding ratio in the BG compared with normal children. Whereas, no significant correlation was found between severity scores of symptoms in children with ADHD and specific/nonspecific DAT binding ratio n the BG. Our findings support complex dysregulation of the dopaminergic neurotransmitter system in children with ADHD.

  1. Dopamine transporter density in the basal ganglia assessed with 123I-IPT SPECT in children with attention deficit hyperactivity disorder

    International Nuclear Information System (INIS)

    Yoo, Y. H.; Cheon, K. A.; Yoon, M. J.; Kim, C. H.; Lee, J. D.; Kim, H. H.; Choi, T. H.

    2002-01-01

    Attention deficit hyperactivity disorder (ADHD) is known as a psychiatric disorder in childhood associated with dopamine dysregulation. We investigated dopamine transporter (DAT) density in children with ADHD in the present study using 123 I-IPT SPECT and postulated that an alteration in DAT density in the basal ganglia (BG) is responsible for dopaminergic dysfunction in children with ADHD. 9 durg-naive children with ADHD and 6 normal children were included in the study. We performed brain SPECT 2 hours after administration of 123 I-IPT and made both quantitative and qualitative analyses for assessment of specific/nonspecific DAT binding ratio in the BG. We investigated the correlation between the severity scores of ADHD symptoms in children with ADHD assessed with ADHD rating scale and specific/nonspecific DAT binding ratio in the BG. Drug-naive children with ADHD showed a significantly incresed specific/nonspecific DAT binding ratio in the BG compared with normal children. Whereas, no significant correlation was found between severity scores of symptoms in children with ADHD and specific/nonspecific DAT binding ratio n the BG. Our findings support complex dysregulation of the dopaminergic neurotransmitter system in children with ADHD

  2. The PKS4 gene in Fusarium graminearum is essential for zearalenone production

    DEFF Research Database (Denmark)

    Lysøe, E.; Klemsdal, S. S.; Bone, K.R.

    2006-01-01

    polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation...

  3. One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

    Directory of Open Access Journals (Sweden)

    Yun-Long Tsai

    Full Text Available Alpha-crystallin-type small heat shock proteins (sHsps are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS. HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence

  4. Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

    Energy Technology Data Exchange (ETDEWEB)

    Bäuerle, Bettina [Institute of Microbiology, University of Stuttgart, 70569 Stuttgart (Germany); Sandalova, Tatyana; Schneider, Gunter [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm (Sweden); Rieger, Paul-Gerhard, E-mail: pg.rieger@imb.uni-stuttgart.de [Institute of Microbiology, University of Stuttgart, 70569 Stuttgart (Germany)

    2006-08-01

    This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.

  5. Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens.

    Science.gov (United States)

    Martínez-Cruz, Jesús; Romero, Diego; de Vicente, Antonio; Pérez-García, Alejandro

    2017-03-01

    The obligate biotrophic fungal pathogen Podosphaera xanthii is the main causal agent of powdery mildew in cucurbit crops all over the world. A major limitation of molecular studies of powdery mildew fungi (Erysiphales) is their genetic intractability. In this work, we describe a robust method based on the promiscuous transformation ability of Agrobacterium tumefaciens for reliable transformation of P. xanthii. The A. tumefaciens-mediated transformation (ATMT) system yielded transformants of P. xanthii with diverse transferred DNA (T-DNA) constructs. Analysis of the resultant transformants showed the random integration of T-DNA into the P. xanthii genome. The integrations were maintained in successive generations in the presence of selection pressure. Transformation was found to be transient, because in the absence of selection agent, the introduced genetic markers were lost due to excision of T-DNA from the genome. The ATMT system represents a potent tool for genetic manipulation of P. xanthii and will likely be useful for studying other biotrophic fungi. We hope that this method will contribute to the development of detailed molecular studies of the intimate interaction established between powdery mildew fungi and their host plants. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  6. Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

    International Nuclear Information System (INIS)

    Bäuerle, Bettina; Sandalova, Tatyana; Schneider, Gunter; Rieger, Paul-Gerhard

    2006-01-01

    This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH 4 ) 2 SO 4 and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na 2 SO 4 and 0.1 M bis-Tris propane pH 8.5

  7. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Atkinson, Sarah C.; Dogovski, Con; Dobson, Renwick C. J.; Perugini, Matthew A.

    2012-01-01

    Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40 Å. Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP-354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40 Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents

  8. X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam (SGX); (BNL)

    2010-01-12

    Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging to this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.

  9. Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2012-01-01

    The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution. (R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2 1 , with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å 3 Da −1 )

  10. Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola.

    Science.gov (United States)

    Flowers, Jennifer L; Vaillancourt, Lisa J

    2005-12-01

    We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation. Various experimental parameters were tested for their effects on transformation efficiencies. The parameters with the greatest influence were the A. tumefaciens strain used and the Ti-plasmid it carried, the ratio of bacterium to fungus during cocultivation, and the length of cocultivation. Southern analysis demonstrated that most transformants (80%) contained tandem integrations of plasmid sequences, and at least 36% had integrations at multiple sites in the genome. In a majority of cases (70%), the whole Ti-plasmid, and not just the T-DNA, had integrated as a series of tandem repeats. Tandem integrations, especially of the whole plasmid, make it difficult to rescue DNA from both flanks of the integrations with standard PCR-based approaches. Thus, ATMT may be unsuitable for insertional mutagenesis of C. graminicola without further modification.

  11. Metabolic modeling of denitrification in Agrobacterium tumefaciens : A tool to study inhibiting and activating compounds for the denitrification pathway

    NARCIS (Netherlands)

    Kampschreur, M.J.; Kleerebezem, R.; Picioreanu, C.; Bakken, L.; Bergaust, L.; De Vries, S.; Jetten, M.S.M.; Van Loosdrecht, M.C.M.

    2012-01-01

    A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study

  12. Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells

    NARCIS (Netherlands)

    Hille, Jacques; Wullems, George; Schilperoort, Rob

    1983-01-01

    A new procedure for site-directed mutagenesis has been applied to the shooting and rooting loci of T-DNA of an octopine Ti-plasmid of Agrobacterium tumefaciens. Mutants have been obtained which induced tumours that either developed shoots or produced more roots than normally observed. Double

  13. Transformation of different barley (Hordeum vulgare L.) cultivars by Agrobacterium tumefaciens infection of in vitro cultured ovules

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted; Brinch-Pedersen, Henrik; Lange, Mette

    2008-01-01

    , and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary...

  14. EXPRESIÓN GUS EN EXPLANTES DE Solanum phureja (Juz. et. Buk Var. Criolla Colombia, TRANSFORMADOS CON Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    IVÁN DARÍO BARRERO-FARFÁN

    2008-01-01

    Full Text Available La expresión transitoria y estable del gen gusA-intron en explantes internodales de papa criolla variedad Criolla Colombia cocultivados con Agrobacterium tumefaciens es reportada. Con el fin de determinar la susceptibilidad de esta variedad a la transformación mediada por A. tumefaciens, explantes internodales de Solanum phureja fueron infectados con la cepa LBA4404 de A. tumefaciens que contiene el plásmido pCAMBIA2301. Este plásmido contiene el gen ntpII que confiere resistencia a kanamicina y el gen reportero gusA-intron. La selección de los explantes potencialmente transgénicos fue realizada en medios con kanamicina. La eficiencia de transformación estable y transitoria fue calculada con base en la actividad GUS (ß-glucuronidasa, detectada por el ensayo histoquímico X-gluc. La expresión transitoria y estable del gen gusA-intron fue observada en células del explante más bien que en tejidos completos. Estos resultados demuestran que la papa criolla (S. phureja Juz. et. Buk variedad Criolla Colombia es susceptible a la infección por A. tumefaciens.

  15. Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Frandsen, Mette; Giese, Nanna Henriette

    2012-01-01

    cloning without the need for subcloning. The cloning efficiency is not always as high as desired, but it still presents an efficient alternative to restriction enzyme and ligase-based cloning systems. The USER technology offers a higher four fragment cloning efficiency than In-Fusion, but depends...

  16. Efficient transformation and expression of gfp gene in Valsa mali var. mali.

    Science.gov (United States)

    Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

    2015-01-01

    Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

  17. Biophysical control of the growth of Agrobacterium tumefaciens using extremely low frequency electromagnetic waves at resonance frequency.

    Science.gov (United States)

    Fadel, M Ali; El-Gebaly, Reem H; Mohamed, Shaimaa A; Abdelbacki, Ashraf M M

    2017-12-09

    Isolated Agrobacterium tumefaciens was exposed to different extremely low frequencies of square amplitude modulated waves (QAMW) from two generators to determine the resonance frequency that causes growth inhibition. The carrier was 10 MHz sine wave with amplitude ±10 Vpp which was modulated by a second wave generator with a modulation depth of ± 2Vpp and constant field strength of 200 V/m at 28 °C. The exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min inhibited the bacterial growth by 49.2%. In addition, the tested antibiotics became more effective against A. tumefaciens after the exposure. Furthermore, results of DNA, dielectric relaxation and TEM showed highly significant molecular and morphological changes due to the exposure to 1.0 Hz QAMW for 90 min. An in-vivo study has been carried out on healthy tomato plants to test the pathogenicity of A. tumefaciens before and after the exposure to QAMW at the inhibiting frequency. Symptoms of crown gall and all pathological symptoms were more aggressive in tomato plants treated with non-exposed bacteria, comparing with those treated with exposed bacteria. We concluded that, the exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min modified its cellular activity and DNA structure, which inhibited the growth and affected the microbe pathogenicity. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division.

    Science.gov (United States)

    Cameron, Todd A; Anderson-Furgeson, James; Zupan, John R; Zik, Justin J; Zambryski, Patricia C

    2014-05-27

    The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l

  19. Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture.

    Science.gov (United States)

    Cornish, A; Greenwood, J A; Jones, C W

    1989-11-01

    Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A

  20. The effect of selected fungicides on survival of Agrobacterium tumefaciens in various kinds of soils

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    Stanisław Barczyński

    2013-12-01

    Full Text Available The effect of 10 fungicides on survival of A. tumefaciens in various types of soils was studied. In fertile, nonsterile soil Dithane M-45 (mancozeb, Euparen 50 WP (tolyfluanid, Kaptan 50 WP (captan and Ridomil Gold 80 WP (metalaxyl at concentration of 1000 ppm showed the highest antibacterial activity. Similar trends in activity of these fungicides occurred in fertile, sterile soil, however a little lower in case of Kaptan and Euparen. In most of investigated soils Befran 25 SL (imimnoctadyne, Syllit 65 WP (dodine and Thiram Granulfo 80 WG (thiram increased bacteria number. In sandy acidic soil (pH 3,5 all tested fungicides totally eliminated bacteria. On the other hand in sandy neutral soil only Dithane, Euparen, Kaptan and Ridomil showed such activity. Ten fold decrease of fungicides concentration generally did not influence Kaptan and Ridomil effectiveness but it decreased the activity of Dithane and Euparen.

  1. Direct fluorescence detection of VirE2 secretion by Agrobacterium tumefaciens.

    Science.gov (United States)

    Yaakov, Noga; Barak, Yoav; Pereman, Idan; Christie, Peter J; Elbaum, Michael

    2017-01-01

    VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation.

  2. INTRODUKSI GEN Sitrat Sintase KE DALAM RUMPUT LAUT Kappaphycus alvarezii MENGGUNAKAN Agrobacterium tumefaciens

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    Ristanti Frinra Daud

    2013-08-01

    ekonomis penting. Ice-ice merupakan penyakit yang paling umum menyerang rumput laut dan menyebabkan menurunnya produksi rumput laut. Penyakit ini disebabkan oleh perubahan salinitas, suhu, dan pencemaran logam berat. Asam sitrat digunakan sebagai pengkelat logam berat. Introduksi gen sitrat sintase ke dalam genom tanaman diketahui dapat mengurangi cekaman oksidatif. Penelitian ini bertujuan untuk mengintroduksi gen sitrat sintase ke dalam genom K. alvarezii menggunakan perantara Agrobacterium tumefaciens. Berdasarkan eksplan yang tahan pada media seleksi higromisin, efisiensi transformasi pada K. alvarezii sebesar 7,5%. Efisiensi regenerasi tunas transgenik putatif sebesar 100%, efisiensi tunas non transgenik sebesar 100%. Analisis molekular menggunakan teknik PCR, satu dari lima K. alvarezii transgenik putatif mengandung transgen PaCS di bawah kendali promoter 35S CaMV.

  3. Direct fluorescence detection of VirE2 secretion by Agrobacterium tumefaciens

    Science.gov (United States)

    Yaakov, Noga; Barak, Yoav; Pereman, Idan; Christie, Peter J.; Elbaum, Michael

    2017-01-01

    VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation. PMID:28403156

  4. Agrobacterium-Mediated Transformation of Tomato Elicits Unexpected Flower Phenotypes with Similar Gene Expression Profiles

    OpenAIRE

    Wang, Yi-Hong; Campbell, Michael A.

    2008-01-01

    BACKGROUND: Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in altered floral structure. METHODOLOGY/PRINCIPAL FINDINGS: Previously we identified two genes (LeTGA1 and SOLly GLB1) induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each...

  5. A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

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    Katholiki Skopelitou

    Full Text Available In the present work, we report a novel class of glutathione transferases (GSTs originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701 with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H. This enzyme (designated as AtuGSTH1-1 was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl-glutathione (Nb-GSH. Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34, an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity.

  6. Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in Agrobacterium tumefaciens

    Science.gov (United States)

    Lin, Jer-Sheng; Wu, Hsin-Hui; Hsu, Pang-Hung; Ma, Lay-Sun; Pang, Yin-Yuin; Tsai, Ming-Daw; Lai, Erh-Min

    2014-01-01

    The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed. PMID:24626341

  7. A Photolyase-Like Protein from Agrobacterium tumefaciens with an Iron-Sulfur Cluster

    Science.gov (United States)

    Oberpichler, Inga; Pierik, Antonio J.; Wesslowski, Janine; Pokorny, Richard; Rosen, Ran; Vugman, Michal; Zhang, Fan; Neubauer, Olivia; Ron, Eliora Z.; Batschauer, Alfred; Lamparter, Tilman

    2011-01-01

    Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster. PMID:22066008

  8. A photolyase-like protein from Agrobacterium tumefaciens with an iron-sulfur cluster.

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    Inga Oberpichler

    Full Text Available Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF that contains an iron-sulfur cluster.

  9. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    Science.gov (United States)

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

  10. Norway spruce (Picea abies) genetic transformation with modified Cry3A gene of Bacillus thuringiensis

    Czech Academy of Sciences Publication Activity Database

    Bříza, Jindřich; Pavingerová, Daniela; Vlasák, Josef; Niedermeierová, Hana

    2013-01-01

    Roč. 60, č. 3 (2013), s. 395-400 ISSN 0001-527X R&D Projects: GA MZe QH71290; GA ČR(CZ) GAP502/11/1471 Institutional support: RVO:60077344 Keywords : Cry3A gene modification * Picea abies * Agrobacterium tumefaciens Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.389, year: 2013

  11. Genetic transformation via Agrobacterium tumefaciens in embryogenic cell suspensions of the plantain hybrid cultivar FHIA 21 (AAAB

    Directory of Open Access Journals (Sweden)

    Boris Chong

    2002-01-01

    Full Text Available The genetic improvement of plantain and banana is of great importance for its level of consumption on a world-wide scale. Genetic transformation constitutes one alternative for genetic improvement and has complemented traditional techniques. In this work some parameters of genetic transformation of plantain were studied by means of transient expression of â-glucoronidase in the hybrid cultivar FHIA-21(AAAB by Agrobacterium tumefaciens. A study with the β-Agrobacterium tumefaciens strains AT-2260 and EHA-105, both with the plasmid pCAMBIA-3301 was done. A comparison between the time of infection and time of co-culture was studied. The strain of better behavior was EHA-105. In the study of the time of infection and co-culture, the best combination resulted to be the one with two hours of infection and six days of co-culture. Key words: At-2260, EHA-105, β-glucoronidase, Musa

  12. Biocontrol activity and patulin-removal effects of Bacillus subtilis, Rhodobacter sphaeroides and Agrobacterium tumefaciens against Penicillium expansum.

    Science.gov (United States)

    Wang, Y; Yuan, Y; Liu, B; Zhang, Z; Yue, T

    2016-11-01

    This study was conducted to evaluate the biocontrol potential of Bacillus subtilis CICC 10034, Rhodobacter sphaeroides CGMCC 1.2182 and Agrobacterium tumefaciens CGMCC 1.2554 against patulin (PAT)-producer Penicillium expansum and their ability to remove PAT. Bacillus subtilis effectively inhibited P. expansum both on apples and in in vitro experiments, which reduced the rot diameter on apples by 38% compared with the control. The reduction was followed by those induced by A. tumefaciens (27·63%) and R. sphaeroides (23·67%). None of the cell-free supernatant (CFS) was able to prevent pathogen growth. Three antagonists could suppress PAT production by P. expansum on apples by 98·5, 93·7 and 94·99% after treatment with B. subtilis, R. sphaeroides and A. tumefaciens respectively. In addition, the three strains led to a 0·56-1·47 log CFU g -1 reduction in colony number of P. expansum on apples. Survival of antagonists on apple wounds revealed their tolerance to PAT. Furthermore, both live and autoclaved cells of three strains efficiently adsorbed artificially spiked PAT from medium. The selected antagonists could be applied before harvesting to control apple infection by PAT-producing fungi and also during processing to act as PAT detoxifiers. Since little information related to the capability of R. sphaeroides and A. tumefaciens to inhibit P. expansum is currently available, the results of this study provide some new perspectives to the biocontrol field. © 2016 The Society for Applied Microbiology.

  13. Metabolic modeling of denitrification in Agrobacterium tumefaciens: A tool to study inhibiting and activating compounds for the denitrification pathway

    OpenAIRE

    Kampschreur, M.J.; Kleerebezem, R.; Picioreanu, C.; Bakken, L.; Bergaust, L.; De Vries, S.; Jetten, M.S.M.; Van Loosdrecht, M.C.M.

    2012-01-01

    A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study the key factors regulating the metabolic response of the denitrification pathway to transition from oxic to anoxic respiration and to find parameter values for the biological processes that were mode...

  14. Plant Transformation by Coinoculation with a Disarmed Agrobacterium tumefaciens Strain and an Escherichia coli Strain Carrying Mobilizable Transgenes

    OpenAIRE

    Pappas, Katherine M.; Winans, Stephen C.

    2003-01-01

    Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was...

  15. Agrobacterium tumefaciens Deploys a Superfamily of Type VI Secretion DNase Effectors as Weapons for Interbacterial Competition In Planta

    Science.gov (United States)

    Ma, Lay-Sun; Hachani, Abderrahman; Lin, Jer-Sheng; Filloux, Alain; Lai, Erh-Min

    2014-01-01

    Summary The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization. PMID:24981331

  16. A novel antibacterial peptide active against peach crown gall (Agrobacterium tumefaciens) isolated from cyanide-tolerant actinomycetes G19.

