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Sample records for agrobacterium tumefaciens strains

  1. (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, ...

  2. Regulation of hydantoin-hydrolyzing enzyme expression in Agrobacterium tumefaciens strain RU-AE01.

    Science.gov (United States)

    Jiwaji, Meesbah; Dorrington, Rosemary Ann

    2009-10-01

    Optically pure D-: amino acids, like D-: hydroxyphenylglycine, are used in the semi-synthetic production of pharmaceuticals. They are synthesized industrially via the biocatalytic hydrolysis of p-hydroxyphenylhydantoin using enzymes derived from Agrobacterium tumefaciens strains. The reaction proceeds via a three-step pathway: (a) the ring-opening cleavage of the hydantoin ring by a D-: hydantoinase (encoded by hyuH), (b) conversion of the resultant D-: N-carbamylamino acid to the corresponding amino acid by a D-: N-carbamoylase (encoded by hyuC), and (c) chemical or enzymatic racemization of the un-reacted hydantoin substrate. While the structure and biochemical properties of these enzymes are well understood, little is known about their origin, their function, and their regulation in the native host. We investigated the mechanisms involved in the regulation of expression of the hydantoinase and N-carbamoylase enzyme activity in A. tumefaciens strain RU-AE01. We present evidence for a complex regulatory network that responds to the growth status of the cells, the presence of inducer, and nitrogen catabolite repression. Deletion analysis and site-directed mutagenesis were used to identify regulatory elements involved in transcriptional regulation of hyuH and hyuC expression. Finally, a comparison between the hyu gene clusters in several Agrobacterium strains provides insight into the function of D-: selective hydantoin-hydrolyzing enzyme systems in Agrobacterium species.

  3. 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 ...

  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. 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...... technical advances has been made within the initial steps of the process, more specifically the efficient construction of plasmids for performing targeted genome modifications. This chapter provides a generic protocol for performing genetic transformation of ascomycetes via A. tumefaciens......-mediated transformation (AMT) and guidelines for optimizing the AMT process with new fungal species. The chapter also includes a highly efficient vector construction system based on Uracil Specific Excisions Reagent (USER) cloning and specific PCR generated building blocks, which can be combined ad hoc to create complex...

  6. 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.

  7. 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)

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Agrobacterium tumefaciens is a diazotrophic bacterium

    Energy Technology Data Exchange (ETDEWEB)

    Kanvinde, L.; Sastry, G.R.K. (Univ. of Leeds (England))

    1990-07-01

    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 {sup 15}N supplied as {sup 15}N{sub 2}. As with most other well-characterized diazotrophic bacteria, the presence of NH{sub 4}{sup +} 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.

  13. 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°

  14. 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...

  15. 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.

  16. 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

  17. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Yukoh eHiei

    2014-11-01

    Full Text Available 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, which are the basis of tissue culture and transformation in dicotyledons, 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 determined 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.

  18. 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

  19. 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

  20. Transient GUS gene expression in cassava (Manihot esculenta Crantz using Agrobacterium tumefaciens leaf infiltration

    Directory of Open Access Journals (Sweden)

    Paula Díaz T.

    2014-09-01

    Full Text Available Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS. A. tumefaciens infiltration (agroinfiltration was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan and CM6438-14 (Vergara, respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

  1. 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)

  2. 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

  3. 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.

  4. 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

  5. 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...

  6. 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...

  7. 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).

  8. 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.

  9. 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

  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. ANALYSIS OF INTERACTION OF PLANT GENOTYPE AND STRAIN AGROBACTERIUM TUMEFACIENS IN BREEDING OF POTATO RESISTANCE TO COLORADO POTATO BEETLE

    Directory of Open Access Journals (Sweden)

    Denis I Bogomaz

    2005-03-01

    Full Text Available Efficiency of potato transformation depends on plant genotype and bacterial strain. Genotypes with high regeneration ability have high transformation ability. It is shown, that transgenosis of Bt gene increases potato resistance to collorado potato beetle, transgenosis of ipt gene does not influence on resistance.

  12. 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.

  13. Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

    Science.gov (United States)

    Matthysse, A G; Wyman, P M; Holmes, K V

    1978-11-01

    Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

  14. Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

    Science.gov (United States)

    Song, Guo-Qing; Sink, K C

    2004-12-01

    Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

  15. 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.

  16. Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens.

    Science.gov (United States)

    Michelmore, R; Marsh, E; Seely, S; Landry, B

    1987-12-01

    Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.

  17. 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.

  18. Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.

    Science.gov (United States)

    Mayo, Kristin J; Gonzales, Barbara J; Mason, Hugh S

    2006-01-01

    This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.

  19. Production of herbicide-resistant coffee plants (Coffea canephora P.) via Agrobacterium tumefaciens-mediated transformation

    OpenAIRE

    Ribas, Alessandra Ferreira; Kobayashi, Adilson Kenji; Pereira, Luiz Filipe Protasio; Vieira, Luiz Gonzaga Esteves

    2006-01-01

    Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained ¼ strength macro salts and half strength micro salts of MS medium, organic constituents of B5 medium and 30 g.L-1 sucrose supp...

  20. 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...

  1. 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

  2. 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...

  3. 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

  4. 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

  5. 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)

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletotrichum sansevieriae.