    Science.gov (United States)

    Wang, Shufang; Ji, Jinglin; Ma, Huanpu; Liu, Zhimin

    2015-01-01

    An antimicrobial peptide was extracted from the antagonistic actinomycetes G19. It was designated as G19-F. By using MALDI-TOF mass spectrometry, the molecular weight of G19-F was determined. The primary structure of the antimicrobial peptide was determined using N-terminal sequencing and mass spectrometry. Results showed that the peptide had eleven amino acids, with the sequence D-V-C-D-G-G-D-G-D-E-D, and a calculated molecular mass of 1,096 Da. G19-F showed antimicrobial activity against peach crown gall caused by Agrobacterium tumefaciens. The antimicrobial peptide maintained its activity after being heated to 100 °C and exhibited stability from pH 4 to 10. Its activity has also remained after ultraviolet irradiation. The mechanism by which G19-F inhibits A. tumefaciens was to increase permeability of the cell membrane and destroy the cell wall structure. Furthermore, as a novel peptide, it has a potential for cure A. tumefaciens infection.

  17. Metabolic modelling of denitrification in Agrobacterium tumefaciens: a tool to study inhibiting and activating compounds for the denitrification pathway

    Directory of Open Access Journals (Sweden)

    Marlies J. Kampschreur

    2012-10-01

    Full Text Available A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study the key factors regulating the metabolic response of the denitrification pathway to transition from oxic to anoxic respiration and to find parameter values for the biological processes that were modelled. The metabolic model was used to test hypotheses that were formulated based on the experimental results and offers a structured look on the processes that occur in the cell during transition in respiration. The main phenomena that were modelled are the inhibition of the cytochrome c oxidase by nitric oxide (NO, the (indirect inhibition of oxygen on the denitrification enzymes. The activation of transcription of nitrite reductase and NO reductase by their respective substrates were hypothesized. The general assumption that nitrite and NO reduction are controlled interdependently to prevent NO accumulation does not hold for A. tumefaciens. The metabolic network model was demonstrated to be a useful tool for unravelling the different factors involved in the complex response of A. tumefaciens to highly dynamic environmental conditions.

  18. Cyanuric acid biodegradation by a mixed bacterial culture of Agrobacterium tumefaciens and Acinetobacter sp. in a packed bed biofilm reactor.

    Science.gov (United States)

    Galíndez-Nájera, S P; Llamas-Martínez, M A; Ruiz-Ordaz, N; Juárez-Ramírez, C; Mondragón-Parada, M E; Ahuatzi-Chacón, D; Galíndez-Mayer, J

    2009-02-01

    Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L(-1)), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L(-1)) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.

  19. Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Clarence I. Kado

    2014-08-01

    Full Text Available The plant tumor disease, known as crown gall, was not called by that name until more recent times. Tumors on plants, particularly on cultivated grapevines, were observed thousands of years ago and recorded in the bible (wine was being made 7000 years ago. Once a cultured bacterium that was first isolated in 1897 by Fridiano Cavara in Napoli, Italy and recognized to be the cause of this disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. The pertinent historical events leading to the identification of a genetic principle that was cleverly inserted into plant host cells and integrated into their chromosome culminated in very detailed studies of Agrobacterium tumefaciens the causal bacterium. Such studies have lead to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing organisms. Knowledge gained from these fundamental discoveries have opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries advanced the genetic engineering of domesticated plants for improved food and fiber.

  20. Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens

    Science.gov (United States)

    Kado, Clarence I.

    2014-01-01

    The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

  1. The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling (IMCB-Singapore); (Indiana)

    2012-02-08

    The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of {gamma}-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe{sup 147}, that are important for SSA association; BlcR{sup F147A} existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcR{sup F147A} retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcR{sup F147A} or BlcR{sup F147A}-DNA. As well as in the SSA-binding site, Phe{sup 147} is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization.

  2. Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.

    2009-06-03

    N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.

  3. Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens.

    Science.gov (United States)

    Ooms, G; Klapwijk, P M; Poulis, J A; Schilperoort, R A

    1980-01-01

    Seven Tn904 insertion mutants of pTi Ach5 affecting Agrobacterium tumefaciens virulence were studied. The mutant character was shown to be plasmid borne. Four of these mutants were avirulent and carried an insertion in restriction endonuclease HpaI fragment 12, a 3.3-megadalton fragment, which therefore appears to be a Ti plasmid region essential for virulence. Two mutants were attenuated in virulence. The inserts mapped close to HpaI fragment 12. One mutant giving rise to small tumors with excessive adventitious root formation on Kalanchoe daigremontiana carried an insertion in the right side of the common sequence in the deoxyribonucleic acid of the Ti plasmid detected in crown gall tumors. The insertion behavior of Tn904 was studied by analyzing 11 independently isolated and randomly chosen mutants. The Tn904 inserts did not affect oncogenicity, tumor morphology, bacterial transfer functions, octopine catabolism functions, or vital parts of the Ti plasmid, such as the origin of replication. Most of the Tn904 inserts were concentrated in a small part of the map. The size of additional deoxyribonucleic acid as a result of Tn904 inserts varied between 5 and 15 megadaltons. In two cases a Ti plasmid was found with two Tn904 insertions at different positions. Images PMID:6252198

  4. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  5. Nodulin gene expression and ENOD2 localization in effective, nitrogen fixing and ineffective, bacteria-free nodules of alfalfa.

    NARCIS (Netherlands)

    Wiel, van de C.C.M.; Nurris, J.H.; Bocheneck, B.; Dickstein, R.; Bisseling, T.; Hirsch, A.M.

    1990-01-01

    Alfalfa plants form bacteria-free nodules in response to a number of agents, including Rhizobium meliloti exo mutants, Agrobacterium tumefaciens transconjugants carrying cloned R. meliloti nodulation genes, and compounds that function as auxin transport inhibitors, N-( 1-naphthyl)phthalamic acid or

  6. Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants

    NARCIS (Netherlands)

    Moravcikova, J.; Matusikova, I.; Libantova, J.; Bauer, M.; Mlynarova, L.

    2004-01-01

    The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) breeding line 116/86 using Agrobacterium tumefaciens. For both transgenes the number of integrated copies and level of RNA expression

  7. The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis

    Czech Academy of Sciences Publication Activity Database

    Sun, J.; Niu, Q. W.; Tarkowski, Petr; Zheng, B.; Tarkowská, Danuše; Sandberg, G.; Chua, N. H.; Zuo, J.

    2003-01-01

    Roč. 131, č. 1 (2003), s. 167-176 ISSN 0032-0889 R&D Projects: GA ČR GA301/02/0475 Grant - others:Natural Science Foundation of China(CN) rant no. NSFC 30270142; Ministry of Science and Technology of China(CN) 2001AA225021 Institutional research plan: CEZ:AV0Z5038910 Keywords : Arabidopsis * Cytokinin Biosynthesis * Isopentenyl Transferase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.634, year: 2003

  8. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  9. Transient expression of β-glucuronidase reporter gene in ...

    African Journals Online (AJOL)

    Agrobacterium tumefaciens strain LBA4404 carrying the pBI.121 binary plasmid was used in transformation to introduce the gus (ß-glucuronidase/GUS) gene into teak shoot-tissues. In vitro regenerated shoots from various teak clones, i.e. the ITB, GT, P97, P96, P75, P20, and P108 clones were vacuum-infiltrated for 5 min in ...

  10. D101 is critical for the function of AttJ, a repressor of quorum quenching system in Agrobacterium tumefaciens.

    Science.gov (United States)

    Wang, Chao; Yan, Chunlan; Gao, Yong-Gui; Zhang, Lian-Hui

    2015-09-01

    The quorum quenching system of Agrobacterium tumefaciens is specifically activated upon entering the stationary phase. Evidence has shown that this system includes two key components: the IclR-type transcriptional factor AttJ (also named as BlcR) and the AHL-lactonase AttM (also named as BlcC). At exponential phase, AttJ binds to the promoter region of attM and thus suppresses the expression of attM. At stationary phase, however, the small molecule SSA directly binds to AttJ and relieves its inhibition of AttJ and thereby triggers the expression of attM. While the regulation of AttM has been extensively investigated, little is known about the regulation of AttJ. In this study, we demonstrated the D101 amino acid of AttJ is essential for the AttJ function. In vitro, the variant protein of AttJD101H appeared to be readily aggregated. In vivo, the D101H mutation in AttJ entirely abolished the inhibitory activity of AttJ and overexpressed attM in A. tumefaciens A6. In addition, D101H mutation led to an overexpression of attJ, indicating an auto-regulatory mechanism for the attJ regulation. Put together, these findings demonstrate that D101 is an important amino acid for the transcription activity of AttJ and the transcription of attJ is regulated by a negative feedback loop. These results expand previous biochemical characterization of AttJ and provide new mechanistic insights into the regulation of quorum quenching in A. tumefaciens.

  11. Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones.

    Science.gov (United States)

    Wolterink-van Loo, Suzanne; Escamilla Ayala, Abril A; Hooykaas, Paul J J; van Heusden, G Paul H

    2015-02-01

    Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 5' end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His6-tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA. © 2015 The Authors.

  12. Virus-Induced Gene Silencing as a Tool to Study Tomato Fruit Biochemistry.

    Science.gov (United States)

    Fantini, Elio; Giuliano, Giovanni

    2016-01-01

    Virus-Induced Gene Silencing (VIGS) is an excellent reverse genetic tool for the study of gene function in plants, based on virus infection. In this chapter, we describe a high-throughput approach based on VIGS for the study of tomato fruit biochemistry. It comprises the selection of the sequence for silencing using bioinformatics tools, the cloning of the fragment in the Tobacco Rattle Virus (TRV), and the agroinfiltration of tomato fruits mediated by Agrobacterium tumefaciens.

  13. Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.

    Science.gov (United States)

    Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui

    2013-03-01

    KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.

  14. Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

    Science.gov (United States)

    Ravanfar, Seyed Ali; Aziz, Maheran Abdul; Saud, Halimi Mohd; Abdullah, Janna Ong

    2015-11-01

    An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

  15. One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

    NARCIS (Netherlands)

    Untergasser, A.; Bijl, G.J.M.; Liu, W.; Bisseling, T.; Schaart, J.G.; Geurts, R.

    2012-01-01

    Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine

  16. Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

    International Nuclear Information System (INIS)

    Mase, Tomoko; Yabuki, Hideya; Okai, Masahiko; Ohtsuka, Jun; Imai, Fabiana Lica; Nagata, Yuji; Tanokura, Masaru

    2012-01-01

    The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution. Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit

  17. Identifikasi Transgene pada Tanaman Padi (oryza sativa var. koshihikari yang Ditransformasi dengan Bantuan Agrobacterium tumefaciens, Menggunakan Metode Tanpa Teknik Kultur Jaringan.

    Directory of Open Access Journals (Sweden)

    I Putu Suparthana

    2015-04-01

    Full Text Available Metode transformasi tanaman dengan bantuan Agrobacterium tumefaciens biasa dilakukan dengan melibatkan teknik kultur jaringan, akan tetapi teknik kultur jaringan memiliki beberapa kelemahan yaitu memerlukan suatu kondisi steril, memakan banyak waktu, sering terjadi mutasi dalam proses kultur in vitro dan sejumlah tanaman bersifat rekalsitran pada tahap regenerasi. Disisi lain metode baru (in planta transformation system yang dikembangkan dalam penelitian ini mampu mengatasi kelemahan-kelemahan tersebut. Penelitian dilakukan pada tanaman padi dengan bantuan A. tumefaciens sebagai inokulum namun tidak melibatkan teknik kultur jaringan pada masa regenerasinya. Biji padi (Oryza sativa L. var. Koshihikari direndam dalam air selama 2 hari, dengan demikian embrionya yang mengandung sel apikal meristem dapat diinokulasi dengan cara menusukkan jarum yang telah dicelupkan dalam inokulum. Biji padi yang telah diinokulasi selanjutnya ditumbuhkan dilapangan selayaknya pembenihan biasa tanpa perlakuan steril. Untuk menentukan keberhasilan teknik ini, dua jenis strain mutan A. tumefaciens (M-21 dan LBA4404 digunakan dalam transformasi. Mutan M-21 mengandung Tn5 tersisip dalam gen iaaM dan mutan LBA4404 membawa binari plasmid vektor. Transgen dari mutan M-21 dapat diidentifikasi dari perubahan fenotipe pada tanaman padi transgenik sedangkan dari mutan LBA4404 dapat diidentifikasi dengan uji histokimia dan ketahanan terhadap antibitik (hygromycin B.Kata kunci: in planta transformation, A. tumefaciens, transformation method

  18. Regulation of Oncogene Expression in T-DNA-Transformed Host Plant Cells

    Science.gov (United States)

    Zhang, Yi; Lee, Chil-Woo; Wehner, Nora; Imdahl, Fabian; Svetlana, Veselova; Weiste, Christoph; Dröge-Laser, Wolfgang; Deeken, Rosalia

    2015-01-01

    Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance

  19. Regulation of oncogene expression in T-DNA-transformed host plant cells.

    Science.gov (United States)

    Zhang, Yi; Lee, Chil-Woo; Wehner, Nora; Imdahl, Fabian; Svetlana, Veselova; Weiste, Christoph; Dröge-Laser, Wolfgang; Deeken, Rosalia

    2015-01-01

    Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance

  20. Regulation of oncogene expression in T-DNA-transformed host plant cells.

    Directory of Open Access Journals (Sweden)

    Yi Zhang

    2015-01-01

    Full Text Available Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase, IaaM (tryptophan monooxygenase and Ipt (isopentenyl transferase, which encode enzymes for the biosynthesis of auxin (IaaH, IaaM and cytokinin (Ipt. Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs between the coding sequences (CDS of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and

  1. Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis

    OpenAIRE

    Liu, Yanbin; Koh, Chong Mei John; Yap, Sihui Amy; Du, Minge; Hlaing, Mya Myintzu; Ji, Lianghui

    2018-01-01

    Background Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. Results We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in t...

  2. Agrobacterium Mediated Transformation of Fld and GUS Genes into Canola for Salinity Stress

    Directory of Open Access Journals (Sweden)

    Niapour, Nazila

    2013-04-01

    Full Text Available Salinity is one of the major abiotic stress which limits wide spread canola cultivation. One way to overcome this problem could be transfection, to produce tolerable species. Cotyledonary and hypocotyls explants obtained from 4 and 7 days old seedling of Elite and RJS003 varieties were utilized in this study. Genetic transformation was implemented through Agrobacterium tumefaciens LBA4404 containing PBI121 plasmid and Agrobacterium tumefaciens C58, LBA4404, AGL0 and EHA 101 strains which contain P6u- ubi- fvt1 construct. The T-DNA region of P6u- Ubi- Fvt1 plasmid included HPT (Hygromycin phosphotransferase plant selectable marker and Fld (flavodoxin gene. PBI121 plasmid had NptII (Neomycin phosphotransferase plant Selectable marker and β-glucuronidase (GUS reporter genes. Transfected explants were analyzed by PCR and histochemical assay for Fld and Gus genes, respectively. Our data indicated that the cotyledonary explants of both cultivars were incompetent to be infected with Fld gens. However, the transformation in Elite hypocotyls explants with Agrobacterium tumefaciens C58 and LBA 4404 strains were confirmed through PCR product and histochemical evaluation for Fld and GUS genes, respectively. Therefore, the result of this manuscript may to certain degree fulfill the endeavor appointed to this oilseed.

  3. Characterization the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation.

    Science.gov (United States)

    Lin, Xinping; Gao, Ning; Liu, Sasa; Zhang, Sufang; Song, Shuang; Ji, Chaofan; Dong, Xiuping; Su, Yichen; Zhao, Zongbao Kent; Zhu, Beiwei

    2017-08-01

    The red yeast Rhodosporidium toruloides is a known lipid producer capable of accumulating large amounts of triacylglycerols and carotenoids. However, it remains challenging to study its carotenoid production profiles owing to limited biochemical information and inefficient genetic tools. Here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) to change its carotenoid production and profiles. We constructed R. toruloides NP11 mutant libraries with ATMT, selected three mutants with different colours, characterized their carotenoid products by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis and assured differences among those strains in terms of carotenoid production and its composition profiles. We then located T-DNA insertion sites using the genome walking technology and provided discussions in terms of the new phenotypes. This study is the first of its kind to change the carotenoid production profiles in R. toruloides. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Inter-organ defense networking: Leaf whitefly sucking elicits plant immunity to crown gall disease caused by Agrobacterium tumefaciens.

    Science.gov (United States)

    Park, Yong-Soon; Ryu, Choong-Min

    2015-01-01

    Plants have elaborate defensive machinery to protect against numerous pathogens and insects. Plant hormones function as modulators of defensive mechanisms to maintain plant resistance to natural enemies. Our recent study suggests that salicylic acid (SA) is the primary phytohormone regulating plant responses to Agrobacterium tumefaciens infection. Tobacco (Nicotiana benthamiana Domin.) immune responses against Agrobacterium-mediated crown gall disease were activated by exposure to the sucking insect whitefly, which stimulated SA biosynthesis in aerial tissues; in turn, SA synthesized in aboveground tissues systemically modulated SA secretion in root tissues. Further investigation revealed that endogenous SA biosynthesis negatively modulated Agrobacterium-mediated plant genetic transformation. Our study provides novel evidence that activation of the SA-signaling pathway mediated by a sucking insect infestation has a pivotal role in subsequently attenuating Agrobacterium infection. These results demonstrate new insights into interspecies cross-talking among insects, plants, and soil bacteria.

  5. Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

    Science.gov (United States)

    Lu, Yuanyuan; Xiao, Shuqin; Wang, Fen; Sun, Jiaying; Zhao, Likun; Yan, Libin; Xue, Chunsheng

    2017-02-01

    An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum L.) cultivar Micro-Tom.

    Science.gov (United States)

    Chetty, V J; Ceballos, N; Garcia, D; Narváez-Vásquez, J; Lopez, W; Orozco-Cárdenas, M L

    2013-02-01

    KEY MESSAGE : Agrobacterium tumefaciens strains differ not only in their ability to transform tomato Micro-Tom, but also in the number of transgene copies that the strains integrate in the genome. The transformation efficiency of tomato (Solanum lycopersicum L.) cv. Micro-Tom with Agrobacterium tumefaciens strains AGL1, EHA105, GV3101, and MP90, harboring the plasmid pBI121 was compared. The presence of the nptII and/or uidA transgenes in regenerated T(0) plants was determined by PCR, Southern blotting, and/or GUS histochemical analyses. In addition, a rapid and reliable duplex, qPCR TaqMan assay was standardized to estimate transgene copy number. The highest transformation rate (65 %) was obtained with the Agrobacterium strain GV3101, followed by EHA105 (40 %), AGL1 (35 %), and MP90 (15 %). The mortality rate of cotyledons due to Agrobacterium overgrowth was the lowest with the strain GV3101. The Agrobacterium strain EHA105 was more efficient than GV3101 in the transfer of single T-DNA insertions of nptII and uidA transgenes into the tomato genome. Even though Agrobacterium strain MP90 had the lowest transformation rate of 15 %, the qPCR analysis showed that the strain MP90 was the most efficient in the transfer of single transgene insertions, and none of the transgenic plants produced with this strain had more than two insertion events in their genome. The combination of higher transformation efficiency and fewer transgene insertions in plants transformed using EHA105 makes this Agrobacterium strain optimal for functional genomics and biotechnological applications in tomato.