    Science.gov (United States)

    Nakamura, Masayuki; Kuwahara, Hideto; Onoyama, Keisuke; Iwai, Hisashi

    2012-08-01

    Agrobacterium tumefaciens-mediated transformation (AtMT) has become a common technique for DNA transformation of yeast and filamentous fungi. In this study, we first established a protocol of AtMT for the phytopathogenic fungus Colletotrichum sansevieriae. Binary T-DNA vector containing the hygromycin B phosphotransferase gene controlled by the Aspergillus nidulans gpdA promoter and the trpC terminator was constructed with pCAMBIA0380 and used with three different strains LBA4404, GV3101, and GV2260 of A. tumefaciens. Transformants were most effectively obtained when GV2260 and C. sansevieriae Sa-1-2 were co-cultivated; there were about 320 transformants per 10(6) spores. When 1,048 transformants were inoculated on Sansevieria trifasciata, three transformants were found to have completely lost their pathogenicity and two transformants displayed reduced pathogenicity. All of the five transformants had a single copy of T-DNA in their genomes. The three pathogenicity-deficient transformants were subjected to thermal asymmetric interlaced polymerase chain reaction and the reaction allowed us to amplify the sequences flanking the left and/or right borders. The flanking sequences of the two transformants, M154 and M875, showed no homology to any sequences in databases, but the sequences of M678 contained motifs of alpha-1,3-glucan synthase, suggesting that the gene might contribute to the pathogenicity of C. sansevieriae. This study describes a useful method for investigating pathogenicity genes in C. sansevieriae.

  15. 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

  16. 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

  17. 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,...

  18. 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

  19. Agrobacterium tumefaciens mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

    NARCIS (Netherlands)

    Zheng, S.J.; Khrustaleva, L.I.; Henken, G.; Sofiari, E.; Jacobsen, E.; Kik, C.; Krens, F.A.

    2001-01-01

    This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development

  20. 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

  1. 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.

  2. 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 ...

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

    KAUST Repository

    Kumar, Nitish; Vijay Anand, K.G.; Pamidimarri, D.V.N. Sudheer; Sarkar, Tanmoy; Reddy, Muppala P.; Radhakrishnan, T.; Kaul, Tanushri; Reddy, M.K.; Sopori, Sudhir K.

    2010-01-01

    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

  4. 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

  5. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

    OpenAIRE

    Metts, J; West, J; Doares, S H; Matthysse, A G

    1991-01-01

    Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was d...

  6. 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.

  7. Production of herbicide-resistant coffee plants (Coffea canephora P. via Agrobacterium tumefaciens-mediated transformation

    Directory of Open Access Journals (Sweden)

    Alessandra Ferreira Ribas

    2006-01-01

    Full Text Available Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained ¼ strength macro salts and half strength micro salts of MS medium, organic constituents of B5 medium and 30 g.L-1 sucrose supplemented with 5µM N6 - (2-isopentenyl-adenine (2-iP. Ten µM ammonium glufosinate was used for putative transgenic somatic embryos selection. Presence and integration of the bar gene were confirmed by PCR and Southern blot analysis. Selected transgenic coffee plants sprayed with up to 1600 mg.L-1 of FinaleTM, a herbicide containing glufosinate as the active ingredient, retained their pigmentation and continued to grow normally during ex vitro acclimation.Plantas transgênicas de Coffea canephora P resistentes ao herbicida glufosinato de amônio foram regeneradas a partir de explantes foliares co-cultivados com Agrobacterium tumefaciens EHA105 contendo o plasmídio pCambia3301 que contém os genes bar e uidA ambos sob controle do promotor 35S. Embriogênese somática direta foi induzida no meio contendo ¼ da concentração de macro, metade da concentração de micronutrientes do meio MS, constituintes orgânicos do meio B5 e 30 g.L-1 de sacarose suplementado com 5µM N6 - (2-isopentenil-adenina (2-iP e 10 µM de glufosinato de amônio para seleção de embriões transgênicos putativos. A presença e a integração do gene bar foram confirmados pelas análises de PCR e Southern blot. As plantas transgênicas selecionadas de café, pulverizadas com 1600 mg.L-1 do herbicida FinaleTM que contém glufosinato como ingrediente ativo, mantiveram a coloração e continuaram crescendo normalmente na aclimatação ex vitro.

  8. Behavior of IncQ Plasmids in Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Schilperoort, Rob

    1981-01-01

    Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

  9. Agrobacterium tumefaciens-mediated transformation of biofuel plant ...

    African Journals Online (AJOL)

    Establishment of an efficient transformation system is a prerequisite for genetic improvement of Jatropha curcas, a promising biodiesel feedstock plant, by transgenic approach. In this study an efficient Agrobacterium-mediated transformation protocol using cotyledon explants from J. curcas seeds was developed.

  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. 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

  12. Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens.

    Science.gov (United States)

    Raharjo, S H; Hernandez, M O; Zhang, Y Y; Punja, Z K

    1996-04-01

    Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

  13. Convergent evolution of Amadori opine catabolic systems in plasmids of Agrobacterium tumefaciens.

    Science.gov (United States)

    Baek, Chang-Ho; Farrand, Stephen K; Lee, Ko-Eun; Park, Dae-Kyun; Lee, Jeong Kug; Kim, Kun-Soo