  7. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    OpenAIRE

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transf...

  8. Proteomics and genetic analyses reveal the effects of arsenite oxidation on metabolic pathways and the roles of AioR in Agrobacterium tumefaciens GW4.

    Science.gov (United States)

    Shi, Kaixiang; Wang, Qian; Fan, Xia; Wang, Gejiao

    2018-04-01

    A heterotrophic arsenite [As(III)]-oxidizing bacterium Agrobacterium tumefaciens GW4 isolated from As(III)-rich groundwater sediment showed high As(III) resistance and could oxidize As(III) to As(V). The As(III) oxidation could generate energy and enhance growth, and AioR was the regulator for As(III) oxidase. To determine the related metabolic pathways mediated by As(III) oxidation and whether AioR regulated other cellular responses to As(III), isobaric tags for relative and absolute quantitation (iTRAQ) was performed in four treatments, GW4 (+AsIII)/GW4 (-AsIII), GW4-ΔaioR (+AsIII)/GW4-ΔaioR (-AsIII), GW4-ΔaioR (-AsIII)/GW4 (-AsIII) and GW4-ΔaioR (+AsIII)/GW4 (+AsIII). A total of 41, 71, 82 and 168 differentially expressed proteins were identified, respectively. Using electrophoretic mobility shift assay (EMSA) and qRT-PCR, 12 genes/operons were found to interact with AioR. These results indicate that As(III) oxidation alters several cellular processes related to arsenite, such as As resistance (ars operon), phosphate (Pi) metabolism (pst/pho system), TCA cycle, cell wall/membrane, amino acid metabolism and motility/chemotaxis. In the wild type with As(III), TCA cycle flow is perturbed, and As(III) oxidation and fermentation are the main energy resources. However, when strain GW4-ΔaioR lost the ability of As(III) oxidation, the TCA cycle is the main way to generate energy. A regulatory cellular network controlled by AioR is constructed and shows that AioR is the main regulator for As(III) oxidation, besides, several other functions related to As(III) are regulated by AioR in parallel. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    Science.gov (United States)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  10. A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii).

    Science.gov (United States)

    Thevissen, K; Cammue, B P; Lemaire, K; Winderickx, J; Dickson, R C; Lester, R L; Ferket, K K; Van Even, F; Parret, A H; Broekaert, W F

    2000-08-15

    We have previously identified a Saccharomyces cerevisiae mutant that is markedly more resistant than wild-type to Dahlia merckii antimicrobial peptide 1 (DmAMP1), an antifungal plant defensin isolated from seeds of dahlia (Dahlia merckii). A complementation approach was followed that consisted of the introduction of a genomic library of DmAMP1-sensitive wild-type yeast into the DmAMP1-resistant yeast mutant and screening for restored sensitivity to DmAMP1. The gene determining sensitivity of S. cerevisiae to DmAMP1 was identified as IPT1, a gene encoding an enzyme involved in the last step of the synthesis of the sphingolipid mannose-(inositol-phosphate)(2)-ceramide. Strains with a nonfunctional IPT1 allele lacked mannose-(inositol-phosphate)(2)-ceramide in their plasma membranes, bound significantly less DmAMP1 compared with wild-type strains, and were highly resistant to DmAMP1-mediated membrane permeabilization. All of these phenotypic deviations could be restored by reintroduction of a functional IPT1 gene. Our data support a model in which membrane patches containing sphingolipids act as binding sites for DmAMP1 or, alternatively, are required to anchor membrane or cell wall-associated proteins, which themselves interact with DmAMP1.

  11. PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens.

    Science.gov (United States)

    Grangeon, Romain; Zupan, John R; Anderson-Furgeson, James; Zambryski, Patricia C

    2015-09-15

    Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole.

  12. Cognitive behavioural therapy (CBT), third-wave CBT and interpersonal therapy (IPT) based interventions for preventing depression in children and adolescents.

    Science.gov (United States)

    Hetrick, Sarah E; Cox, Georgina R; Witt, Katrina G; Bir, Julliet J; Merry, Sally N

    2016-08-09

    Depression is common in young people. It has a marked negative impact and is associated with self-harm and suicide. Preventing its onset would be an important advance in public health. This is an update of a Cochrane review that was last updated in 2011. To determine whether evidence-based psychological interventions (including cognitive behavioural therapy (CBT), interpersonal therapy (IPT) and third wave CBT)) are effective in preventing the onset of depressive disorder in children and adolescents. We searched the specialised register of the Cochrane Common Mental Disorders Group (CCMDCTR to 11 September 2015), which includes relevant randomised controlled trials from the following bibliographic databases: The Cochrane Library (all years), EMBASE (1974 to date), MEDLINE (1950 to date) and PsycINFO (1967 to date). We searched conference abstracts and reference lists of included trials and reviews, and contacted experts in the field. We included randomised controlled trials of an evidence-based psychological prevention programme compared with any comparison control for young people aged 5 to 19 years, who did not currently meet diagnostic criteria for depression. Two authors independently assessed trials for inclusion and rated their risk of bias. We adjusted sample sizes to take account of cluster designs and multiple comparisons. We contacted trial authors for additional information where needed. We assessed the quality of evidence for the primary outcomes using GRADE. We included 83 trials in this review. The majority of trials (67) were carried out in school settings with eight in colleges or universities, four in clinical settings, three in the community and four in mixed settings. Twenty-nine trials were carried out in unselected populations and 53 in targeted populations.For the primary outcome of depression diagnosis at medium-term follow-up (up to 12 months), there were 32 trials with 5965 participants and the risk of having a diagnosis of depression was

  13. The yield difference between wild-type cotton and transgenic cotton that expresses IPT depends on when water-deficit stress is applied

    Science.gov (United States)

    Drought is the No. 1 factor that limits agricultural production in the world, thus, making crops more drought tolerant is a major goal in agriculture. Many genes with functions in abiotic stress tolerance were identified, and overexpression of these genes confers increased drought tolerance in trans...

  14. Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Ernandes Manfroi

    2015-01-01

    Full Text Available AbstractLow transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037. Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciensovergrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.

  15. Development of disease-resistant marker-free tomato by R/RS site-specific recombination.

    Science.gov (United States)

    Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

    2011-06-01

    The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).

  16. Loss of PopZ At activity in Agrobacterium tumefaciens by Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects.

    Science.gov (United States)

    Grangeon, Romain; Zupan, John; Jeon, Yeonji; Zambryski, Patricia C

    2017-11-14

    Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZ At deletion strain (Δ popZ At ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like Δ popZ At cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ At IMPORTANCE Agrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth

  17. Characterization of the photolyase-like iron sulfur protein PhrB from Agrobacterium tumefaciens by Mössbauer spectroscopy

    Science.gov (United States)

    Bauer, T. O.; Graf, D.; Lamparter, T.; Schünemann, V.

    2014-04-01

    High field Mössbauer spectroscopy has been used to characterize the [4Fe-4S] 2 +cluster of the protein PhrB from Agrobacterium tumefaciens which belongs to the cryptochrome/photolyase family (CPF) and which biological function has previously been shown to be DNA repair. Mössbauer spectra taken of the as prepared protein reveal δ = 0. 42 mms - 1, and Δ E Q = 1. 26 mms - 1as well as an asymmetry parameter of η = 0. 8. These parameters are characteristic for a ferredoxin-type [4Fe-4S] 2 +cluster. In order to investigate whether this cluster is involved in DNA-repair the protein has also been studied in its photoactivated state during DNA binding. The so obtained data sets exhibit essentially the same Mössbauer parameters as those of the non-activated PhrB. This indicates that during DNA repair the [4Fe-4S] 2 +cluster of PhrB has no significant amounts of transition states which have conformational changes compared to the resting state of the protein and which have life times of several seconds or longer.

  18. Theoretical Study of Molecular Determinants Involved in Signal Binding to the TraR Protein of Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    N. Kumar

    2005-10-01

    Full Text Available N-acylated homoserine lactone (AHL mediated cell-cell communication in bacteria is dependent on the recognition of the cognate signal by its receptor. This interaction allows the receptor-ligand complex to act as a transcriptional activator, controlling the expression of a range of bacterial phenotypes, including virulence factor expression and biofilm formation. One approach to determine the key features of signal- binding is to model the intermolecular interactions between the receptor and ligand using computational-based modeling software (LigandFit. In this communication, we have modeled the crystal structure of the AHL receptor protein TraR and its AHL signal N-(3- oxooctanoyl-homoserine lactone from Agrobacterium tumefaciens and compared it to the previously reported antagonist behaviour of a number of AHL analogues, in an attempt to determine structural constraints for ligand binding. We conclude that (i a common conformation of the AHL in the hydrophobic and hydrophilic region exists for ligand-binding, (ii a tail chain length threshold of 8 carbons is most favourable for ligand-binding affinity, (iii the positive correlation in the docking studies could be used a virtual screening tool.

  19. Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

    Science.gov (United States)

    Thomashow, M F; Karlinsey, J E; Marks, J R; Hurlbert, R E

    1987-01-01

    We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent. Images PMID:3597321

  20. Development of a simple and effective protocol for Agrobacterium tumefaciens mediated leaf disc transformation of commercial tomato cultivars.

    Science.gov (United States)

    Van, Dang Thi; Ferro, Noel; Jacobsen, Hans-Jörg

    2010-01-01

    The transformation of tomato (Solanum lycopersicum) through Agrobacterium tumefaciens is still far from being routine, particularly when it comes to commercial varieties. In the present paper, we present an efficient and simple protocol for leaf disc transformation of three Vietnamese tomato cultivars (DM8, MTS, FM372C) by comparing shoot regeneration media for expanding leaves and examining different parameters of inoculation, co-culture and selection conditions. The present transformation method requires neither feeder layers of cell suspension cultures nor pre-culture. The data clearly show that appropriate cytokinin- and auxin combinations and concentrations provide competent tissues for transformation. Supplementing of 8 µM trans-zeatin and 5 µM indoleacetic acid (IAA) into pre-treatment, inoculation and co-culture media resulted in higher frequency of transformation and stronger GUS-expression than that of media supplemented with 4 µM trans-zeatin and 2 µM IAA. The experiments also exhibited that tomato leaf tissues were more sensitive to glufosinate after inoculation with Agrobacteria compared to the untreated controls, so a more sophisticated scheme for the glufosinate selection had to be established.

  1. RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis

    Science.gov (United States)

    Escobar, Matthew A.; Civerolo, Edwin L.; Summerfelt, Kristin R.; Dandekar, Abhaya M.

    2001-01-01

    Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show ≈90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens, transgenic Arabidopsis lines averaged 0.0–1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor–ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks. PMID:11687652

  2. Studi perbandingan metode transformasi DNA menggunakan vektor Agrobacterium tumefaciens pada tanaman tebu (Sacharum hybrid

    Directory of Open Access Journals (Sweden)

    Sri Setyati

    2012-02-01

    Full Text Available In order to compare transient expression of gus gene driven by CaMV 35S and rice ubiquitin RUBQ2 promoters, a DNA transformation was conducted using embryogenic callus and suspension cultures of sugarcane. The transient gus expression was observed by histochemical staining method. The histochemical observation of GUS activity after co-cultivation showed that RUBQ2 promoter produced high level of clear blue spots both in embryogenic callus and suspension cultures, while the CaMV35S promoter was not detected. The suspension cultures slightly increased transient gus gene expression compared to embryogenic callus. However, the histochemical analysis of regenerated putative transformant plants after 5 successive cycles on the selection medium showed no blue spots of gus gene expression. PCR amplification of DNA for CaMV35 or nptII in putative transformant plants confirmed that there was no integration of the transformed gene in the genome DNA. The results suggested a possibility of somaclonal variation with callus propagation, thus did not produce transformed plants. To avoid the somaclonal variation, the transformation was conducted using in vitro plants and multiple shoots without intervening callus phase. Histochemical observation of infected materials after co-cultivation showed that almost all of the infected materials partially exhibited blue color in the basal region. In case of in vitro plants, they rapidly grow and multiplied in the selection medium, thus the method provided an excellent system for the transformation in sugarcane. The results suggest that in vitro plants as well as multiple shoots need further investigation to be used as target tissues for Agrobacteriummediated transformation in sugarcane.

  3. Maize (Zea mays L.) transformation by Agrobacterium tumefaciens infection of pollinated ovules.

    Science.gov (United States)

    Chen, Liang; Cong, Yuanyuan; He, Hongxia; Yu, Ying

    2014-02-10

    A novel transformation system was established for maize using Agrobacterium infection of in vitro cultured ovules. The maize ovules were isolated 24h after pollination and infected with Agrobacterium. The embryos were isolated from the pollinated ovules 2-3 weeks after Agrobacterium infection, regenerated to plantlets and investigated for transgene expression and inheritance. Experimental evaluations were focused on the four main aspects. Firstly, through the introduction of gus gene for monitoring transformation and development of embryo, it was confirmed that transgenic plants can be generated from in vitro cultured maize ovules infected with Agrobacterium. Secondly, in order to standardize the transformation protocol, several important factors that affected transformation efficiency were optimized. They included Agrobacterium delivery approach, surfactant, AS concentration, and cocultivation duration. Thirdly, stable expression and Mendelian inheritance of the introduced genes were analyzed in independent lines over two generations. Fourthly, the pollinated ovule culture-regeneration potential and transformation efficiency of five maize inbred lines were investigated to confirm the genotype independence of this transformation system. We conclude that the transformation system established in this study can be used to generate high-quality transgenic maize plants rapidly and directly. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Optimization of Factors Influencing Agrobacterium tumefaciens-Mediated Transformation of Tomato

    Directory of Open Access Journals (Sweden)

    L Jeddi

    2011-01-01

    Full Text Available Abstract Tomato is a plant which has highly important nutrition, economic and scientific aspects. Due to its susceptibility to biotic and abiotic stresses, breeding through genetic engineering of this plant is an effective method for enhancement of its tolerance to stresses. In this experiment, factors influencing the transformation of cultivated tomato, were studied. The high shoot regeneration was observed in Peto earlyCH and Early urbanaY cultivars, respectively and the most transformation rate was obtained in Early urbanaY cultivar. Preculture of explants for 1 day and addition of 100 - 150 µM acetosyringone into inoculation solution had a positive effect on transformation efficency. The optimum inoculation and cocultivation time for explants was 15 minutes and 2 days respectively. Putative transgenic plants were confirmrd using PCR and specific primers of both genes (nptII and codA genes. These plants were transferred to the soil for further molecular and bioassay experiments. Keywords: Agrobacterium, Cocultivation, Preculture, Regeneration, Transformation, Tomato

  5. Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions.

    Science.gov (United States)

    Bhubhanil, Sakkarin; Chamsing, Jareeya; Sittipo, Panida; Chaoprasid, Paweena; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2014-05-01

    Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)-vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

  6. Producción de plantas transgénicas de Solanum phureja variedad yema de huevo clon 1 mediada por Agrobacteriun tumefaciens

    OpenAIRE

    Chaparro Giraldo Alejandro; Díazgranados Daza Elba Cristina

    2006-01-01

    El objetivo general de este proyecto fue la producción de plantas transformadas genéticamente de S. phureja
    variedad Yema de Huevo Clon 1 que sean potencialmente tolerantes a insectos, rasgo conferido por la inserción
    en su genoma del gen mirl2 derivado del pomelo (Citrus paradisi) que codifica para un inhibidor de proteasas de
    tipo Serina. Se trabajó con el sistema de transformación mediado por Agrobacterium tumefaciens utilizando la cepa
    LBA4404 q...

  7. Producción de plantas transgénicas de Solanum phureja variedad yema de huevo clon 1 mediada por Agrobacteriun tumefaciens

    OpenAIRE

    Elba Cristina Díazgranados Daza; Alejandro Chaparro Giraldo

    2006-01-01

    El objetivo general de este proyecto fue la producción de plantas transformadas genéticamente de S. phureja variedad Yema de Huevo Clon 1 que sean potencialmente tolerantes a insectos, rasgo conferido por la inserción en su genoma del gen mirl2 derivado del pomelo (Citrus paradisi) que codifica para un inhibidor de proteasas de tipo Serina. Se trabajó con el sistema de transformación mediado por Agrobacterium tumefaciens utilizando la cepa LBA4404 que contiene el plásmido resident...

  8. Expresión transitoria del gen GUS en caña de azúcar usando Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Martha Liliana Bonilla Betancourt

    2008-10-01

    Full Text Available En el estudio se desarrolló una metodología de transformación genética mediante Agrobacterium tumefaciens en cultivares colombianos de caña de azúcar. La transformación se evaluó mediante la expresión del gen GUS. Callos embriogénicos y explantes meristemáticos de los genotipos CC85-92, CC84-75 y CC87-505 se transformaron usando tres cepas (AGL-1, LBA4404 y EHA105 con el plásmido pCambia 1305.2 y dos (EHA105 y LBA4404 con pCambia 2301. Se usó el medio de infiltración (IM con acetosiringona y se evaluó el tiempo de cocultivo y la densidad óptica de la bacteria al momento de la inducción. Los genotipos mostraron respuesta diferencial con las combinaciones cepa-plásmido: obtuvieron mayor expresión del gen GUS cuando el genotipo CC85-92 se transformó con la cepa AGL-1-pCambia 1305.2. CC84-75 y CC87-505 mostraron mayor expresión cuando se transformaron con la cepa EHA105-pCambia 1305.2. Mayor eficiencia en la expresión se obtuvo cuando la bacteria se indujo en IM después de siete días de cocultivo y cuando la densidad óptica de la bacteria fue de 0.2(600nm al momento de la inducción. Se demostró superioridad de los explantes en la eficiencia de transformación.

  9. The Essential Role of Spermidine in Growth of Agrobacterium tumefaciens Is Determined by the 1,3-Diaminopropane Moiety.

    Science.gov (United States)

    Kim, Sok Ho; Wang, Yi; Khomutov, Maxim; Khomutov, Alexey; Fuqua, Clay; Michael, Anthony J

    2016-02-19

    The ubiquitous polyamine spermidine is indispensable for eukaryotic growth and cell proliferation. A conserved vital function of spermidine across eukaryotes is conferred by its aminobutyl group that is transferred to a single lysine in translation factor eIF5A to form the essential hypusine post-translational modification required for cellular translation. In direct contrast, although spermidine is absolutely essential for growth of α-proteobacterial plant pathogen Agrobacterium tumefaciens, we have found, by employing a suite of natural polyamines and synthetic methylated spermidine analogues together with spermidine biosynthetic mutants, that it is solely the 1,3-diaminopropane moiety of spermidine that is required for growth. Indeed, any polyamine containing an intact terminal 1,3-diaminopropane moiety can replace spermidine for growth, including the simple diamine 1,3-diaminopropane itself, a paradigm shift in understanding polyamine function in bacteria. We have identified for the first time a spermidine retroconversion activity in bacteria, producing diamine putrescine from triamine spermidine; however, exogenously supplied tetraamine spermine is resistant to retroconversion. When spermidine levels are pharmacologically decreased, synthesis of spermine from spermidine is induced via the same biosynthetic enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase that produce spermidine from putrescine, the first identification of a spermine biosynthetic pathway in bacteria. This also suggests that spermidine represses spermine biosynthesis, but when spermidine levels decrease, it is then converted by carboxyspermidine dehydrogenase and decarboxylase enzymes to spermine, which is resistant to retroconversion and constitutes a sequestered pool of protected 1,3-diaminopropane modules required for growth. We also identify an efficient N-acetylspermidine deacetylase activity, indicative of a sophisticated bacterial polyamine homeostasis system.