    2003-01-01

    Deoxyfructosyl glutamine (DFG, referred to elsewhere as dfg) is a naturally occurring Amadori compound found in rotting fruits and vegetables. DFG also is an opine and is found in tumors induced by chrysopine-type strains of Agrobacterium tumefaciens. Such strains catabolize this opine via a pathway coded for by their plasmids. NT1, a derivative of the nopaline-type A. tumefaciens strain C58 lacking pTiC58, can utilize DFG as the sole carbon source. Genes for utilization of DFG were mapped to the 543-kb accessory plasmid pAtC58. Two cosmid clones of pAtC58 allowed UIA5, a plasmid-free derivative of C58, harboring pSa-C that expresses MocC (mannopine [MOP] oxidoreductase that oxidizes MOP to DFG), to grow by using MOP as the sole carbon source. Genetic analysis of subclones indicated that the genes for utilization of DFG are located in a 6.2-kb BglII (Bg2) region adjacent to repABC-type genes probably responsible for the replication of pAtC58. This region contains five open reading frames organized into at least two transcriptional soc (santhopine catabolism) groups: socR and socABCD. Nucleotide sequence analysis and analyses of transposon-insertion mutations in the region showed that SocR negatively regulates the expression of socR itself and socABCD. SocA and SocB are responsible for transport of DFG and MOP. SocA is a homolog of known periplasmic amino acid binding proteins. The N-terminal half of SocB is a homolog of the transmembrane transporter proteins for several amino acids, and the C-terminal half is a homolog of the transporter-associated ATP-binding proteins. SocC and SocD could be responsible for the enzymatic degradation of DFG, being homologs of sugar oxidoreductases and an amadoriase from Corynebacterium sp., respectively. The protein products of socABCD are not related at the amino acid sequence level to those of the moc and mot genes of Ti plasmids responsible for utilization of DFG and MOP, indicating that these two sets of genes and their

  14. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

    Science.gov (United States)

    Metts, J; West, J; Doares, S H; Matthysse, A G

    1991-02-01

    Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

  15. Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)].

    Science.gov (United States)

    Karthik, Sivabalan; Pavan, Gadamchetty; Sathish, Selvam; Siva, Ramamoorthy; Kumar, Periyasamy Suresh; Manickavasagam, Markandan

    2018-04-01

    Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301- bar was used for transformation. The two-stage selection was carried out using 4 and 250 mg l -1 BASTA ® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.

  16. Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

    Science.gov (United States)

    Norzagaray-Valenzuela, Claudia D; Germán-Báez, Lourdes J; Valdez-Flores, Marco A; Hernández-Verdugo, Sergio; Shelton, Luke M; Valdez-Ortiz, Angel

    2018-05-16

    Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD 600  = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

    Science.gov (United States)

    González-Mula, Almudena; Lang, Julien; Grandclément, Catherine; Naquin, Delphine; Ahmar, Mohammed; Soulère, Laurent; Queneau, Yves; Dessaux, Yves; Faure, Denis

    2018-07-01

    Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  18. 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......-regulation of gene expression. This review summarizes the technical advances within the field from 1998 to the summer of 2011, focusing on the development of binary vectors that are compatible with fungal transformation (over 180 general vectors) and methods for constructing binary vectors for targeted integration...

  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. Optimization of Agrobacterium tumefaciens-Mediated Transformation Systems in Tea Plant (Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Qianru LV

    2017-05-01

    Full Text Available In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C. sinensis were used as the receptors for transformation by Agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 µmol⋅L−1 acetosyringone (AS. The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg⋅L−1 spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.

  1. 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.

  2. Attachment study of Agrobacterium tumefaciens to yam spp

    African Journals Online (AJOL)

    Charles S.Wortmann

    ISOLATES FROM CLINICAL AND ENVIRONMENTAL SOURCES OF ... The antimicrobial susceptibility test result shows that all E. coli,. Klebsiella, and ... indicated the prevalence of multi-drug resistant bacterial strains from in- patients and ..... flora: longitudinal community based surveillance of children from urban. Mexico.

  3. Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s).

    Science.gov (United States)

    Hibbing, Michael E; Fuqua, Clay

    2012-06-01

    Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

  4. 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

  5. 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

    -step cloning strategies for construction of vectors for Agrobacterium tumefaciens-mediated transformation (ATMT). Targeted genome modifications require integration by a homologous double crossover event, which is achieved by placing target sequences on either side of a selection marker gene in the vector....... Protocols are given for two single-step vector construction techniques. The In-Fusion cloning technique is independent of compatible restriction enzyme sites in the vector and the fragment to be cloned. The method can be directly applied to any vector of choice and it is possible to carry out four fragment...... 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    Abbas El Sahili

    2015-08-01

    Full Text Available 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

  10. 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

  11. 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

  12. 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 )

  13. INTRODUKSI GEN METALLOTHIONEIN TIPE II KE DALAM RUMPUT LAUT Kappaphycus alvarezii MENGGUNAKAN Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Ulia Fajriah

    2014-12-01

    Full Text Available Kappaphycus alvarezii adalah jenis alga merah yang memproduksi kappa karagenan yang sangat penting untuk industri makanan, farmasi, dan kosmetik. Untuk meningkatkan produksi, diperlukan ketersediaan bahan baku yang baik. Salah satu yang memengaruhi ketersediaan bahan baku adalah kondisi ingkungan perairan untuk budidaya. Metallothionein (MT adalah protein yang memiliki kemampuan untuk mengikat ion logam seperti Cd, Zn, dan Cu. Tujuan penelitian ini adalah untuk mengintroduksi gen Metallothionein Tipe II (MaMt2 ke dalam genom K. alvarezii menggunakan Agrobacterium tumefaciens. Talus rumput laut diinokulasi dengan A. tumefaciens mengandung plasmid pIG6-SMt2 yang membawa gen MaMt2, selanjutnya dilakukan seleksi bertingkat menggunakan higromisin 10 mg/L dan 20 mg/L. Hasil efisiensi transformasi yang diperoleh adalah 27,4%, efisiensi regenerasi tunas transgenik adalah 27,6%. Analisis molekuler dengan PCR menunjukkan bahwa 13 tunas transgenik mengandung gen MaMt2. Tunas transgenik putatif ditumbuhkan hingga menjadi talus baru dan dapat dilakukan uji tantang pada penelitian selanjutnya.