  10. Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from Agrobacterium tumefaciens showing high thermal stability and catalytic efficiency.

    Directory of Open Access Journals (Sweden)

    Ana Belén Martínez-Moñino

    Full Text Available NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organismal homeostasis. Among the enzymes involved in its recycling, nicotinamide mononucleotide (NMN deamidase is one of the key players in the bacterial pyridine nucleotide cycle, where it catalyzes the conversion of NMN into nicotinic acid mononucleotide (NaMN, which is later converted to NAD+ in the Preiss-Handler pathway. The biochemical characteristics of bacterial NMN deamidases have been poorly studied, although they have been investigated in some firmicutes, gamma-proteobacteria and actinobacteria. In this study, we present the first characterization of an NMN deamidase from an alphaproteobacterium, Agrobacterium tumefaciens (AtCinA. The enzyme was active over a broad pH range, with an optimum at pH 7.5. Moreover, the enzyme was quite stable at neutral pH, maintaining 55% of its activity after 14 days. Surprisingly, AtCinA showed the highest optimal (80°C and melting (85°C temperatures described for an NMN deamidase. The above described characteristics, together with its high catalytic efficiency, make AtCinA a promising biocatalyst for the production of pure NaMN. In addition, six mutants (C32A, S48A, Y58F, Y58A, T105A and R145A were designed to study their involvement in substrate binding, and two (S31A and K63A to determine their contribution to the catalysis. However, only four mutants (C32A, S48A Y58F and T105A showed activity, although with reduced catalytic efficiency. These results, combined with a thermal and structural analysis, reinforce the Ser/Lys catalytic dyad mechanism as the most plausible among those proposed.

  11. Involvement of the Acr3 and DctA anti-porters in arsenite oxidation in Agrobacterium tumefaciens 5A.

    Science.gov (United States)

    Kang, Yoon-Suk; Shi, Zunji; Bothner, Brian; Wang, Gejiao; McDermott, Timothy R

    2015-06-01

    Microbial arsenite (AsIII) oxidation forms a critical piece of the arsenic cycle in nature, though our understanding of how and why microorganisms oxidize AsIII remains rudimentary. Our model organism Agrobacterium tumefaciens 5A contains two distinct ars operons (ars1 and ars2) that are similar in their coding region content. The ars1 operon is located nearby the aio operon that is essential for AsIII oxidation. The AsIII/H(+) anti-porters encoded by acr3-1 and acr3-2 are required for maximal AsIII and antimonite (SbIII) resistance, but acr3-1 (negatively regulated by ArsR-1) appears more active in this regard and also required for AsIII oxidation and expression of aioBA. A malate-phosphate anti-porter DctA is regulated by RpoN and AsIII, and is required for normal growth with malate as a sole carbon source. Qualitatively, a ΔdctA mutant was normal for AsIII oxidation and AsIII/SbIII resistance at metalloid concentrations inhibitory to the Δacr3-1 mutant; however, aioBA induction kinetics was significantly phase-shift delayed. Acr3 involvement in AsIII/SbIII resistance is reasonably well understood, but the role of Acr3 and DctA anti-porters in AsIII oxidation and its regulation is unexpected, and suggests that controlled AsIII trafficking across the cytoplasmic membrane is important to a process understood to occur in the periplasm. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Co-ordinate regulation of cytokinin gene family members during flag leaf and reproductive development in wheat.

    Science.gov (United States)

    Song, Jiancheng; Jiang, Lijun; Jameson, Paula Elizabeth

    2012-06-06

    As the global population continues to expand, increasing yield in bread wheat is of critical importance as 20% of the world's food supply is sourced from this cereal. Several recent studies of the molecular basis of grain yield indicate that the cytokinins are a key factor in determining grain yield. In this study, cytokinin gene family members in bread wheat were isolated from four multigene families which regulate cytokinin synthesis and metabolism, the isopentenyl transferases (IPT), cytokinin oxidases (CKX), zeatin O-glucosyltransferases (ZOG), and β-glucosidases (GLU). As bread wheat is hexaploid, each gene family is also likely to be represented on the A, B and D genomes. By using a novel strategy of qRT-PCR with locus-specific primers shared among the three homoeologues of each family member, detailed expression profiles are provided of family members of these multigene families expressed during leaf, spike and seed development. The expression patterns of individual members of the IPT, CKX, ZOG, and GLU multigene families in wheat are shown to be tissue- and developmentally-specific. For instance, TaIPT2 and TaCKX1 were the most highly expressed family members during early seed development, with relative expression levels of up to 90- and 900-fold higher, respectively, than those in the lowest expressed samples. The expression of two cis-ZOG genes was sharply increased in older leaves, while an extremely high mRNA level of TaGLU1-1 was detected in young leaves. Key genes with tissue- and developmentally-specific expression have been identified which would be prime targets for genetic manipulation towards yield improvement in bread wheat breeding programmes, utilising TILLING and MAS strategies.

  13. Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes

    OpenAIRE

    Aarrouf, Jawad; Mallard, Stephanie; Caromel, Bernard; Lizzi, Y.; Lefebvre, Véronique

    2012-01-01

    Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated wit...

  14. Robust regeneration protocol for the Agrobacterium tumefaciens mediated transformation of Solanum tuberosum

    International Nuclear Information System (INIS)

    Abbasi, A.; Bilal, M.; Hussain, J.; Shah, M. M.; Hassan, A.

    2016-01-01

    Plant genetic transformation requires robust regeneration system. Plant growth regulators (PGRs) such as cytokinins (CKs) play a pivotal role in organogenesis; however, CKs are the most expensive PGRs. In the current study, an efficient yet economical protocol for regeneration of potato plant was developed. Stem inter-nodal and leaf explants were cultured on different regeneration media supplemented with varying concentration of different CKs such as kinetin and zeatin. Murashige- Skoog media added with zeatin (1, 1.5 mg/L) was designated as RZ1, RZ1.5, respectively or kinetin (1.5, 2 mg/L) was designated as RK1.5 and RK2, respectively, however, concentrations of other hormones such as NAA (1-Naphthaleneacetic acid) and GA3 (Gibberellic acid A3) were kept same. RZ1 and RZ1.5 gave significantly better Results as compared to RK-type media in all aspects studied such as callus initiation, days to first shoot emergence, number of shoots per explants. RZ1 medium was then selected as regeneration media for Agrobacterium-mediated transformation of potato plants with cyanobacterial phosphoenol pyruvate carboxylase gene, which provided multiple putative transformants on selection media. The transformants were further confirmed through PCR. The current protocol is found to be cost effective and efficient for the regeneration of Solanum tuberosum and can be successfully implied for the Agrobacterium-mediated transformation. (author)

  15. Highly efficient transformation system for Malassezia furfur and Malassezia pachydermatis using Agrobacterium tumefaciens-mediated transformation.

    Science.gov (United States)

    Celis, A M; Vos, A M; Triana, S; Medina, C A; Escobar, N; Restrepo, S; Wösten, H A B; de Cock, H

    2017-03-01

    Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7days at 19°C and 24°C, respectively. Transformation efficiency was 0.75-1.5% for M. furfur and 0.6-7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. An Improved Binary Vector and Escherichia coli Strain for Agrobacterium tumefaciens-Mediated Plant Transformation

    Directory of Open Access Journals (Sweden)

    Michael R. Watson

    2016-07-01

    Full Text Available The plasmid vector pGreenII is widely used to produce plant transformants via a process that involves propagation in Escherichia coli. However, we show here that pGreenII-based constructs can be unstable in E. coli as a consequence of them hampering cell division and promoting cell death. In addition, we describe a new version of pGreenII that does not cause these effects, thereby removing the selective pressure for mutation, and a new strain of E. coli that better tolerates existing pGreenII-based constructs without reducing plasmid yield. The adoption of the new derivative of pGreenII and the E. coli strain, which we have named pViridis and MW906, respectively, should help to ensure the integrity of genes destined for study in plants while they are propagated and manipulated in E. coli. The mechanism by which pGreenII perturbs E. coli growth appears to be dysregulation within the ColE1 origin of replication.

  17. An Improved Binary Vector and Escherichia coli Strain for Agrobacterium tumefaciens-Mediated Plant Transformation.

    Science.gov (United States)

    Watson, Michael R; Lin, Yu-Fei; Hollwey, Elizabeth; Dodds, Rachel E; Meyer, Peter; McDowall, Kenneth J

    2016-07-07

    The plasmid vector pGreenII is widely used to produce plant transformants via a process that involves propagation in Escherichia coli However, we show here that pGreenII-based constructs can be unstable in E. coli as a consequence of them hampering cell division and promoting cell death. In addition, we describe a new version of pGreenII that does not cause these effects, thereby removing the selective pressure for mutation, and a new strain of E. coli that better tolerates existing pGreenII-based constructs without reducing plasmid yield. The adoption of the new derivative of pGreenII and the E. coli strain, which we have named pViridis and MW906, respectively, should help to ensure the integrity of genes destined for study in plants while they are propagated and manipulated in E. coli The mechanism by which pGreenII perturbs E. coli growth appears to be dysregulation within the ColE1 origin of replication. Copyright © 2016 Watson et al.

  18. Regulierung der Expression der Onkogene aus Agrobakterien in Wirtspflanzen

    OpenAIRE

    Zhang, Yi

    2014-01-01

    Virulente Bakterien des Stamms Agrobakterium tumefaciens, transferieren und integrieren einen Teil ihrer DNA, die T-DNA aus dem Tumor induzierenden Plasmid (Ti), in das Pflanzengenom. Dadurch wird die Tumorbildung induziert und die Krankheit bricht aus. Die wichtigsten Gene, die für die Entwicklung eines Tumors benötigt werden, sind auf der T-DNA lokalisierte Onkogene: IaaH (indole-3-aceetamide hydrolase), IaaM (tryptophan monooxygenase) für die Auxin Biosynthese und Ipt (isopentenyl transfer...

  19. Enhanced Virulence Gene Activity of Agrobacterium in Muskmelon (Cucumis melo L. cv. ‘Birdie’

    Directory of Open Access Journals (Sweden)

    Abul K.M. MOHIUDDIN

    2011-05-01

    Full Text Available Muskmelon (Cucumis melo L. cultivar ‘Birdie’, was evaluated for its response to the tumorigenic Agrobacterium tumefaciens and the oncogenic A. rhizogenes strains. Stem and petiole of three week-old in vitro-grown muskmelon plants were inoculated with five strains of A. tumefaciens and A. rhizogenes each and observed phenotypic expressions i.e. induction of crown galls and hairy roots. This phenotypic expression was efficaciously increased when virulence gene activity of different strains of two Agrobacterium species was enhanced. Intensive studies on enhancement of virulence gene activity of Agrobacterium found to be correlated to the appropriate light intensity (39.3 μmol m-2 s-1 with a specific concentration of monocyclic phenolic compound, acetosyringone (20 μM. The gene activity was also influenced by several other physical factors e.g. plant tissue type, Agrobacterium species and their strains, and plant tissue-Agrobacterium interaction. Among the different A. tumefaciens strains, LBA4404 showed the best virulence gene activity in both stem and petiole through the formation of higher rate of crown galls. On the other hand, strain 15834 of A. rhizogenes showed better gene activity in stem and 8196 in petiole through the formation of higher rate of hairy roots as well as higher average number of hairy roots. Among the two different types of explants, petiole was more susceptible to both Agrobacterium species. Thus it was concluded that future muskmelon transformation study can efficiently be carried out with LBA4404, 15834 and 8196 strains using petiole explants by adding 20 μM of acetosyringone in the medium.

  20. The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis of Agrobacterium tumefaciens-Induced Crown Galls on Tomato Stems12

    Science.gov (United States)

    Aloni, Roni; Wolf, Asnat; Feigenbaum, Pua; Avni, Adi; Klee, Harry J.

    1998-01-01

    We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis. PMID:9662526

  1. Efficacy of Eucalyptus cinerea as a Source of Bioactive Compounds for Curative Biocontrol of Crown Gall Caused by Agrobacterium tumefaciens Strain B6.

    Science.gov (United States)

    Kahla, Yosra; Zouari-Bouassida, Karama; Rezgui, Fatma; Trigui, Mohamed; Tounsi, Slim

    2017-01-01

    This research investigated the Eucalyptus cinerea leaves efficiency in the Agrobacterium tumefaciens biocontrol, the causative agent of crown gall. GC-MS analysis of the essential oil (EO) showed that the main components were 1,8-cineole (61%) and camphene (15.13%). Thanks to its polyphenols, flavonoids, quinones, terpenoids, alkaloids, and tannins richness, the EtOAc-F exhibited the most potent antibacterial activity in vitro. Indeed, compared to the other fractions, it has the lowest MIC and MBC values of 0.312 mg/mL and 2.5 mg/mL, respectively. The GC-MS analysis of EtOAc-F confirmed its richness in antibacterial compounds including gallic acid (7.18%), shikimic acid (5.07%), and catechin (3.12%). The time-kill curve assay of EtOAc-F (2.5 mg/mL) showed a potent bactericidal effect after 20 min of direct contact with A. tumefaciens . In planta experiments, gall weights were significantly reduced when EtOAc-F was applied at 0.625 and 2.5 mg/wounds. Besides, the disease reduction rates in gall weight were 95% and 97.5%, respectively. Interestingly, no phytotoxic effect was observed since tomato seeds germinated in the presence of the different concentrations of EtOAc-F. These results suggest that EtOAc-F has a good potential to be a curative biocontrol agent for crown gall disease.

  2. The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

    NARCIS (Netherlands)

    Zheng Sijun, S.J.; Henken, B.; Ahn, Y.K.; Krens, F.A.; Kik, C.

    2004-01-01

    This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus

  3. SacB-SacR gene cassette as the negative selection marker to suppress Agrobacterium overgrowth in Agrobacterium-mediated plant transformation

    Directory of Open Access Journals (Sweden)

    Yiming Liu

    2016-10-01

    Full Text Available Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transfor-mation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium sup¬plemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of Agrobacterium tumefaciens in the plant tissue culture process. We generated a mutant Agrobacterium tumefaciens strain GV2260 (recA-SacB/R that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcrip¬tion factor.

  4. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    Energy Technology Data Exchange (ETDEWEB)

    Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Yamasaki, Masayuki; Mikami, Bunzo [Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru; Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2006-05-01

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2{sub 1} and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°.

  5. Binding events of (S )-N -(3-oxo-octanoyl)-homoserine lactone with agrobacterium tumefaciens mutant cells studied by saturation transfer difference NMR

    International Nuclear Information System (INIS)

    Cabeca, Luis Fernando; Pomini, Armando Mateus; Cruz, Pedro Luiz R.; Marsaioli, Anita J.

    2011-01-01

    Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. (author)

  6. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    International Nuclear Information System (INIS)

    Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2006-01-01

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2 1 and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°

  7. Binding events of (S )-N -(3-oxo-octanoyl)-homoserine lactone with agrobacterium tumefaciens mutant cells studied by saturation transfer difference NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cabeca, Luis Fernando; Pomini, Armando Mateus; Cruz, Pedro Luiz R.; Marsaioli, Anita J. [University of Campinas (UNICAMP), SP (Brazil). Chemistry Inst.

    2011-07-01

    Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. (author)

  8. Efficacy of Eucalyptus cinerea as a Source of Bioactive Compounds for Curative Biocontrol of Crown Gall Caused by Agrobacterium tumefaciens Strain B6

    Directory of Open Access Journals (Sweden)

    Yosra Kahla

    2017-01-01

    Full Text Available This research investigated the Eucalyptus cinerea leaves efficiency in the Agrobacterium tumefaciens biocontrol, the causative agent of crown gall. GC-MS analysis of the essential oil (EO showed that the main components were 1,8-cineole (61% and camphene (15.13%. Thanks to its polyphenols, flavonoids, quinones, terpenoids, alkaloids, and tannins richness, the EtOAc-F exhibited the most potent antibacterial activity in vitro. Indeed, compared to the other fractions, it has the lowest MIC and MBC values of 0.312 mg/mL and 2.5 mg/mL, respectively. The GC-MS analysis of EtOAc-F confirmed its richness in antibacterial compounds including gallic acid (7.18%, shikimic acid (5.07%, and catechin (3.12%. The time-kill curve assay of EtOAc-F (2.5 mg/mL showed a potent bactericidal effect after 20 min of direct contact with A. tumefaciens. In planta experiments, gall weights were significantly reduced when EtOAc-F was applied at 0.625 and 2.5 mg/wounds. Besides, the disease reduction rates in gall weight were 95% and 97.5%, respectively. Interestingly, no phytotoxic effect was observed since tomato seeds germinated in the presence of the different concentrations of EtOAc-F. These results suggest that EtOAc-F has a good potential to be a curative biocontrol agent for crown gall disease.

  9. Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus

    Directory of Open Access Journals (Sweden)

    Giuseppe Ianiri

    2016-11-01

    Full Text Available The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species.

  10. Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance.

    Science.gov (United States)

    Galambos, A; Zok, A; Kuczmog, A; Oláh, R; Putnoky, P; Ream, W; Szegedi, E

    2013-11-01

    Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance. Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.

  11. Experimental Report: ORNL Proposal ID IPTS 8937

    Energy Technology Data Exchange (ETDEWEB)

    Mirmelstein, A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2014-02-03

    Neutron scattering experiment was performed using fine-resolution Fermi chopper spectrometer “SEQUOIA” installed at the Spallation Neutron Source, ORNL. Although this spectrometer is designed to measure inelastic neutron scattering spectra, during experiments a signal of elastic scattering is also recorded. The coherent nuclear component of this elastic scattering provides Bragg diffraction pattern of a sample, i.e., CeNi single crystal in our case. Therefore, it is possible to follow the CeNi structural variations as a function of pressure and to register structural phase transition. Measurements were performed at the temperature of 15 K under pressure of zero (ambient pressure at 15 K), 400, 800, and 2200 bars.

  12. Development of a rapid and simple Agrobacterium tumefaciens mediated transformation system for the fungal pathogen Heterobasidion annosum

    Science.gov (United States)

    Nicklas Samils; Malin Elfstrand; Daniel L. Lindner Czederpiltz; Jan Fahleson; Ake Olson; Christina Dixelius; Jan Stenlid

    2006-01-01

    Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at...

  13. A twelve-week randomized controlled study of the cognitive-behavioral Integrated Psychological Therapy program: positive effect on the social functioning of schizophrenic patients Estudo controlado randomizado de 12 semanas do programa cognitivo-comportamental IPT (Terapia Psicológica Integrada com efeito positivo sobre o funcionamento social em pacientes com esquizofrenia

    Directory of Open Access Journals (Sweden)

    Marilene Zimmer

    2007-06-01

    Full Text Available OBJECTIVES: The present study was designed to evaluate the effect of twelve weekly sessions of the cognitive-behavioral program originally known as the Integriertes Psychologisches Therapieprogramm für Schizophrene Patienten, designated the Integrated Psychological Therapy (IPT program in English, on cognition, social adjustment and quality of life in schizophrenic outpatients, comparing it to the effect of treating such patients as usual. METHOD: Fifty-six adult outpatients (from 18 to 65 years of age with ICD-10-based diagnoses of schizophrenia were randomly assigned to two different groups: active intervention (IPT group; and treatment as usual (control group. Outcome measures were quality of life (as determined using the WHOQOL-Bref, cognition (Mini-Mental State Examination and Word Recall Test, global functioning (DSM-IV Global Assessment of Functioning Scale, social functioning (Social and Occupational Functioning Assessment Scale and social adjustment (Social Adjustment Scale. RESULTS: The findings suggest that, in comparison with treatment as usual (control group, the twelve-session IPT program had a positive effect on several outcome measures: cognition in the domains of spatiotemporal orientation (p = 0.051 and memory (p = 0.031; overall social adjustment (p = 0.037, leisure/social life (p = 0.051 and family relations (p = 0.008; overall functioning (p = 0.000; social-occupational functioning (p = 0.000; and quality of life in the psychological domain (p = 0.021. CONCLUSIONS: The twelve-session cognitive-behavioral IPT intervention demonstrated superiority over treatment as usual in its effects on cognition, social adjustment and quality of life. Studies involving larger samples, longer follow-up periods and additional outcome measures are needed in order to assess the specific effects on dimensions of social functioning, cognitive functioning and quality of life in patients with schizophrenia.OBJETIVOS: O presente estudo foi

  14. Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene

    Science.gov (United States)

    Powell Abel, Patricia; Nelson, Richard S.; de, Barun; Hoffmann, Nancy; Rogers, Stephen G.; Fraley, Robert T.; Beachy, Roger N.

    1986-05-01

    A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

  15. Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene.

    Science.gov (United States)

    Azadi, Pejman; Otang, Ntui Valentaine; Supaporn, Hasthanasombut; Khan, Raham Sher; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2011-06-01

    Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

  16. Genes and Gene Therapy

    Science.gov (United States)

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  17. A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens.

    Science.gov (United States)

    Feirer, Nathan; Xu, Jing; Allen, Kylie D; Koestler, Benjamin J; Bruger, Eric L; Waters, Christopher M; White, Robert H; Fuqua, Clay

    2015-06-30

    The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins. Pathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have

  18. Transformation of heat shock protein gene (HspB-C) of helicobacter pylori into sweet potato varieties

    International Nuclear Information System (INIS)

    Wu Jie; Yan Wenzhao; Zhou Yu; Zhang Xuemei

    2010-01-01

    Sweet potato which is one of the most important crops in the world has many advantages as a new bioreactor. Helicobacter pylori, as a kind of cancer-causing factor by the World Health Organization, has a strong immunogenicity, and its monoclonal antibody has bactericidal activity, which has the possibility as the vaccine components. In this research, we have constructed the plant expression vector with heat shock protein gene (HspB-C) of Helicobacter pylori. This vector was transformed by agrobactrium tumefaciens EHA105 into four sweet potato varieties. After callus-induction and re-differentiation, we got the transgenic plants from sweet potato variety of Nancy holl. (authors)

  19. Construction and Application of R Prime Plasmids, Carrying Different Segments of an Octopine Ti Plasmid from Agrobacterium tumefaciens, for Complementation of vir Genes

    NARCIS (Netherlands)

    Hille, Jacques; Klasen, Ina; Schilperoort, Rob

    1982-01-01

    Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with

  20. The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens

    Science.gov (United States)

    Plants defend themselves against potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs). However, the molecular mechanisms underlying this PAMP triggered immunity (PTI) are largely unknown. In this study, we show that tomato HP1/DDB1, coding for a key component of ...

  1. A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

    Science.gov (United States)

    Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

  2. Development of marker-free insect-resistant indica rice by Agrobacterium tumefaciens-mediated co-transformation

    Directory of Open Access Journals (Sweden)

    Fei Ling

    2016-10-01

    Full Text Available Agrobacterium-mediated co-transformation is an efficient strategy to generate marker-free transgenic plants. In this study, the vectors pMF-2A* containing a synthetic cry2A* gene driven by maize ubiquitin promoter and pCAMBIA1301 harboring hygromycin phosphotransferase gene (hpt were introduced into Minghui86 (Oryza sativa L. ssp. indica, an elite indica restorer line. Two independent transformants containing both the cry2A* gene and hpt gene were regenerated. Several homozygous marker-free transgenic progenies were derived from family 2AH2, and three of them were selected for further insect bioassay in the laboratory and field. Insect-resistance assays revealed that all the three transgenic lines were highly resistant to striped stem borer (Chilo suppressalis, yellow stem borer (Tryporyza incertulas and rice leaf folder (Cnaphalocrocis medinalis. The measurement of Cry2A protein concentration showed that Cry2A protein was stably expressed in leaves and stems of homozygous transgenic lines and their hybrids. The yields of the marker-free homozygous transgenic lines and their hybrids were not significantly different from those of their corresponding controls. Furthermore, the results of flanking sequence isolation showed that the T-DNA in line 8-30 was integrated into the intergenic region of chromosome 2 (between Os02g43680 and Os02g43690. These results indicate that the marker-free transgenic rice has the potential for commercial production.

  3. Delineation of polar localization domains of Agrobacterium tumefaciens type IV secretion apparatus proteins VirB4 and VirB11.

    Science.gov (United States)

    Das, Aditi; Das, Anath

    2014-10-01

    Agrobacterium tumefaciens transfers DNA and proteins to a plant cell through a type IV secretion apparatus assembled by the VirB proteins. All VirB proteins localized to a cell pole, although these conclusions are in dispute. To study subcellular location of the VirB proteins and to identify determinants of their subcellular location, we tagged two proteins, VirB4 and VirB11, with the visual marker green fluorescent protein (GFP) and studied localization of the fusion proteins by epifluorescence microscopy. Both GFP-VirB4 and GFP-VirB11 fusions localized to a single cell pole. GFP-VirB11 was also functional in DNA transfer. To identify the polar localization domains (PLDs) of VirB4 and VirB11, we analyzed fusions of GFP with smaller segments of the two proteins. Two noncontiguous regions in VirB4, residues 236-470 and 592-789, contain PLDs. The VirB11 PLD mapped to a 69 amino acid segment, residues 149-217, in the central region of the protein. These domains are probably involved in interactions that target the two proteins to a cell pole. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  4. Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens.

    Science.gov (United States)

    Park, Chang-Su; Park, Chul-Soon; Shin, Kyung-Chul; Oh, Deok-Kun

    2016-02-01

    The specific activity of recombinant Escherichia coli cells expressing the double-site variant (I33L-S213C) d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was highest at 24 h of cultivation time in Terrific Broth (TB) medium among the media tested. The contents of crude protein and DPEase in recombinant cells at 24 h were 37.0 and 8.6% (w/w), respectively, indicating that the enzyme was highly expressed. The reaction conditions for the production of d-psicose from d-fructose by whole recombinant cells with the highest specific activity were optimal at 60°C, pH 8.5, 4 g/l cells, and 700 g/l d-fructose. Under these conditions, whole recombinant cells produced 230 g/l d-psicose after 40 min, with a conversion yield of 33% (w/w), a volumetric productivity of 345 g/l/h, and a specific productivity of 86.2 g/g/h. These are the highest conversion yield and volumetric and specific productivities of d-psicose from d-fructose by cells reported thus far. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    Science.gov (United States)

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  6. Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.

    Science.gov (United States)

    Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju

    2014-05-01

    Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS + 1 mg/l BAP + 1 mg/l NAA, while indirect regeneration from callus was obtained on MS + 1 mg/l BAP + 2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling.

  7. Hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA

    Energy Technology Data Exchange (ETDEWEB)

    Hood, E.E.; Helmer, G.L.; Fraley, R.T.; Chilton, M.D.

    1986-12-01

    A binary-vectory strategy was used to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. Plasmids were constructed (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs were tested in trans with complementary each of regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results the authors suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

  8. (FIE) gene from soybean

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-04-17

    Apr 17, 2012 ... Reproduction of angiosperm plants initiates from two fertilization events: an egg fusing with a sperm to form an embryo and a second sperm fusing with the central cell to generate an endosperm. The tryptophan-aspartate .... to transform Agrobacterium tumefaciens (strain GV3101) by freeze- thaw methods ...

  9. Production of engineered long-life and male sterile Pelargonium plants

    Directory of Open Access Journals (Sweden)

    García-Sogo Begoña

    2012-08-01

    Full Text Available Abstract Background Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. Results The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3–4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. Conclusion The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.

  10. A preliminary attempt for efficient genetic transformation and regeneration of legume Mucuna pruriens L. mediated by Agrobacterium tumefaciens

    OpenAIRE

    SATHYANARAYANA, Raghavendra; KUMAR, Vadlapudi; RAMESH, Chapeyil Kumaran

    2014-01-01

    Mucuna pruriens belongs to the family Papillonaceae. The seeds and leaves of these plants contain a large amount of L-DOPA. L-DOPA (L-3,4-dihydroxyphenylalanine) is one of the most widely used drugs in the treatment of Parkinson disease. Development of an efficient gene transfer method is an absolute requirement for genetically improving Mucuna pruriens and creating plants with more desirable traits. A simple protocol was developed for the Agrobacterium-mediated stable genetic transformation ...

  11. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae

    Energy Technology Data Exchange (ETDEWEB)

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the [beta]-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate [beta]-carboxy-cis,cis-muconate. [beta]-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for [beta]-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to [beta]-carboxy-cis,cis-muconate.

  12. Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority and Rule 23a, Note 1 as applied to the corresponding specific epithets. Opinion 94. Judicial Commission of the International Committee on Systematics of Prokaryotes.

    Science.gov (United States)

    Tindall, B J

    2014-10-01

    The Judicial Commission affirms that, according to the Rules of the International Code of Nomenclature of Bacteria (including changes made to the wording), the combination Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over the combination Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority as applied to the corresponding specific epithets. The type species of the genus is Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942, even if treated as a later heterotypic synonym of Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942. Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 is typified by the strain defined on the Approved Lists of Bacterial Names and by strains known to be derived from the nomenclatural type. IUMS.

  13. Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit

    OpenAIRE

    Alves, G. S. C.; Torres, L. F.; Dechamp, E.; Breitler, J. C.; Joët, Thierry; Gatineau, F.; Andrade, A. C.; Bertrand, B.; Marraccini, P.; Etienne, H.

    2017-01-01

    Despite the importance of the DREB1D gene (also known as CBF4) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three CcDREB1D promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and drought-sensitive clones of Coffea canephora was carried out in plants of C. arabica stably transformed by Agrobacterium tumefaciens by analysing their ability to regula...

  14. Purification, crystallization and structural elucidation of D-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation.

    Science.gov (United States)

    Vetting, Matthew W; Bouvier, Jason T; Gerlt, John A; Almo, Steven C

    2016-01-01

    Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is D-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, D-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-L-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6 Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. D-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

  15. ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58.

    Science.gov (United States)

    Wichelecki, Daniel J; Vetting, Matthew W; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T; Almo, Steven C; Gerlt, John A

    2015-11-27

    Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Understanding the role of residues around the active site tunnel towards generating a glucose-tolerant β-glucosidase from Agrobacterium tumefaciens 5A.

    Science.gov (United States)

    Goswami, Shubhasish; Das, Shibashis; Datta, Supratim

    2017-07-01

    Most β-glucosidases are subjected to inhibition by the final hydrolysis product glucose resulting in the accumulation of cellobiose and oligosaccharides. This accumulated cellobiose and oligosaccharides further inhibit the activities of endoglucanase and cellobiohydrolases, resulting in the inhibition of cellulose degradation and a more expensive biofuel. To elucidate the mechanism(s) of glucose tolerance, we designed and characterised six mutations of a moderately glucose-tolerant β-glucosidase (H0HC94) from the mesophilic bacterium Agrobacterium tumefaciens 5A. The hydrophobicity and steric were varied across non-conserved residues in specific regions of the active site tunnel. In contrast to the uncompetitive inhibition of WT enzyme by glucose, C174V and H229S are competitively inhibited pointing towards a possible glucose-binding site in the protein at these positions. Increasing hydrophobicity at the +1 subsite and increasing hydrophobicity and steric at +2 subsites seemed to be critical for glucose tolerance for this BG. Additionally, in L178E, specific activity was 1.8 times higher on the natural substrate cellobiose while both W127F and L178E mutants showed an enhancement in thermostability. The kinetic stability of W127F, V176A, L178A and L178E also increased between 2- and 3-folds compared to WT. Our results indicate that while the structure between subsites +1 and +2 is critical for the glucose tolerance, the specific residues may not be identical across such enzymes. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58*

    Science.gov (United States)

    Wichelecki, Daniel J.; Vetting, Matthew W.; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T.; Almo, Steven C.; Gerlt, John A.

    2015-01-01

    Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. PMID:26472925

  18. A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants.

    Science.gov (United States)

    Anderson, J M; Palukaitis, P; Zaitlin, M

    1992-01-01

    Nicotiana tabacum cv. Turkish Samsun NN plants were transformed with a modified and truncated replicase gene encoded by RNA-2 of cucumber mosaic virus strain Fny. The replicase gene had been modified by deleting a 94-base-pair region spanning nucleotides 1857-1950; the deletion also caused a shift in the open reading frame, resulting in a truncated translation product approximately 75% as large as the full-length protein. Upon transformation via Agrobacterium tumefaciens, transgenic plants were obtained that were resistant to virus disease when challenged with either cucumber mosaic virus virions or RNA at concentrations up to 500 micrograms/ml or 50 micrograms/ml, respectively, the highest concentrations tested. This resistance was absolute, as neither symptoms nor virus could be detected in uninoculated leaves, even after prolonged incubation (120 days after inoculation). These data suggest, therefore, that such a "replicase-mediated" resistance strategy may be applicable to other plant and animal viruses. Images PMID:1528890

  19. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mihail Kantor

    2013-01-01

    Full Text Available The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9 in transgenic tomato plants from the T0 and T1 generations.

  20. ISOLATION AND CHARACTERIZATION OF CHITINASE GENE FROM THE UNTRADITIONAL PLANT SPECIES

    Directory of Open Access Journals (Sweden)

    Dominika Ďurechová

    2013-02-01

    Full Text Available Round-leaf sundew (Drosera rotundifolia L. from Droseraceae family belongs among a few plant species with strong antifungal potential. It was previously shown that chitinases of carnivorous plant species may play role during the insect prey digestion, when hard chitin skeleton is being decomposed. As many phytopathogenic fungi contain chitin in their cell wall our attention in this work was focused on isolation and in silico characterization of genomic DNA sequence of sundew chitinase gene. Subsequently this gene was fused to strong constitutive CaMV35S promoter and cloned into the plant binary vector pBinPlus and tested in A. tumefaciens LBA 4404 for its stability. Next, when transgenic tobacco plants are obtained, increasing of their antifungal potential will be tested.

  1. The role of plant bio-technologies with P4502E1 gene to remove radioactive contamination

    International Nuclear Information System (INIS)

    Mohammed, R. S.; Ibrahim, K.; Ali, N.; Al-Daoude, A.

    2012-12-01

    The aim of this research is to attempt removing pollution caused by some radioactive materials in polluted soils as a result exposure to radioactive particularly radioactive bombardments or misuse of atomic reactors, causing soil, water and air pollution. Two plant species were used in this study namely, sabins grandiflora and Arabidopsis thaliana. The bacterium victor Agrobacterium tumefaciens was used to transform the two plant species with used to transform the two plant species with the cytochrome gene P4502E1 which has been isolated from rabbit liver. The presence of the gene was investigated in plants using polymers chain reaction (PCR) after design of primers for this purpose. Transformation was proved after gene expression of P4502E1. Results showed that the plants were examined after transformation for their ability in removing the uranium, cesium and strontium in soils polluted with the three pollutants. (Author)

  2. Evaluation of two gene-silencing constructs for resistance to tomato yellow leaf curl viruses in Nicotiana benthamiana plants.

    Science.gov (United States)

    Gharsallah Chouchane, S; Gorsane, F; Nakhla, M K; Salus, M; Martin, C T; Maxwell, D P; Marrakchi, M; Fakhfakh, H

    2008-01-01

    Infiltration of Agrobacterium tumefaciens into intact plant leaves of N. benthamiana was used to test the efficiency of two virus-based silencing constructs conferring resistance to the closely related begomoviruses. The constructs contained the most conserved sequences of the coat protein (CP) gene and replication-associated protein (Rep) gene of Tomato yellow leaf curl Sardinia virus (Sicily strain) (TYLCSV-[Sic]). Both constructs formed a hairpin structure that enhanced the post-transcriptional gene-silencing mechanism. When agro-infiltrated plants were challenged separately with infectious viruses TYLCSV-[Sic] and Tomato yellow leaf curl virus (TYLCV), the plants showed resistance to TYLCSV-[Sic], but not to the related TYLCV.

  3. Identification of a splicing coactivator gene that affects the production of ochratoxin a in Aspergillus carbonarius

    Directory of Open Access Journals (Sweden)

    Lígia Uno Lunardi

    2009-11-01

    Full Text Available Ochratoxin A is a mycotoxin produced by some fungi species. Among them, Aspergillus carbonarius is considered a powerful producer. Genes involved in the ochratoxin A biosynthesis pathway have been identified in some producer species. However, there are few studies that purpose to identify these genes in A. carbonarius. The use of insertion mutants to identify genes associated with certain properties has been increased in the literature. In this work, the region of T-DNA integration was investigated in one A. carbonarius ochratoxin-defective mutant previously obtained by Agrobacterium tumefaciens-mediated transformation, in order to find an association between interrupted gene and the biosynthesis of ochratoxin A. The integration occurred in a gene that possibly encodes a splicing coactivator protein. The analysis of the relative expression of the splicing coativator gene from A. carbonarius wild type strain in four different media showed high correlation between the transcript levels and the ochratoxin A production.A ocratoxina A é uma micotoxina frequentemente encontrada em uma grande variedade de produtos alimentares e apresenta efeitos nefrotóxicos e potencial carcinogênico para animais e humanos. É naturalmente produzida por algumas espécies fúngicas, como Aspergillus carbonarius, que é considerado um potente produtor. Apesar disso, o número de estudos que visam identificar genes que são essenciais para a biossíntese de ocratoxina em A. carbonarius é ainda reduzido. Um mutante de A. carbonarius com baixa produção de ocratoxina A previamente obtido por transformação mediada por Agrobacterium tumefaciens foi investigado com o objetivo de encontrar uma associação entre o gene interrompido e a biossíntese desta micotoxina. Os resultados mostraram a ocorrência de uma junção não exata entre o T-DNA e o DNA genômico do fungo durante o evento de integração. A integração do T-DNA no genoma do mutante T188 provocou dele

  4. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato.