  14. 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.

  15. 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...

  16. 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.

  17. BINARY VECTOR CONSTRUCTION OF KAPPA(κ-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

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    Muh Alias L. Rajamuddin

    2016-11-01

    Full Text Available Increasing of kappa (κ-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of  κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes.  The κ-Carrageenase (κ-Car gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced.                                                                                   Keywords:  Agrobacterium tumefaciens, kappa(κ-Carrageenase gene, transgenesis, vector

  18. 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.

  19. 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.

  20. 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.

  1. 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.

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

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    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.

  3. 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.

  4. Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

    International Nuclear Information System (INIS)

    Burr, T.J.; Norelli, J.L.; Katz, B.H.; Bishop, A.L.

    1990-01-01

    Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32 P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10 6 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains

  5. 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.

  6. Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens.

    Science.gov (United States)

    Kim, K S; Baek, C H; Lee, J K; Yang, J M; Farrand, S K

    2001-06-01

    pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1. NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR. In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine. Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine. When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited. In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58. These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly. The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours. Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium. Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain. Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form. MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms. Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition. Genetic and physiological analyses

  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. 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

  9. 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...

  10. Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens.

    Science.gov (United States)

    Skopelitou, Katholiki; Muleta, Abdi W; Pavli, Ourania; Skaracis, Georgios N; Flemetakis, Emmanouil; Papageorgiou, Anastassios C; Labrou, Nikolaos E

    2012-03-01

    In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

  11. 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

  12. Sonication assisted Agrobacterium -mediated transformation of ...

    African Journals Online (AJOL)

    In this study, a protocol was developed to obtain stable lines of the Spring Dendrobium cultivar 'Sanya' via sonication assisted Agrobacterium-mediated transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector AG205 containing the chalcone ...

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Efficacy of the Non-Pathogenic Agrobacterium Strains K84 and K1026 against Crown Gall in Tunisia

    Directory of Open Access Journals (Sweden)

    A. Rhouma

    2004-08-01

    Full Text Available The non-pathogenic Agrobacterium radiobacter strain K84 and its genetically modified (GEM strain K1026 were tested for their effectiveness against local Tunisian strains and two reference strains (C58 and B6 of the crown gall bacterium Agrobacterium tumefaciens. Tests in planta were carried out on herbaceous plants (tomato and tagetes and on some sensitive rootstocks (bitter almond, peach almond hybrid GF677 and quince BA29. In vitro tests showed that both K84 and K1026 were effective and that the difference between these strains was not statistically significant. On tomato and tagetes, strain K84 was effective against all crown gall isolates with the exception of the A. tumefaciens reference strain B6. GEM strain K1026 was very effective against all isolates from Tunisia and against the reference strains. Both antagonistic strains significantly reduced the percentage of galled plants as well as the number of galls per plant. Under field conditions, both antagonists controlled crown gall effectively. Best results were obtained on the bitter almond-tree rootstock. Antagonist effectiveness was less evident on quince BA29 and peach almond GF677 rootstocks. The genetically modified strain K1026 is of interest in controlling crown gall disease in Tunisia.

  18. 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

  19. 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.

  20. The BlcC (AttM) lactonase of Agrobacterium tumefaciens does not quench the quorum-sensing system that regulates Ti plasmid conjugative transfer.

    Science.gov (United States)

    Khan, Sharik R; Farrand, Stephen K

    2009-02-01

    The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

  1. Complementation of a threonine dehydratase-deficient Nicotiana plumbaginifolia mutant after Agrobacterium tumefaciens-mediated transfer of the Saccharomyces cerevisiae ILV1 gene.

    OpenAIRE

    Colau, D; Negrutiu, I; Van Montagu, M; Hernalsteens, J P

    1987-01-01

    The Saccharomyces cerevisiae ILV1 gene, encoding threonine dehydratase (EC 4.2.1.16) was fused to the transferred DNA nopaline synthase promoter and the 3' noncoding region of the octopine synthase gene. It was introduced, by Agrobacterium tumefaciens-mediated gene transfer, into an isoleucine-requiring Nicotiana plumbaginifolia auxotroph deficient in threonine dehydratase. Functional complementation by the ILV1 gene product was demonstrated by the selection of several transformed lines on a ...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Agrobacterium-mediated transformation of in vitro cultured barley ovules is an attractive alternative to well-established barley transformation methods of immature embryos. The ovule culture system can be used for transformation with and without selection and has successfully been used to transfo...

  3. Role of calcium-depositing bacteria Agrobacterium tumefaciens and its influence on corrosion of different engineering metals used in cooling water system.