    Science.gov (United States)

    Velásquez, André C; Chakravarthy, Suma; Martin, Gregory B

    2009-06-10

    RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host. Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript. Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS. Inoculation of Nicotiana benthamiana and tomato seedlings

  5. Characterization and functional analysis of Calmodulin and Calmodulin-like genes in Fragaria vesca

    Directory of Open Access Journals (Sweden)

    Kai Zhang

    2016-12-01

    Full Text Available Calcium is a universal messenger that is involved in the modulation of diverse developmental and adaptive processes in response to various stimuli. Calmodulin (CaM and calmodulin-like (CML proteins are major calcium sensors in all eukaryotes, and they have been extensively investigated for many years in plants and animals. However, little is known about CaMs and CMLs in woodland strawberry (Fragaria vesca. In this study, we performed a genome-wide analysis of the strawberry genome and identified 4 CaM and 36 CML genes. Bioinformatics analyses, including gene structure, phylogenetic tree, synteny and three-dimensional model assessments, revealed the conservation and divergence of FvCaMs and FvCMLs, thus providing insight regarding their functions. In addition, the transcript abundance of four FvCaM genes and the four most related FvCML genes were examined in different tissues and in response to multiple stress and hormone treatments. Moreover, we investigated the subcellular localization of several FvCaMs and FvCMLs, revealing their potential interactions based on the localizations and potential functions. Furthermore, overexpression of five FvCaM and FvCML genes could not induce a hypersensitive response, but four of the five genes could increase resistance to Agrobacterium tumefaciens in Nicotiana benthamiana leaves. This study provides evidence for the biological roles of FvCaM and CML genes, and the results lay the foundation for future functional studies of these genes.

  6. Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles.

    Science.gov (United States)

    Wang, Yi-Hong; Campbell, Michael A

    2008-08-13

    Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in altered floral structure. Previously we identified two genes (LeTGA1 and SOLly GLB1) induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected. This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.

  7. [Construction of transgenic tobacco expressing tomato GGPS2 gene and analysis of its low light tolerance].

    Science.gov (United States)

    Li, Cuiping; Dong, Weihua; Zhang, Xingguo

    2015-05-01

    To explore the influence of low light on the synthesis of carotenoids, chlorophyll and the adaptability of transgenic plants with tomato Solanum lycopersicon L. GGPS2 gene, we constructed a vector containing a GGPS2 gene with green fluorescent protein (GFP) as report gene under the control of a cauliflower mosaic virus 35S promoter and introduced it into tobacco Nicotiana tabacum L. cv. Wisconsin 38 by Agrobacterium tumefaciens-mediated transformation. PCR analysis of the DNA from kanamycin resistant tobacco indicated that the transgenic tobacco containing the nptII gene, SlaGGPS2 gene and without contamination of Agrobacterium. We also detected the root tip of kanamycin resistant tobacco showing characteristic fluorescence. The contents of carotenoid, chlorophyll and photosynthesis of transgenic tobacco increased in comparison with wild tobacco after low light treatment. In addition, leaf mass per unit area, total dry weight, ratio of root to shoot in transgenic tobacco were all higher than that of the wild tobacco, which proved that the transgenic tobacco could increase the accumulation of biomass and promote it transport to root. The transgenic tobacco with SlaGGPS2 gene can increase the contents of carotenoid, chlorophyll, enhance the photosynthetic rate, promote the biomass accumulation and its distribution to root. Hence, the transgenic tobacco with SlaGGPS2 gene had increased low light tolerance and the SlaGGPS2 gene maybe can be used in other crops.

  8. Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles.

    Directory of Open Access Journals (Sweden)

    Yi-Hong Wang

    Full Text Available BACKGROUND: Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in altered floral structure. METHODOLOGY/PRINCIPAL FINDINGS: Previously we identified two genes (LeTGA1 and SOLly GLB1 induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected. CONCLUSION: This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.

  9. GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange Expressão do gene GUS controlado por promotor de citros em plantas transgênicas de tabaco e laranja 'Valência'

    Directory of Open Access Journals (Sweden)

    Fernando Alves de Azevedo

    2006-11-01

    Full Text Available The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL gene promoter (CsPP. Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.O objetivo deste trabalho foi realizar a transformação de plantas de tabaco e laranja 'Valência' com o gene GUS controlado pelo promotor do gene da fenilalanina amônia-liase (PAL de citros (CsPP. Foi realizada transformação genética por meio do co-cultivo de explantes de tabaco e laranja 'Valência' com Agrobacterium tumefaciens que continha o vetor binário CsPP-GUS/2201. Após a transformação e a regeneração, a detecção da atividade de GUS por ensaios histoquímicos revelou que o promotor CsPP, preferencialmente, mas não exclusivamente, confere expressão gênica em tecidos do xilema de tabaco. Expressão mais baixa de GUS também foi detectada na região de tecido de pecíolo, em plantas transgênicas (CsPP de tabaco e laranja 'Valência'.

  10. Detection of transformed cells in crown gall tumors using the GUS reporter gene and correlation of GUS stained cells with T-DNA gene activity

    International Nuclear Information System (INIS)

    Black, R.C.; Labriola, J.; Binns, A.N.

    1990-01-01

    Crown gall tumors are a mixture of transformed hormone producing cells and normal cells. Until now it has not been possible to directly visualize these cell types in situ. We have constructed strains of Agrobacterium tumefaciens that carry the 35S-β-glucuronidase (GUS) reporter gene in either wild type or mutant Ti plasmids. Using histochemical staining for GUS activity, blue (GUS positive) sectors are observed in tumor sections. In order to demonstrate that the blue sectors actually represent cells expressing other T-DNA genes, we have looked for T-DNA gene encoded enzyme activity in the stained and unstained sectors. The blue sectors accumulate octopine (a product of the octopine synthase gene on the T-DNA) while the white (GUS negative) sectors do not. We conclude that the use of the GUS reporter gene provides a sensitive and reliable method for visualizing transformation events in plant tissues. A comparison of the proportion of transformed and nontransformed cells in wild type tumors vs. tumors deficient in auxin or cytokinin encoding genes will be discussed

  11. Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis.

    Science.gov (United States)

    Cai, Zhiying; Li, Guohua; Lin, Chunhua; Shi, Tao; Zhai, Ligang; Chen, Yipeng; Huang, Guixiu

    2013-07-19

    To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to identify mutants of C. gloeosporioides impaired in pathogenicity. An ATMT library of 4128 C. gloeosporioides transformants was generated. Transformants were screened for defects in pathogenicity with a detached copper brown leaf assay. 32 mutants showing reproducible pathogenicity defects were obtained. Southern blot analysis showed 60.4% of the transformants had single-site T-DNA integrations. 16 Genomic sequences flanking T-DNA were recovered from mutants by thermal asymmetric interlaced PCR, and were used to isolate the tagged genes from the genome sequence of wild-type C. gloeosporioides by Basic Local Alignment Search Tool searches against the local genome database of the wild-type C. gloeosporioides. One potential pathogenicity genes encoded calcium-translocating P-type ATPase. Six potential pathogenicity genes had no known homologs in filamentous fungi and were likely to be novel fungal virulence factors. Two putative genes encoded Glycosyltransferase family 28 domain-containing protein and Mov34/MPN/PAD-1 family protein, respectively. Five potential pathogenicity genes had putative function matched with putative protein of other Colletotrichum species. Two known C. gloeosporioides pathogenicity genes were also identified, the encoding Glomerella cingulata hard-surface induced protein and C. gloeosporioides regulatory subunit of protein kinase A gene involved in cAMP-dependent PKA signal transduction pathway. Copyright © 2013 Elsevier GmbH. All rights reserved.

  12. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    Directory of Open Access Journals (Sweden)

    Kristensen Matilde B

    2008-08-01

    Full Text Available Abstract Background The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results Here, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.

  13. Transformation of Inhibitor of Meristem Activity (IMA Gene into Jatropha curcas L.

    Directory of Open Access Journals (Sweden)

    Asri Pirade Paserang

    2015-09-01

    Full Text Available Jatropha is one of the many biodiesel plants developed in tropical countries. Efforts to increase its productivity can be done using various methods of breeding. One of the breeding methods is the introduction of genes into the Jatropha plant. The aim of this study is to assess the success of genetic transformation using the Inhibitor of Meristem Activity (IMA gene in Jatropha curcas. The research procedures included inoculation of explants with Agrobacterium tumefaciens, callus induction, screening test of selection media, regeneration, and gene expression analysis using Polymerase Chain Reaction (PCR. IMA is one of the genes that controls flowering genes and ovule development. It was first isolated from tomato plants and has been successfully overexpressed in these plants using the Cauliflower Mosaic Virus (CaMV 35S promoter. In this experiment, plant transformation was performed on J. curcas as the target. Explant callus formation in both the control and treated samples was good, but shoot formation decreased dramatically in the treated explants. PCR analysis indicated that IMA genes can be inserted into J. curcas with the size of the IMA gene is 500 bp.

  14. Studying Genes

    Science.gov (United States)

    ... NIGMS NIGMS Home > Science Education > Studying Genes Studying Genes Tagline (Optional) Middle/Main Content Area PDF Version (382 KB) Other Fact Sheets What are genes? Genes are segments of DNA that contain instructions ...

  15. Vitreoscilla hemoglobin gene ( vgb) improves lutein production in Chlorella vulgaris

    Science.gov (United States)

    Ma, Ruijuan; Lin, Xiangzhi

    2014-03-01

    Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene ( vgb) into Chlorella vulgaris by Agrobacterium tumefaciens -mediated transformation (ATMT). PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.

  16. Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement.

    Science.gov (United States)

    Kozák, László; Szilágyi, Zoltán; Vágó, Barbara; Kakuk, Annamária; Tóth, László; Molnár, István; Pócsi, István

    2018-04-01

    The hypocrealean fungus Claviceps paspali is a parasite of wild grasses. This fungus is widely utilized in the pharmaceutical industry for the manufacture of ergot alkaloids, but also produces tremorgenic and neurotoxic indole-diterpene (IDT) secondary metabolites such as paspalitrems A and B. IDTs cause significant losses in agriculture and represent health hazards that threaten food security. Conversely, IDTs may also be utilized as lead compounds for pharmaceutical drug discovery. Current protoplast-mediated transformation protocols of C. paspali are inadequate as they suffer from inefficiencies in protoplast regeneration, a low frequency of DNA integration, and a low mitotic stability of the nascent transformants. We adapted and optimized Agrobacterium tumefaciens-mediated transformation (ATMT) for C. paspali and validated this method with the straightforward creation of a mutant strain of this fungus featuring a targeted replacement of key genes in the putative IDT biosynthetic gene cluster. Complete abrogation of IDT production in isolates of the mutant strain proved the predicted involvement of the target genes in the biosynthesis of IDTs. The mutant isolates continued to produce ergot alkaloids undisturbed, indicating that equivalent mutants generated in industrial ergot producers may have a better safety profile as they are devoid of IDT-type mycotoxins. Meanwhile, ATMT optimized for Claviceps spp. may open the door for the facile genetic engineering of these industrially and ecologically important organisms.

  17. Gene Therapy

    Science.gov (United States)

    Gene therapy Overview Gene therapy involves altering the genes inside your body's cells in an effort to treat or stop disease. Genes contain your ... that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds a new ...

  18. TaWRKY68 responses to biotic stresses are revealed by the orthologous genes from major cereals

    Directory of Open Access Journals (Sweden)

    Bo Ding

    2014-01-01

    Full Text Available WRKY transcription factors have been extensively characterized in the past 20 years, but in wheat, studies onWRKY genes and their function are lagging behind many other species. To explore the function of wheat WRKY genes, we identified a TaWRKY68 gene from a common wheat cultivar. It encodes a protein comprising 313 amino acids which harbors 19 conserved motifs or active sites. Gene expression patterns were determined by analyzing microarray data of TaWRKY68 in wheat and of orthologous genes from maize, rice and barley using Genevestigator. TaWRKY68 orthologs were identified and clustered using DELTA-BLAST and COBALT programs available at NCBI. The results showed that these genes, which are expressed in all tissues tested, had relatively higher levels in the roots and were up-regulated in response to biotic stresses. Bioinformatics results were confirmed by RT-PCR experiments using wheat plants infected by Agrobacterium tumefaciens and Blumeria graminis, or treated with Deoxynivalenol, a Fusarium graminearum-induced mycotoxin in wheat or barley. In summary,TaWRKY68 functions differ during plant developmental stages and might be representing a hub gene function in wheat responses to various biotic stresses. It was also found that including data from major cereal genes in the bioinformatics analysis gave more accurate and comprehensive predictions of wheat gene functions.

  19. The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ).

    Science.gov (United States)

    Małecki, Jędrzej; Dahl, Helge-André; Moen, Anders; Davydova, Erna; Falnes, Pål Ø

    2016-04-29

    Human METTL20 is a mitochondrial, lysine-specific methyltransferase that methylates the β-subunit of electron transfer flavoprotein (ETFβ). Interestingly, putative METTL20 orthologues are found in a subset of α-proteobacteria, including Agrobacterium tumefaciens Using an activity-based approach, we identified in bacterial extracts two substrates of recombinant METTL20 from A. tumefaciens (AtMETTL20), namely ETFβ and the ribosomal protein RpL7/L12. We show that AtMETTL20, analogous to the human enzyme, methylates ETFβ on Lys-193 and Lys-196 both in vitro and in vivo ETF plays a key role in mediating electron transfer from various dehydrogenases, and we found that its electron transferring ability was diminished by AtMETTL20-mediated methylation of ETFβ. Somewhat surprisingly, AtMETTL20 also catalyzed monomethylation of RpL7/L12 on Lys-86, a common modification also found in many bacteria that lack METTL20. Thus, we here identify AtMETTL20 as the first enzyme catalyzing RpL7/L12 methylation. In summary, here we have identified and characterized a novel bacterial lysine-specific methyltransferase with unprecedented dual substrate specificity within the seven β-strand class of lysine-specific methyltransferases, as it targets two apparently unrelated substrates, ETFβ and RpL7/L12. Moreover, the present work establishes METTL20-mediated methylation of ETFβ as the first lysine methylation event occurring in both bacteria and humans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

    Science.gov (United States)

    Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

    2014-07-01

    Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

  1. Production of transgenic brassica juncea with the synthetic chitinase gene (nic) conferring resistance to alternaria brassicicola

    International Nuclear Information System (INIS)

    Munir, I.; Hussan, W.; Kazi, M.; Mian, A.

    2016-01-01

    Brassica juncea is an important oil seed crop throughout the world. The demand and cultivation of oil seed crops has gained importance due to rapid increase in world population and industrialization. Fungal diseases pose a great threat to Brassica productivity worldwide. Absence of resistance genes against fungal infection within crossable germplasms of this crop necessitates deployment of genetic engineering approaches to produce transgenic plants with resistance against fungal infections. In the current study, hypocotyls and cotyledons of Brassica juncea, used as explants, were transformed with Agrobacterium tumefacien strain EHA101 harboring binary vector pEKB/NIC containing synthetic chitinase gene (NIC), an antifungal gene under the control of cauliflower mosaic virus promoter (CaMV35S). Bar genes and nptII gene were used as selectable markers. Presence of chitinase gene in trangenic lines was confirmed by PCR and southern blotting analysis. Effect of the extracted proteins from non-transgenic and transgenic lines was observed on the growth of Alternaria brassicicola, a common disease causing pathogen in brassica crop. In comparison to non-transgenic control lines, the leaf tissue extracts of the transgenic lines showed considerable resistance and antifungal activity against A. brassicicola. The antifungal activity in transgenic lines was observed as corresponding to the transgene copy number. (author)

  2. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993

    Energy Technology Data Exchange (ETDEWEB)

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the {beta}-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate {beta}-carboxy-cis,cis-muconate. {beta}-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for {beta}-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to {beta}-carboxy-cis,cis-muconate.

  3. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

    Directory of Open Access Journals (Sweden)

    Tuttle John

    2012-08-01

    Full Text Available Abstract Background We previously developed a virus-induced gene silencing (VIGS vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV. The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens. We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

  4. [Fruit-specific RNAi-mediated Lcy gene enhances content of lycopene in tomatoes silencing].

    Science.gov (United States)

    Wan, Qun; Zhang, Xing-Guo; Song, Ming

    2007-05-01

    Tomatoes ( Lycopersicon esculentum Mill.) are the principal dietary source of Lycopene which is one of carotenoid and is highly beneficial in preventing some diseases such as the cancer and the heart disease. Suppressing the expression of Lcy gene, the main gene regulating the transformation of the lycopene, is a convenient and effective way to enhance the content of lycopene. The primers were designed according to the gene sequence(U46919)and (X86452) in GenBank. The fruit-specific promoter--phytoene desaturase gene(Pds) promoter and the DNA segment of the Lcy gene were isolated from the genome DNA of tomatoes. The 3'end of Lcy DNA segment was connected together by an intron to inform the RNA interferential segment then which was inserted in the expression vector with the Pds promoter to inform the fruit-specific expression vector. The vector was transformed into the tomatoes through the Agrobacterium tumefaciens. Five transformants were obtained. And the PCR proved that the extra-gene was integrated into the tomato genome. The lycopene in the transgenic tomatoes fruit was increased significantly through analysing the contents of lycopene. These results show that regutating biosynthetic enzyme in carotenoid pathway by RNAi can improve the lycopene content of plant-derived products.

  5. Transient Expression of cor Gene in Papaver somniferum

    Directory of Open Access Journals (Sweden)

    Bahman Hosseini

    2011-12-01

    Full Text Available Introduction: Papaver somniferum is the commercial source of morphine and codeine. The isolation of effective genes involved in the morphine biosynthesis of P. somniferum is very important in the production of specific metabolites achieved using metabolic engineering techniques. In this pathway, the key enzyme COR is involved in the conversion of codeinone to codeine and morphinone to morphine. Methods: the gene encoding of this enzyme was isolated using primers designed on the base of gene sequence available on (NCBI for P. somniferum. This gene correct size around (960 bp was first subcloned into pTZ57RIT vector then cloned into expression vectors (pBI121 between BamHI and SacI sites to allow the expression of cor gene driven by the cauliflower mosaic virus 35S promoter. The result was confirmed through different molecular methods e.g. PCR and enzyme digestion by BamHI and SacI. The recombinant plasmid was transformed into the E. coli strain DH5α using a freeze-thaw method. Having selected positive colones on selection medium, plasmid was extracted by miniprep method and recombinant plasmids were selected based on PCR and digestion. The construct was then mobilized in Agrobacterium tumefaciens C58/pGV3850 (KmR RifR. After gene transformation to P. somniferum plants, the agroinfiltration method was also used for transient expression of COR enzyme. Results: evaluation results showed that morphine and codeine were detectable in the leaves of transgenic plants containing cor transgene and there was significant difference in the final production. After completing this experiment for three times, results showed that in 11 sets from 15 sets of leaves experiment tested, main alkaloids (codeine, morphine, papaverin, noscapine and thebaine were detectable. Conclusion: Whereas no signal was detected in non-infiltrated control leaves or in leaves infiltrated with non-recombinant bacteria for morphine and codeine, others such as thebaine and papaverine

  6. Targeting fungal genes by diced siRNAs: a rapid tool to decipher gene function in Aspergillus nidulans.

    Science.gov (United States)

    Kalleda, Natarajaswamy; Naorem, Aruna; Manchikatla, Rajam V

    2013-01-01

    Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans. Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression. We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.

  7. Identification and Plant Interaction of a Phyllobacterium sp., a Predominant Rhizobacterium of Young Sugar Beet Plants

    Science.gov (United States)

    Lambert, Bart; Joos, Henk; Dierickx, Sabine; Vantomme, Robert; Swings, Jean; Kersters, Karel; Van Montagu, Marc

    1990-01-01

    The second most abundant bacterium on the root surface of young sugar beet plants was identified as a Phyllobacterium sp. (Rhizobiaceae) based on a comparison of the results of 39 conventional identification tests, 167 API tests, 30 antibiotic susceptibility tests, and sodium dodecyl sulfate-polyacrylamide gel electrophoretic fingerprints of total cellular proteins with type strains of Phyllobacterium myrsinacearum and Phyllobacterium rubiacearum. It was found on 198 of 1,100 investigated plants between the 2nd and 10th leaf stage on three different fields in Belgium and one field in Spain. Densities ranged from 2 × 104 to 2 × 108 CFU/g of root. Five isolates exerted a broad-spectrum in vitro antifungal activity. DNA-DNA hybridizations showed that Phyllobacterium sp. does not contain DNA sequences that are homologous with the attachment genes chvA, chvB, the transferred-DNA (T-DNA) hormone genes iaaH and ipt from Agrobacterium tumefaciens, iaaM from A. tumefaciens and Pseudomonas savastanoi, or the nitrogenase genes nifHDK from Klebsiella pneumoniae. Phyllobacterium sp. produces indolylacetic acid in in vitro cultures and induces auxinlike effects when cocultivated with callus tissue of tobacco. When Phyllobacterium sp. was transformed with a Ti plasmid derivative, it gained the capacity to induce tumors on Kalanchoe daigremontiana. The potential role of Phyllobacterium sp. in this newly recognized niche is discussed. Images PMID:16348158

  8. gene structure, gene expression

    Indian Academy of Sciences (India)

    and seedling leaves were sampled at 6 h after the treatment. For cold stress, the seedlings were transferred to 4◦C growth chamber for 30 min. Control seedlings were exposed to none of these treatments. To examine the expression patterns of these predicted genes in Poplar and to further confirm their stress responsive-.

  9. Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

    Directory of Open Access Journals (Sweden)

    Sebahattin Çürük

    2012-01-01

    Full Text Available The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP and neomycin phosphotransferase II (NPT II genes to the watermelon 'Crimson Sweet'(CS genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

  10. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    Directory of Open Access Journals (Sweden)

    Yu-Ju Lin

    Full Text Available Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.

  11. The novel gene VpPR4-1 from Vitis pseudoreticulata increases powdery mildew resistance in transgenic Vitis vinifera L.

    Directory of Open Access Journals (Sweden)

    Lingmin eDai

    2016-05-01

    Full Text Available Pathogenesis-related proteins (PRs can lead to increased resistance of the whole plant to pathogen attack. Here, we isolate and characterize a PR-4 protein from a wild Chinese grape Vitis pseudoreticulata which shows greatly elevated transcription following powdery mildew infection. Its expression profiles under a number of abiotic stresses were also investigated. The PR-4 gene was overexpressed in regenerated V. vinifera cv. Red Globe via Agrobacterium tumefaciens-mediated transformation and verified by the Western blot. The transgenic grapevines exhibited higher expression levels of PR-4 protein content than wild-type vines and also repressed the growth of powdery mildew. The PR gene responds differently to different stresses in the PR-4 transformants. This study demonstrates that PR-4 protein in grapes plays a vital role in defense against powdery mildew invasion.

  12. Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments Transformação genética em Citrus sinensis e Citrus limonia mediada por Agrobacterium tumefaciens a partir de segmentos de epicótilo

    Directory of Open Access Journals (Sweden)

    Weliton Antonio Bastos de Almeida

    2003-02-01

    Full Text Available Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck and Rangpur lime (Citrus limonia L. Osbeck. Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm; co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone.A transformação genética permite produzir cultivares com características específicas e pode, dessa forma ser associada a programas de melhoramento de citros. O objetivo deste trabalho foi estabelecer protocolos de transformação genética para as laranjas doce 'Valência' e 'Natal' (Citrus sinensis L. Osbeck, bem como para o limão 'Cravo'(Citrus limonia L. Osbeck. Segmentos de epicótilo de plântulas germinadas in vitro (três semanas no

  13. Genetic Transformation of Transcription Factor (35S-oshox4 Gene into Rice Genome and Transformant Analysis of hpt Gene by PCR and Hygromycin Resistance Test

    Directory of Open Access Journals (Sweden)

    INEZ HORTENZE SLAMET-LOEDIN

    2009-04-01

    Full Text Available Global warming, climate change and crop extensification in marginal dryland areas are related to long dry season and water deficit. The water availability is an important factor in improving plant production. Application of drought tolerant rice cultivars is one of several options that might be used. Genetic engineering at the level of transcription factors is particularly promising strategy to develop drought tolerant rice varieties. Transcription factors regulate a wide range of target genes in which of them contribute to stress tolerance. HD Zip genes are transcription factor that potential in the adaptation of plants to some environment stresses including water deficit. HD-ZIP oshox4 (oryza sativa homeobox gene controlled by 35S promotor is inserted into pCAMBIA 1300 vector with hpt (hygromycin gene as a selectable marker. The aim of this research is to obtain transgenic rice plant from transformation with 35S-oshox4 plasmid, segregation analysis of marker gene (hpt by PCR method at T0 and T1 generation, and hygromycin resistance analysis of seeds. Recombinant plasmid was transformed into rice genome of IRAT 112 and rojolele cultivars using Agrobacterium tumefaciens. The results showed that transformation efficiency of IRAT 112 is 5.7-13.6% and 26-66,7% for rojolele. While regeneration efficiency for IRAT 112 is 4.7-43.7% and 23-44.1% for rojolele. The result of hygromycin resistance test at T1 seeds were obtained 14 lines cv. rojolele segregation Mendelian for hpt gene. The PCR analysis using specific primers for hpt gene at the parent (T0 from 14 lines showed that 7 lines contain the gene. At the second generation (T1, PCR analysis using hpt primers showed that 3 from 4 lines were followed Mendelian segregation pattern by the presence of specific band.

  14. Genetic transformation of the tomato pathogen Pyrenochaeta lycopersici allowed gene knockout using a split-marker approach.

    Science.gov (United States)

    Aragona, Maria; Valente, Maria Teresa

    2015-05-01

    Pyrenochaeta lycopersici, as other soil-transmitted fungal pathogens, generally received little attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and important fruit yield reduction of agronomic relevant crops. The scope of this study was to develop a system allowing to investigate the functional role of P. lycopersici genes putatively involved in the corky root rot of tomato. A genetic transformation system based on a split-marker approach was developed and tested to knock out a P. lycopersici gene encoding for a lytic polysaccharide monooxygenase (Plegl1) induced during the disease development. The regions flanking Plegl1 gene were fused with the overlapping parts of hygromycin marker gene, to favour homologous recombination. We were able to obtain four mutants not expressing the Plegl1 gene though, when tested on a susceptible tomato cultivar, Plegl1 mutants showed unaltered virulence, compared with the wild-type strain. The strategy illustrated in the present work demonstrated for the first time that homologous recombination occurs in P. lycopersici. Moreover, a transformation system mediated by Agrobacterium tumefaciens was established and stable genetic transformants have been obtained. The transformation systems developed represent important tools for investigating both the role of genes putatively involved in P. lycopersici interaction with host plant and the function of other physiological traits which emerged to be genetically expanded from the recent genome sequencing of this fungus.

  15. The Yeast HAL1 Gene Improves Salt Tolerance of Transgenic Tomato1

    Science.gov (United States)

    Gisbert, Carmina; Rus, Ana M.; Bolarín, M. Carmen; López-Coronado, J. Miguel; Arrillaga, Isabel; Montesinos, Consuelo; Caro, Manuel; Serrano, Ramon; Moreno, Vicente

    2000-01-01

    Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K+ concentration and decreasing intracellular Na+ during salt stress. In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo. Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene. In addition, measurement of the intracellular K+ to Na+ ratios showed that transgenic lines were able to retain more K+ than the control under salt stress. Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast. PMID:10806256

  16. [Double-antisense ACC oxidase and ACC synthase fusion gene introduced into tomato by Agrobacterium-mediated transformation and analysis the ethylene production of transgenic plants].

    Science.gov (United States)

    Xiong, Ai Sheng; Yao, Quan Hong; Li, Xian; Fan, Hui Qin; Peng, Ri He

    2003-12-01

    The tomato fruit-specific promoter 2A11 was amplified from tomato genomic DNA using PCR techniques. Total RNA was isolated from ripen fruit of tomato, then ACC oxidase gene and ACC synthase gene were obtained using reverse-transcription polymerase chain reaction. The fusion encoding ACC oxidase and ACC synthase gene was obtained through ACC oxidase gene and ACC synthase gene ligation. The fusion gene was then inserted into a plant binary vector pYPX145 in an inverted orientation. Finally, the binary plant expression vector pOSACC was constructed in which the double-antisense fusion gene was controlled by fruit-specific 2A11 promoter. By using hypocotyls and cotyledon petioles as explants, the unit of double-antisense fusion gene was successfully introduced into tomato (Lycopersicon esculentum Mill) cultivar "Hezuo 903" by Agrobacterium tumefaciens-mediated transformation. 105 transgenic plants were obtained through 200 mg/L kanamycin selection and GUS assay. Two lines of DR-1 and DR-2 were obtained through selecting the characteristics of prolonged shelf life and agriculture. The transgenic plants showed the characteristics of prolonged shelf life over 50 d. The amount of ethylene released from DR-1 and DR-2 fruits were reduced significantly to about 9.5% of that released by non-transformed controls.

  17. Research Activities at IPT, DTU on Resistance Projection Welding

    DEFF Research Database (Denmark)

    Bay, Niels

    2000-01-01

    Resistance welding processes and among these especially the resistance projection welding is considered an industrially strategic process with increasing applications as alternative to other welding processes, soldering, brazing and mechanical assembling. This is due to increasing requirements as...... as regards quality assurance and the special possibilities of joining complex metal combinations and geometries using resistance projection welding.......Resistance welding processes and among these especially the resistance projection welding is considered an industrially strategic process with increasing applications as alternative to other welding processes, soldering, brazing and mechanical assembling. This is due to increasing requirements...

  18. New cytokinin metabolites in IPT transgenic Arabidopsis thaliana plants

    Czech Academy of Sciences Publication Activity Database

    Werner, Tomáš; Hanuš, Jan; Holub, Jan; Schmülling, T.; Onckelen, H. V.; Strnad, Miroslav

    2003-01-01

    Roč. 118, č. 1 (2003), s. 127-137 ISSN 0031-9317 R&D Projects: GA ČR GA301/02/0475 Grant - others: Volkswagen Stiftung(DE) I/76 86:153100008; - - -(BE) IUAP P5/13 Institutional research plan: CEZ:AV0Z5038910 Keywords : transgenic Arabidopsis thaliana * cytokinin * zeatin-O-glucoside Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.767, year: 2003

  19. A tomato mutant that shows stunting, wilting, progressive necrosis and constitutive expression of defence genes contains a recombinant Hcr9 gene encoding an autoactive protein.

    Science.gov (United States)

    Barker, Claire L; Talbot, Stephen J; Jones, Jonathan D G; Jones, David A

    2006-05-01

    The tomato Cf-9 gene confers resistance to races of the leaf mould fungus Cladosporium fulvum that carry the Avr9 avirulence gene. Cf-9 resides at a locus containing five paralogous genes and was isolated by transposon tagging using a modified maize Dissociation (Ds) element. The tagging experiment generated an allelic series of Ds-induced mutations of Cf-9, most of which were wild type in appearance. However, one mutant, designated M205, showed stunted growth, wilting, progressive leaf chlorosis and necrosis and constitutive expression of defence genes. The phenotype of M205 was caused by a semidominant, Avr9-independent mutation that co-segregated with a Ds element insertion at the Cf-9 locus. Molecular genetic analysis indicated that the Cf-9 locus of M205 had undergone recombination, generating a chimeric gene, designated Hcr9-M205, that comprised an in-frame fusion between the 5' coding region of the Cf-9 paralogue, Hcr9-9A, and the 3' coding region of Cf-9. The presence of a possible excision footprint adjacent to the junction between Hcr9-9A and Cf-9, and a Ds insertion at the homologous position in the downstream paralogue Hcr9-9D, is consistent with recombination between Hcr9-9A and Cf-9 promoted by transposition of Ds from Cf-9 into Hcr9-9D. Agrobacterium tumefaciens-mediated transient expression of Hcr9-M205 in Nicotiana tabacum caused chlorosis and the accumulation of defence gene transcripts, indicating that the protein encoded by this novel Hcr9 gene is autoactive.

  20. Inducible Expression of Agrobacterium Virulence Gene VirE2 for Stringent Regulation of T-DNA Transfer in Plant Transient Expression Systems.

    Science.gov (United States)

    Denkovskienė, Erna; Paškevičius, Šarūnas; Werner, Stefan; Gleba, Yuri; Ražanskienė, Aušra

    2015-11-01

    Agrotransfection with viral vectors is an effective solution for the transient production of valuable proteins in plants grown in contained facilities. Transfection methods suitable for field applications are desirable for the production of high-volume products and for the transient molecular reprogramming of plants. The use of genetically modified (GM) Agrobacterium strains for plant transfections faces substantial biosafety issues. The environmental biosafety of GM Agrobacterium strains could be improved by regulating their T-DNA transfer via chemically inducible expression of virE2, one of the essential Agrobacterium virulence genes. In order to identify strong and stringently regulated promoters in Agrobacterium strains, we evaluated isopropyl-β-d-thiogalactoside-inducible promoters Plac, Ptac, PT7/lacO, and PT5/lacOlacO and cumic acid-inducible promoters PlacUV5/CuO, Ptac/CuO, PT5/CuO, and PvirE/CuO. Nicotiana benthamiana plants were transfected with a virE2-deficient A. tumefaciens strain containing transient expression vectors harboring inducible virE2 expression cassettes and containing a marker green fluorescent protein (GFP) gene in their T-DNA region. Evaluation of T-DNA transfer was achieved by counting GFP expression foci on plant leaves. The virE2 expression from cumic acid-induced promoters resulted in 47 to 72% of wild-type T-DNA transfer. Here, we present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a novel approach to address environmental biosafety concerns in agrobiotechnology.

  1. Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus.

    Science.gov (United States)

    Ianiri, Giuseppe; Averette, Anna F; Kingsbury, Joanne M; Heitman, Joseph; Idnurm, Alexander

    2016-11-29

    The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species. Species in the genus Malassezia are a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culture in vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species of Malassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals. Copyright © 2016 Ianiri et al.

  2. Tomato (Solanum lycopersicum L.) SlIPT3 and SlIPT4 isopentenyltransferases mediate salt stress response in tomato

    Czech Academy of Sciences Publication Activity Database

    Žižková, Eva; Dobrev, Petre; Muhovski, Y.; Hošek, Petr; Hoyerová, Klára; Haisel, Daniel; Procházková, Dagmar; Lutts, S.; Motyka, Václav; Hichri, I.

    2015-01-01

    Roč. 15, MAR 12 (2015) ISSN 1471-2229 R&D Projects: GA ČR(CZ) GAP506/11/0774 Institutional support: RVO:61389030 Keywords : Cytokinin * Isopentenyltransferase * Salt stress Subject RIV: EF - Botanics Impact factor: 3.631, year: 2015

  3. The impact of plasmid on regeneration and expression efficiencies of gfp gene in tobacco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Niko PAVLIN

    2015-12-01

    Full Text Available Tobacco leaf explants were transformed by bacteria Agrobacterium tumefaciens (A. t. and plasmid pBIN mgfp5-ER, which has a single copy of the green fluorescent gfp gene and A. t.-pART27 2mgfp5-ER, which has two copies of the gfp gene. Both plasmids have a built-in selection nptII gene for resistance to the antibiotic kanamycin. The presence of the green fluorescent mGFP-ER protein was detected with the epifluorescent microscope in the individual cells 3 days after transformation with A. t.-pART27 2mgfp5-ER and after 6 days in cells transformed with A.t.-pBIN mgfp5-ER. After infection by A. t.-pART27 2mgfp5-ER, in most cases the regeneration was direct, without intermediate stages of callus and faster, as the first globular structures were formed 10–12 days after transformation and a 204 % regeneration was achieved, while the first globular structure, after infection with A. t.-pBIN mgfp5-ER, occurred after 18 days and formed more callus and the regeneration was only 78.4 %. The duplex PCR analysis, performed on all 149 resulting regenerants, confirmed the presence of fragments of length 650 bp specific to the selection nptII gene and length of 422 bp specific for gfp marker gene.

  4. Genetic engineering of cotton with a novel cry2AX1 gene to impart insect resistance against Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Karunamurthy Dhivya

    2016-09-01

    Full Text Available Embryogenic calli of cotton (Coker310 were cocultivated with the Agrobacterium tumefaciens strain LBA4404 harbouring the codon-optimised, chimeric cry2AX1 gene consisting of sequences from cry2Aa and cry2Ac genes isolated from Indian strains of Bacillus thuringiensis. Forty-eight putative transgenic plants were regenerated, and PCR analysis of these plants revealed the presence of the cry2AX1 gene in 40 plants. Southern blot hybridisation analysis of selected transgenic plants confirmed stable T-DNA integration in the genome of transformed plants. The level of Cry2AX1 protein expression in PCR positive plants ranged from 4.9 to 187.5 ng g-1 of fresh tissue. A transgenic cotton event, TP31, expressing the cry2AX1 gene showed insecticidal activity of 56.66 per cent mortality against Helicoverpa armigera in detached leaf disc bioassay. These results indicate that the chimeric cry2AX1 gene expressed in transgenic cotton has insecticidal activity against H. armigera.

  5. A primary study of high performance transgenic rice through maize UBI-1 promoter fusing selective maker gene

    International Nuclear Information System (INIS)

    Shen, J.; Cai, P.; Qing, F.