    Science.gov (United States)

    Narenkumar, Jayaraman; Sathishkumar, Kuppusamy; Selvi, Adikesavan; Gobinath, Rajagopalan; Murugan, Kadarkarai; Rajasekar, Aruliah

    2017-12-01

    The present investigation deals with the role of calcium-depositing bacterial community on corrosion of various engineering metals, namely, brass alloy (BS), copper (Cu), stainless steel (SS) and mild steel (MS). Based on the corrosion behavior, Agrobacterium tumefaciens EN13, an aerobic bacterium is identified as calcium-depositing bacteria on engineering metals. The results of the study are supported with biochemical characterization, 16S rRNA gene sequencing, calcium quantification, weight loss, electrochemical (impedance and polarization) and surface analysis (XRD and FTIR) studies. The calcium quantification study showed carbonate precipitation in abiotic system/biotic system as 50 and 700 ppm, respectively. FTIR results too confirmed the accumulation of calcium deposits from the environment on the metal surface by EN13. Electrochemical studies too supported the corrosion mechanism by showing a significant increase in the charge transfer resistance ( R ct ) of abiotic system (44, 33.6, 45, 29.6 Ω cm 2 ) than compared to biotic system (41, 10.1 29 and 25 Ω cm 2 ). Hence, the outcome of the present study confirmed the enhanced bioaccumulation behavior of calcium by the strain, EN13.

  4. 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.

  5. Draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659

    Science.gov (United States)

    This work reports the draft genome of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, and is composed of 1 circular chromosome, the Ri virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild type strain cau...

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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)

  11. 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.

  12. Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.

    Science.gov (United States)

    Vinoth, S; Gurusaravanan, P; Jayabalan, N

    2013-02-01

    A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

  13. Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens.

    Science.gov (United States)

    Manfroi, Ernandes; Yamazaki-Lau, Elene; Grando, Magali F; Roesler, Eduardo A

    2015-12-01

    Low 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. tumefaciens overgrowth 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.

  14. 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.

  15. 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

  16. Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White)

    Science.gov (United States)

    Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-gl...

  17. 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.

  18. 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

  19. [Agrobacterium rubi strains from blueberry plants are highly diverse].

    Science.gov (United States)

    Abrahamovich, Eliana; López, Ana C; Alippi, Adriana M

    2014-01-01

    The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  20. Regeneration and Agrobacterium -mediated transformation studies ...

    African Journals Online (AJOL)

    Leaf explants of carnation (Dianthus caryophyllus L. cv. Turbo) were used for the transformation of gene performed by the EHA 105 strain of Agrobacterium tumefaciens harboring the binary vector, pGA482GG. This vector carries the marker genes, neomycin phosphotansferase II (npt II) that determine resistance to ...

  1. Agrobacterium-mediated transformation of watermelon ( Citrullus ...

    African Journals Online (AJOL)

    Transformation of watermelon (Citrullus lanatus cv. Zaojia) using Agrobacterium tumefaciens strain EHA105 containing the plasmid pRD400 carrying Pti4 gene was studied in this work. Proximal cotyledons as explants were pre-cultivated for two day in the dark and it was found that the best condition for transformation of ...

  2. Improvement of Agrobacterium-mediated transformation and rooting of black cherry

    Science.gov (United States)

    Ying Wang; Paula M. Pijut

    2014-01-01

    An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105...

  3. Agrobacterium-medicated transformation of mature Prunus serotina (black cherry) and regeneration of trangenic shoots

    Science.gov (United States)

    Xiaomei Liu; Paula Pijut

    2010-01-01

    A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced...

  4. 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

  5. Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution.

    Science.gov (United States)

    Kosloff, Mickey; Han, Gye Won; Krishna, S Sri; Schwarzenbacher, Robert; Fasnacht, Marc; Elsliger, Marc-André; Abdubek, Polat; Agarwalla, Sanjay; Ambing, Eileen; Astakhova, Tamara; Axelrod, Herbert L; Canaves, Jaume M; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; DiDonato, Michael; Duan, Lian; Feuerhelm, Julie; Grittini, Carina; Grzechnik, Slawomir K; Hale, Joanna; Hampton, Eric; Haugen, Justin; Jaroszewski, Lukasz; Jin, Kevin K; Johnson, Hope; Klock, Heath E; Knuth, Mark W; Koesema, Eric; Kreusch, Andreas; Kuhn, Peter; Levin, Inna; McMullan, Daniel; Miller, Mitchell D; Morse, Andrew T; Moy, Kin; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Page, Rebecca; Paulsen, Jessica; Quijano, Kevin; Reyes, Ron; Rife, Christopher L; Sims, Eric; Spraggon, Glen; Sridhar, Vandana; Stevens, Raymond C; van den Bedem, Henry; Velasquez, Jeff; White, Aprilfawn; Wolf, Guenter; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2006-11-15

    Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies. (c) 2006 Wiley-Liss, Inc.

  6. Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones.

    Science.gov (United States)

    De Block, M

    1990-07-01

    Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25 degrees C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO(3) (-)/NH(4) (+) ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba x P. tremula and of the poplar hybrid Populus trichocarpa x P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not.

  7. 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.

  8. 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...

  9. Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

    Science.gov (United States)

    Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

    2010-08-09

    Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system

  10. Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants

    Directory of Open Access Journals (Sweden)

    Dan Yinghui

    2010-08-01

    Full Text Available Abstract Background Impatiens (Impatiens walleriana is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. Results In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892 bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. Conclusion We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained

  11. 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.

  12. Effective Agrobacterium –mediated transformation of pineapple with ...

    African Journals Online (AJOL)

    cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant expression vector pUHA1 containing a human cytochrome P4501A1 (CYP1A1) gene for 3 days. Then, the infected calli were transferred to differentiation medium.

  13. 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.

  14. [Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

    Science.gov (United States)

    Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

    2006-05-01

    The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement.