    2012-01-01

    Based on the expression vector pBI121, we successfully constructed a plant over-expression vector of Hspa4 gene fusing with selective maker gene (hygromycin-resistance gene) driven by the Ubi-1 promoter (pBI121-Ubi-Hpt-Hspa4, p121UHH). The plant expression vectors p121UHH and pCAMBIA1301-Ubi-Hspa4 (p1301UH) were transformed into the rice callus, mediated by Agrobacterium tumefaciens. We screened 17 p121UHH-positive transgenic plants and 15 p1301UH-positive transgenic plants by the hygromycin-resistance gene. The pick-up rate of the resistance callus was 51.7% and 42.5%, respectively, and the rate of regeneration for the resistance callus was 51.2% and 49.1%, respectively. The result of polymerase chain reaction (P CR) identification indicated that the pick-up rate of positive transgenic plants was 51.7% and 42.5% and the total transformation efficiency was 16.5% and 6.2%, and the former was 2.66 time s of the later. The results of the experiment indicate that the possibility of the appearance of false positive results in the fusing of a plant over-expression vector with a selective maker gene is much less. (author)

  6. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea.

    Science.gov (United States)

    Ding, Zhong-Tao; Zhang, Zhi; Luo, Di; Zhou, Jin-Yan; Zhong, Juan; Yang, Jie; Xiao, Liang; Shu, Dan; Tan, Hong

    2015-05-06

    The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.

  7. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  8. Functional analysis of agrobacterium virulence genes

    NARCIS (Netherlands)

    Niu, Xiaolei

    2013-01-01

    Agrobacterium tumefaciens is a gram-negative soil bacterium that induces plant tumors by transferring a segment of DNA, called T-DNA, into plant cells. Under laboratory conditions, Agrobacterium can also transform many different non-plant organisms such as the yeast Saccharomyces cerevisiae. During

  9. Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

    Science.gov (United States)

    Tang, Wei; Newton, Ronald J; Weidner, Douglas A

    2007-01-01

    An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

  10. Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

    Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

  11. Testing Transgenic Aspen Plants with bar Gene for Herbicide Resistance under Semi-natural Conditions.

    Science.gov (United States)

    Lebedev, V G; Faskhiev, V N; Kovalenko, N P; Shestibratov, K A; Miroshnikov, A I

    2016-01-01

    Obtaining herbicide resistant plants is an important task in the genetic engineering of forest trees. Transgenic European aspen plants (Populus tremula L.) expressing the bar gene for phosphinothricin resistance have been produced using Agrobacterium tumefaciens-mediated transformation. Successful genetic transformation was confirmed by PCR analysis for thirteen lines derived from two elite genotypes. In 2014-2015, six lines were evaluated for resistance to herbicide treatment under semi-natural conditions. All selected transgenic lines were resistant to the herbicide Basta at doses equivalent to 10 l/ha (twofold normal field dosage) whereas the control plants died at 2.5 l/ha. Foliar NH4-N concentrations in transgenic plants did not change after treatment. Extremely low temperatures in the third ten-day period of October 2014 revealed differences in freeze tolerance between the lines obtained from Pt of f2 aspen genotypes. Stable expression of the bar gene after overwintering outdoors was confirmed by RT-PCR. On the basis of the tests, four transgenic aspen lines were selected. The bar gene could be used for retransformation of transgenic forest trees expressing valuable traits, such as increased productivity.

  12. Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation.

    Science.gov (United States)

    Onyilo, Francis; Tusiime, Geoffrey; Chen, Li-Hung; Falk, Bryce; Stergiopoulos, Ioannis; Tripathi, Jaindra N; Tushemereirwe, Wilberforce; Kubiriba, Jerome; Changa, Charles; Tripathi, Leena

    2017-01-01

    Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijie nsis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1 -silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

  13. Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation

    Directory of Open Access Journals (Sweden)

    Francis Onyilo

    2017-05-01

    Full Text Available Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs. The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

  14. Conserved RXLR Effector Genes of Phytophthora infestans Expressed at the Early Stage of Potato Infection Are Suppressive to Host Defense

    Directory of Open Access Journals (Sweden)

    Junliang Yin

    2017-12-01

    Full Text Available Late blight has been the most devastating potato disease worldwide. The causal agent, Phytophthora infestans, is notorious for its capability to rapidly overcome host resistance. Changes in the expression pattern and the encoded protein sequences of effector genes in the pathogen are responsible for the loss of host resistance. Among numerous effector genes, the class of RXLR effector genes is well-known in mediating host genotype-specific resistance. We therefore performed deep sequencing of five genetically diverse P. infestans strains using in planta materials infected with zoospores (12 h post inoculation and focused on the identification of RXLR effector genes that are conserved in coding sequences, are highly expressed in early stages of plant infection, and have defense suppression activities. In all, 245 RXLR effector genes were expressed in five transcriptomes, with 108 being co-expressed in all five strains, 47 of them comparatively highly expressed. Taking sequence polymorphism into consideration, 18 candidate core RXLR effectors that were conserved in sequence and with higher in planta expression levels were selected for further study. Agrobacterium tumefaciens-mediated transient expression of the selected effector genes in Nicotiana benthamiana and potato demonstrated their potential virulence function, as shown by suppression of PAMP-triggered immunity (PTI or/and effector-triggered immunity (ETI. The identified collection of core RXLR effectors will be useful in the search for potential durable late blight resistance genes. Analysis of 10 known Avr RXLR genes revealed that the resistance genes R2, Rpi-blb2, Rpi-vnt1, Rpi-Smira1, and Rpi-Smira2 may be effective in potato cultivars. Analysis of 8 SFI (Suppressor of early Flg22-induced Immune response RXLR effector genes showed that SFI2, SFI3, and SFI4 were highly expressed in all examined strains, suggesting their potentially important function in early stages of pathogen infection.

  15. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    DEFF Research Database (Denmark)

    Sorensen, Lisette Quaade; Lysøe, Erik; Larsen, Jesper Erup

    2014-01-01

    The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infe......The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize...... single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results: The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated...... fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the Delta...

  16. BraLTP1, a lipid transfer protein gene involved in epicuticular wax deposition, cell proliferation and flower development in Brassica napus.

    Directory of Open Access Journals (Sweden)

    Fang Liu

    Full Text Available Plant non-specific lipid transfer proteins (nsLTPs constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

  17. The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates.

    Science.gov (United States)

    Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J; Sarkar, Mayukh K; Li, Feng; Christie, Peter J

    2015-07-01

    Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here

  18. Propuesta de un sistema de transformación de plantas de papa (Solanum tuberosum sp. andigena var. Pastusa suprema mediado por Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    López Alfredo

    2007-06-01

    Full Text Available

    Se ha demostrado que la transformación de papa (Solanum tuberosum mediada por Agrobacterium tumefaciens es dependiente del genotipo y que la mayoría de protocolos de transformación reportados son ineficientes al aplicarlos en la subespecie andigena. En esta propuesta se manejaron los procesos iniciales de mejoramiento genético de la nueva variedad colombiana de papa Pastusa suprema (Solanum tuberosum sp. andigena que es altamente androestéril, característica de gran importancia para los organismos modificados genéticamente. Esta variedad resultó de la hibridación interespecífica de tres especies de papa (Solanum stoloniferum, Solanum phureja var. Yema de huevo y Solanum tuberosum sp. andigena var. Parda pastusa. Se transformaron explantes internodales mediante el vector pCambia2301 que posee un gen reportero de la β-glucoronidasa y un gen de resistencia a la kanamicina. Se obtuvo un porcentaje de transformación inicial de 31 ± 2,5%, que se expresó mediante formación de callo sobre medios de selección y una frecuencia final con base en el ensayo GUS de 30%. Este es el primer reporte de transformación de un híbrido interespecífico de tres especies diferentes.

  19. Cloning and heterologous expression of a hydrophobin gene Ltr.hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis

    Directory of Open Access Journals (Sweden)

    Dongmei Liu

    2018-03-01

    Full Text Available Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier. Keywords: Agrobacterium tumefaciens, Emulsifier, Expression vector, Filamentous fungi, Gel electrophoresis, Glyceraldehyde-3-phosphate dehydrogenase, Heterogenous expression, Hydrophobin, Quantitative real-time PCR, Southern blot, Surface activity

  20. CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines.

    Science.gov (United States)

    Yu, Qing-Hui; Wang, Baike; Li, Ning; Tang, Yaping; Yang, Shengbao; Yang, Tao; Xu, Juan; Guo, Chunmiao; Yan, Peng; Wang, Qiang; Asmutola, Patiguli

    2017-09-19

    Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T 0 transgenic plants respectively. None of homozygous mutation was detected in T 0 transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T 1 generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T 1 generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.

  1. Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease.

    Science.gov (United States)

    Ban, Huifang; Chai, Xinli; Lin, Yongjun; Zhou, Ying; Peng, Donghai; Zhou, Yi; Zou, Yulan; Yu, Ziniu; Sun, Ming

    2009-12-01

    Amorphophallus konjac is an important economic crop widely used in health products and biomaterials. However, this monocotyledonous plant's production is seriously restricted by soft rot disease. Some Bacillus thuringiensis strains generate an endocellular acyl homoserine lactonase (AiiA), which has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain transgenic A. konjac expressing AiiA protein and exhibiting resistance to soft rot. But till now, there is not any report about exogenous gene transformation in A. konjac. In this research, an Agrobacterium-mediated genetic transformation system was constructed. An aiiA gene was synthesized according to the codon usage in A. konjac. Embryogenic callus was infected with the A. tumefaciens strain EHA105 harboring the plant transformation plasmid pU1301 plus synthesized aiiA gene. After antibiotics screening, 34 plants were obtained. PCR analysis showed that positive amplified fragments were present in 21 out of these 34 lines. Southern blot analysis indicated that aiiA gene had integrated into the genome of A. konjac. Western blotting demonstrated that the target protein of interest was reactive with the antibody against AiiA. Further disease resistance detection revealed that all of the tested transgenic A. konjac lines exhibited high resistance to soft rot bacteria Erwinia carotovora subsp. Carotovora (Ecc) SCG1. The protocol is useful for the quality improvement of A. konjac through genetic transformation.

  2. Trichoderma genes

    Science.gov (United States)

    Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  3. pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants.

    Directory of Open Access Journals (Sweden)

    Dipak Kumar Sahoo

    Full Text Available We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71 was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24 of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3'rbcsE9 terminator, replication functions for Escherichia coli (ColE1 and Agrobacterium tumefaciens (pRK2-OriV and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII and ampicillin resistance (bla. The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP and β-glucuronidase (GUS both transiently (agro-infiltration, protoplast electroporation and biolistic and stably in plant systems (Arabidopsis and tobacco using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses.

  4. Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced abiotic stress tolerance.

    Directory of Open Access Journals (Sweden)

    Zhi Wang

    Full Text Available Alfalfa (Medicago sativa L., a perennial forage crop with high nutritional content, is widely distributed in various environments worldwide. We recently demonstrated that the sweetpotato Orange gene (IbOr is involved in increasing carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In this study, in an effort to improve the nutritional quality and environmental stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv. Xinjiang Daye under the control of an oxidative stress-inducible peroxidase (SWPA2 promoter through Agrobacterium tumefaciens-mediated transformation. Among the 11 transgenic alfalfa lines (referred to as SOR plants, three lines (SOR2, SOR3, and SOR8 selected based on their IbOr transcript levels were examined for their tolerance to methyl viologen (MV-induced oxidative stress in a leaf disc assay. The SOR plants exhibited less damage in response to MV-mediated oxidative stress and salt stress than non-transgenic plants. The SOR plants also exhibited enhanced tolerance to drought stress, along with higher total carotenoid levels. The results suggest that SOR alfalfa plants would be useful as forage crops with improved nutritional value and increased tolerance to multiple abiotic stresses, which would enhance the development of sustainable agriculture on marginal lands.

  5. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    Directory of Open Access Journals (Sweden)

    Liping Ke

    Full Text Available Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel. In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L and bentazon (4.2 µmol. A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

  6. Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    Directory of Open Access Journals (Sweden)

    Tengale Dipak Bhauso

    2014-01-01

    Full Text Available In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L. crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

  7. Transgenic Alfalfa Plants Expressing the Sweetpotato Orange Gene Exhibit Enhanced Abiotic Stress Tolerance

    Science.gov (United States)

    Wang, Zhi; Ke, Qingbo; Kim, Myoung Duck; Kim, Sun Ha; Ji, Chang Yoon; Jeong, Jae Cheol; Lee, Haeng-Soon; Park, Woo Sung; Ahn, Mi-Jeong; Li, Hongbing; Xu, Bingcheng; Deng, Xiping; Lee, Sang-Hoon; Lim, Yong Pyo; Kwak, Sang-Soo

    2015-01-01

    Alfalfa (Medicago sativa L.), a perennial forage crop with high nutritional content, is widely distributed in various environments worldwide. We recently demonstrated that the sweetpotato Orange gene (IbOr) is involved in increasing carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In this study, in an effort to improve the nutritional quality and environmental stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv. Xinjiang Daye) under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter through Agrobacterium tumefaciens-mediated transformation. Among the 11 transgenic alfalfa lines (referred to as SOR plants), three lines (SOR2, SOR3, and SOR8) selected based on their IbOr transcript levels were examined for their tolerance to methyl viologen (MV)-induced oxidative stress in a leaf disc assay. The SOR plants exhibited less damage in response to MV-mediated oxidative stress and salt stress than non-transgenic plants. The SOR plants also exhibited enhanced tolerance to drought stress, along with higher total carotenoid levels. The results suggest that SOR alfalfa plants would be useful as forage crops with improved nutritional value and increased tolerance to multiple abiotic stresses, which would enhance the development of sustainable agriculture on marginal lands. PMID:25946429

  8. Enhanced resistance to early blight in transgenic tomato lines expressing heterologous plant defense genes.

    Science.gov (United States)

    Schaefer, Scott C; Gasic, Ksenija; Cammue, Bruno; Broekaert, Willem; van Damme, Els J M; Peumans, Willy J; Korban, Schuyler S

    2005-11-01

    Genes coding for an iris ribosomal-inactivating protein (I-RIP), a maize beta-glucanase (M-GLU), and a Mirabilis jalapa antimicrobial peptide (Mj-AMP1) were separately introduced into tomato (Lycopersicon esculentum cv. Sweet Chelsea) cotyledons via Agrobacterium tumefaciens-mediated transformation. Transgenic lines carrying each of the transgenes were confirmed for integration into the tomato genome using Southern blot hybridization. Transcription of I-RIP, M-GLU, and Mj-AMP1 genes in various transgenic lines was determined using Northern blot analysis. Plants of selected transgenic lines were inoculated with a 2-3x10(4) conidial spores/ml suspension of the fungal pathogen Alternaria solani, the causal agent of tomato early blight. Compared to control (non-transformed) plants, two transgenic lines carrying either a M-GLU or Mj-AMP1 showed enhanced resistance to early blight disease. None of the four lines carrying the I-RIP transgene showed increased resistance to early blight.

  9. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum Plantas transgênicas de tomate expressando o gene do antígeno PfCP-2.9 de Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mihail Kantor

    2013-01-01

    Full Text Available The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9 in transgenic tomato plants from the T0 and T1 generations.O objetivo deste trabalho foi obter plantas transgênicas de tomate que expressem a proteína PfCP-2.9 (uma quimera dos antígenos MSP1 e AMA1 de Plasmodium falciparum. Cotilédones de tomate, cultivar Summers, com sete dias de idade, foram transformados via Agrobacterium tumefaciens. A expressão transgênica nas plantas T0 foi verificada no DNA extraído dos frutos. A análise por PCR foi utilizada para testar a presença do gene de interesse na geração T1. A evidência da expressão do gene no RNA foi constatada por meio da PCR de transcriptase reversa, e a análise "Western blot" confirmou a presença da proteína de interesse nas plantas T1. Este é o primeiro relato de transformação bem sucedida com a expressão de um antígeno da malária (PfCP-2,9 em plantas transgênicas de tomate da geração T0 e T1.

  10. A fungal transcription factor gene is expressed in plants from its own promoter and improves drought tolerance.

    Science.gov (United States)

    Martínez, Félix; Arif, Anjuman; Nebauer, Sergio G; Bueso, Eduardo; Ali, Rashid; Montesinos, Consuelo; Brunaud, Veronique; Muñoz-Bertomeu, Jesús; Serrano, Ramón

    2015-07-01

    A fungal gene encoding a transcription factor is expressed from its own promoter in Arabidopsis phloem and improves drought tolerance by reducing transpiration and increasing osmotic potential. Horizontal gene transfer from unrelated organisms has occurred in the course of plant evolution, suggesting that some foreign genes may be useful to plants. The CtHSR1 gene, previously isolated from the halophytic yeast Candida tropicalis, encodes a heat-shock transcription factor-related protein. CtHSR1, with expression driven by its own promoter or by the Arabidopsis UBQ10 promoter, was introduced into the model plant Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation and the resulting transgenic plants were more tolerant to drought than controls. Fusions of the CtHSR1 promoter with β-glucuronidase reporter gene indicated that this fungal promoter drives expression to phloem tissues. A chimera of CtHSR1 and green fluorescence protein is localized at the cell nucleus. The physiological mechanism of drought tolerance in transgenic plants is based on reduced transpiration (which correlates with decreased opening of stomata and increased levels of jasmonic acid) and increased osmotic potential (which correlates with increased proline accumulation). Transcriptomic analysis indicates that the CtHSR1 transgenic plants overexpressed a hundred of genes, including many relevant to stress defense such as LOX4 (involved in jasmonic acid synthesis) and P5CS1 (involved in proline biosynthesis). The promoters of the induced genes were enriched in upstream activating sequences for water stress induction. These results demonstrate that genes from unrelated organisms can have functional expression in plants from its own promoter and expand the possibilities of useful transgenes for plant biotechnology.

  11. Immunoglobulin genes

    National Research Council Canada - National Science Library

    Honjo, T; Alt, F. W; Rabbitts, T. H

    1989-01-01

    ... Cataloguing in Publication Data Immunoglobulin genes 1. Vertebrates. Immunoglobulins 1. Honjo, T. II. Alt, F.W. III. Rabbitts, T.H. 612'. 118223 ISBN 0-12-354865-9 This book is printed on acid-free paper ( T...

  12. Ageing genes

    DEFF Research Database (Denmark)

    Rattan, Suresh

    2018-01-01

    The idea of gerontogenes is in line with the evolutionary explanation of ageing as being an emergent phenomenon as a result of the imperfect maintenance and repair systems. Although evolutionary processes did not select for any specific ageing genes that restrict and determine the lifespan...... of an individual, the term ‘gerontogenes’ primarily refers to any genes that may seem to influence ageing and longevity, without being specifically selected for that role. Such genes can also be called ‘virtual gerontogenes’ by virtue of their indirect influence on the rate and process of ageing. More than 1000...... virtual gerontogenes have been associated with ageing and longevity in model organisms and humans. The ‘real’ genes, which do influence the essential lifespan of a species, and have been selected for in accordance with the evolutionary life history of the species, are known as the longevity assurance...

  13. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

    Science.gov (United States)

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level o