  15. 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.

  16. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

    Science.gov (United States)

    Srinivasan, Ramachandran; Gothandam, Kodiveri Muthukalianan

    2016-01-01

    An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

  17. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

    Directory of Open Access Journals (Sweden)

    Ramachandran Srinivasan

    Full Text Available An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404. In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells followed by GV3101 (128 ± 5.29 cfu per 106 cells and EHA105 (61 ± 5.03 cfu per 106 cells. However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

  18. 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

  19. 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

  20. Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium

    NARCIS (Netherlands)

    Ooms, G.; Burrell, M.M.; Karp, A.; Bevan, M.; Hille, J.

    1987-01-01

    Derivatives of potato (Solanum tuberosum cv.'s 'Maris Bard' and 'Desiree') transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixed-cultures

  1. Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

    Science.gov (United States)

    Ningxia Du; Paula M. Pijut

    2009-01-01

    A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion...

  2. Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains.

    Science.gov (United States)

    Shah, Jasmine M; Ramakrishnan, Anantha Maharasi; Singh, Amit Kumar; Ramachandran, Subalakshmi; Unniyampurath, Unnikrishnan; Jayshankar, Ajitha; Balasundaram, Nithya; Dhanapal, Shanmuhapreya; Hyde, Geoff; Baskar, Ramamurthy

    2015-08-26

    Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies. For plants co-cultivated for 48 h with Agrobacterium, if the strain contained vir genes, then the rates of transversions, SHRs and FSMs (measured 2 weeks later) were lower than those of uninfected controls. In contrast, co-cultivation for 48 h with any of the Agrobacterium strains raised the transposition rates above control levels. The multiple time-point study showed that in seedlings co-cultivated with wild type Ach5, the reduced rates of transversions and SHRs after 48 h co-cultivation represent an apparent suppression of an earlier short-lived increase in mutation rates (peaking for plants co-cultivated for 3 h). An increase after 3 h co-cultivation was also seen for rates of transversions (but not SHR) in seedlings exposed to the strain lacking vir genes, oncogenes and T-DNA. However, the mutation rates in plants co-cultivated for longer times with this strain subsequently

  3. Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

    Science.gov (United States)

    Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min

    2015-07-01

    Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  4. 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

  5. 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...

  6. Draft genome sequence of Agrobacterium sp. strain R89-1, a morphine alkaloid-biotransforming bacterium

    Czech Academy of Sciences Publication Activity Database

    Zahradník, Jiří; Kyslíková, Eva; Kyslík, Pavel

    2016-01-01

    Roč. 4, č. 2 (2016), e00196-16 ISSN 2169-8287 Institutional support: RVO:61388971 Keywords : Agrobacterium sp. strain R89-1 * codeine/morphine * phylogenetic lineage Subject RIV: EE - Microbiology, Virology

  7. 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.

  8. Isolation and Characterization of Agrobacterium Strains from Soil: A Laboratory Capstone Experience

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    Kim R. Finer

    2016-12-01

    Full Text Available In this investigation, the students’ goal was to isolate and characterize Agrobacterium strains from soil. Following selection and enrichment on 1A-t medium, putative Agrobacterium isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA. Primer sets included AGRH to identify isolates that were members of the Rhizobiaceae, BIOVAR1 primers to identify members of Agrobacterium biovar group I, and a third set, VIRG, to determine presence of virG (only present in pathogenic Agrobacterium strains. During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of Agrobacterium–plant interactions (p ≤ 0.001 and molecular biology (p ≤ 0.01. The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores (p ≤ 0.0001 and attainment of capstone experience learning outcomes.

  9. Agrobacterium and Tumor Induction: A Model System.

    Science.gov (United States)

    Lennox, John E.

    1980-01-01

    The author offers laboratory procedures for experiments using the bacterium, Agrobacterium tumefaciens, which causes crown gall disease in a large number of plants. Three different approaches to growing a culture are given. (SA)

  10. Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor.

    Science.gov (United States)

    Kim, K S; Farrand, S K

    1996-06-01

    Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

  11. Optimisation de la transformation génétique de la pomme de terre par Agrobacterium tumefaciens. Utilisation de la résistance à l'hygromycine comme marqueur sélectif

    Directory of Open Access Journals (Sweden)

    Rouvière C.

    2003-01-01

    Full Text Available Optimization of potato genetic transformation by Agrobacterium tumefaciens using hygromycin resistance as selective marker. The objective of this work is the optimization of a genetic transformation and a regeneration protocol of transgenic plants in Solanum tuberosum cv Désirée using hygromycin resistance as selective marker. The gene Cat2 of Nicotiana plumbaginifolia and the gene SU2 of Gossypium hirsutum, both coding for catalases, have been used. Two antibiotic concentrations (5 and 10 mg . l -1 of culture medium combined with two preculture periods (5 and 20 days on non-selective medium were tested. Coculture medium was supplemented with 10 mg . l -1 of acetosyringone to test its effect on potato transformation. The addition of this phenolic compound to the coculture medium affected positively the regeneration aptitude of transgenic SU2 plants. Only the combination of 5 mg . l -1 of hygromycin and 20 days of preculture without antibiotic allowed the obtention of plants transformed with the gene SU2. Screening of the rooted shoots with PCR showed 45% of transgenic plants for both molecular constructions used.

  12. 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)

  13. Hairy Root Induction on Justicia gendarussa by Various Density of Agrobacterium rhizogenes strain LB 510

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    Dwi Kusuma Wahyuni

    2017-04-01

    Full Text Available Gandarusa (Justicia gendarussa Burm.f. is an Indonesian medicinal plant that has many benefits as drug and male contracetive. For industrial needs, Gandarusa must be  available in large quantity. Hairy root culture is one of methode to produce phytochemistry compound. The objective of the study was to examine the effect of various density of Agrobacterium rhizogenes strain LB510 on hairy roots induction of gandarusa (Justicia gendarussa Burm.f. leaf plant. Leaf explants were inoculated in MS liquid medium with various density of OD600 = 0.1; 0.2; 0.3; 0.4; and 0.5. Explants were co-cultivated for 2 days on MS solid medium without any hormone then sub-cultured on MS solid medium containing antibiotic cefotaxim 300 ppm, in dark condition. The data were analyzed descriptively and statistically. The results showed that various density of Agrobacterium rhizogenes strain LB510 was affected the lenght of hairy roots induction of J. gendarussa Burm.f., but these was not effected toward lenght formation time and number of hairy root. The treatment of OD600 0.2 was the best treatment for hairy root induction on Justicia gendarussa Burm. f. This data could be used for optimized the quality of methode of hairy root induction. 

  14. 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

  15. A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus.

    Science.gov (United States)

    Kimura, Mitsuhiro; Cutler, Sean; Isobe, Sachiko

    2015-01-01

    Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

  16. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Fuzhou [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Wang, Chao [Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore); National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610 (Singapore); Fu, Qinqin [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Zhang, Lian-hui [Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore); Gao, Yong-gui, E-mail: ygao@ntu.edu.sg [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore)

    2015-08-25

    The crystallization of the novel virulence factors SghA and SghR is reported. Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.

  17. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Ye, Fuzhou; Wang, Chao; Fu, Qinqin; Zhang, Lian-hui; Gao, Yong-gui

    2015-01-01

    The crystallization of the novel virulence factors SghA and SghR is reported. Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction

  18. Transformación de Agrobacterium tumefaciens CEPA LBA4404 con el vector binario pNOV022, selección in vitro y caracterización molecular

    Directory of Open Access Journals (Sweden)

    Janneth Fabiola Santos Rodríguez

    2002-01-01

    Full Text Available La caracterización molecular de la infección causada por Agrobacterium tumefaciensy el des-cubrimiento de que una parte del plásmido inductor de tumores (T-DNA es transferido algenoma de las plantas que infecta, han permitido utilizar esta bacteria como sistema detransformación vegetal. La utilización de esta tecnología ha permitido a los científicos resolverproblemas con relación a especies cultivables. En el presente trabajo se realizó la transfor-mación de las cepas LBA4404 de Agrobacterium tumefaciensy DH5a de Escherichia coli. Para tal finse utilizó la técnica de choque térmico incorporando el vector binario pNOV022, que contenía6868Resúmenes la construcción p35S-IP-t35S - pUBQ-PMI-tNos (promotor 35S del CaMV, inhibidor de protea-sas, terminador 35S del CaMV, promotor ubiquitina 3, fosfomanosa isomerasa y terminadorde la nopalinsintetasa. Igualmente se realizó selección de clones bacterianos transformados(basado en la resistencia a espectinomicina y/o estreptomicina y su caracterización molecularpor medio de PCR y un perfil de restricción. Este trabajo apuntó a producir un vector adecuadopara la transformación estable de papa criolla (Solanum phureja y de papa (Solanum tuberosum,un gen que confiere resistencia a insectos. Los resultados de las metodologías empleados evi-denciaron la introducción exitosa del plásmido recombinante en E. coliy A. tumefaciens. Si bienla eficiencia de transformación fue baja (0,032 x 10-8, los resultados de la PCR mostraron laamplificación de una secuencia de 500 pb del gen inhibidor de proteasa aislado a partir de lascepas transformadas. En cuanto al perfil de restricción los resultados no fueron los esperados,sin embargo, permiten diferenciar la cepa de A. tumefacienstransformada. Adicionalmente seestandarizó las condiciones de los medios de selección para diferenciar cepas transformadas.

  19. 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.

  20. Transformation of Mortierella alpina (fatty acid supplier myceliums via AMT system (Agrobacterium Mediated Transformation

    Directory of Open Access Journals (Sweden)

    Aida Javanmard

    2016-09-01

    Full Text Available Introduction: Mortierella alpina is one of the most important fungi in food industry because of having ability of synthesizing unsaturated fatty acids, particularly Arashidonic Acid. This is a precursor of Eicosanoidregulate-lipoprotein metabolism which is involved in blood rheology, platelet activation and leukocyte-function, and the functional characteristics of the cell membrane. Materials and methods: In this study genetic transformation of M. alpina CBS754.68 fungus was evaluated via Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacteriums containing pBI121 vector were used for transformation of three days of old mycelia. Three days old hyphae were exposed to the bacteria with three level of time (one, two and three hours in the present of acetosyringone. Mitotic stability of the third generation of transgenic (T2 was confirmed by GUS assay and amplification of CaMV 35S promoter by polymerase chain reaction. Results: The highest percentage of transformation and mitotic stability were obtained by using A. tumefaciens and A. rhizogenese, respectively. Discussion and conclusion: The results showed that to obtain more efficient and more stable transformation, the fundamental factor is the use of suitable species of Agrobacterium. It is the first report for transformation of autothroph strain of M. alpine via Agrobacterium.

  1. Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid.

    Science.gov (United States)

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sato, Yukari; Momota, Naoto; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2012-07-01

    Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  2. Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons

    Science.gov (United States)

    Epicotyl and internodal stem segments provide the predominantly used explants for regeneration of transgenic citrus plants following co-cultivation with Agrobacterium. Previous reports using epicotyls segments from Mexican lime have shown low affinity for Agrobacterium tumefaciens infection which re...

  3. CrdR function in a curdlan-producing Agrobacterium sp. ATCC31749 strain.

    Science.gov (United States)

    Yu, Xiaoqin; Zhang, Chao; Yang, Liping; Zhao, Lamei; Lin, Chun; Liu, Zhengjie; Mao, Zichao

    2015-02-10

    Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis. Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred

  4. Beta Glucan Production from Two Strains of Agrobacterium sp in Medium Containing of Molases and Uracil Combine

    Directory of Open Access Journals (Sweden)

    KUSMIATI

    2007-04-01

    Full Text Available Production of β-glucan by Agrobacterium sp is influenced by the composition of nutrition in the fermentation media. Molases has been used successfully by others in the fermentation media of S. cerevisiae to increase the yield of -glucan, and similarly, uracil has been used in the fermentation media of Agrobacterium sp to increase the yield of -glucan. Investigations to increase the yield of -glucan by two strains of Agrobacterium sp, i.e. A1.5 (reference and B4.4 (local strain, have been carried out by addition of various combination of molases and uracil into fermentation media, i.e. 5%(v/v molase-0,05%(b/v uracil; 5% molase-0,025% uracil; 10% molase-0,05% uracil; and 10% molase-0,025% uracil. The β-1,3-glucan and β-1,2-glucan fractions were separated by extraction method. Beta-glucan concentration was determined as the glucose monomer using the phenol-sulphate spectrophotometric method at 490 nm. The protein content was determined by a modified Lowry-spectrophotometric method at 750 nm. The results showed that all combination of molases and uracil in the fermentation media of Agrobacterium sp A1.5 and B4.4 strains have increased both the dry-weight yield of β-glucan (crude and the β¬glucan content, with the highest was in a medium containing 10% molases-0,025% uracil combination. In the above medium, the A1.5 strain produced the highest β-glucan (7,5% with the lowest protein content ( 8,4% in the β-1,3-glucan fraction, while the β-glucan content in the β-1,2-glucan fraction were all lower than in the control media, while the protein content were all higher than in the control media. In the above media, the B4.4 strain produced the highest β-glucan, 7,2% in the β-1,3-glucan fraction, and 13,1% in β-1,2-glucan fraction, while the lowest protein content ( 8,4% was in the β-1,3-glucan fraction. In conclusion, fermentation media of Agrobacterium sp A1.5 strain or B4.4 strain containing molase and uracil combination have increased both

  5. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

    Science.gov (United States)

    Sparks, Caroline A; Doherty, Angela; Jones, Huw D

    2014-01-01

    The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).

  6. Assessment of the genetic and phenotypic diversity among rhizogenic Agrobacterium biovar 1 strains infecting solanaceous and cucurbit crops.

    Science.gov (United States)

    Bosmans, Lien; Álvarez-Pérez, Sergio; Moerkens, Rob; Wittemans, Lieve; Van Calenberge, Bart; Kerckhove, Stefan Van; Paeleman, Anneleen; De Mot, René; Rediers, Hans; Lievens, Bart

    2015-08-01

    Rhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. 77 FR 60431 - Agrobacterium radiobacter strains K84/Kerr-84 and K1026; Notice of Availability

    Science.gov (United States)

    2012-10-03

    ... pesticide specific information, contact: Ann Sibold, Biopesticides and Pollution Prevention Division (7511P... in seed, root, and stem treatments of nonbearing fruit, nut, and ornamental plants, Agrobacterium..., Pesticides and pests. Dated: September 25, 2012. Keith A. Matthews, Director, Biopesticides and Pollution...

  8. Barley Transformation Using Agrobacterium-Mediated Techniques

    Science.gov (United States)

    Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.

    Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.

  9. Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

    Science.gov (United States)

    Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

    2015-01-01

    To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

  10. Agrobacterium mediated transformation of Tunisian Cucumis melo ...

    African Journals Online (AJOL)

    Transgenic Cucumis melo cv. Maazoun containing the neomycin phosphotransferase II (NPT II) chimeric gene conferring resistance to kanamycin were obtained from cotyledons explants inoculated with Agrobacterium tumefaciens (GV3101) that contained the binary vector plasmid pADI. Transformed shoots were obtained ...

  11. Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

    Science.gov (United States)

    Han, J-S; Kim, C K; Park, S H; Hirschi, K D; Mok, I- G

    2005-03-01

    We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

  12. The efficiency of Arabidopsis thaliana floral dip transformation is determined not only by the Agrobacterium strain used but also by the physiology and the ecotype of the dipped plant.

    Science.gov (United States)

    Ghedira, Rim; De Buck, Sylvie; Nolf, Jonah; Depicker, Ann

    2013-07-01

    To evaluate the chromosomal background of different Agrobacterium strains on floral dip transformation frequency, eight wild-type Agrobacterium strains, provided by Laboratorium voor Microbiologie Gent (LMG) and classified in different genomic groups, were compared with the commonly used Agrobacterium strains C58C1 Rif(r) (pMP90) and LBA4404 in Arabidopsis thaliana Columbia (Col-0) and C24 ecotypes. The C58C1 Rif(r) chromosomal background in combination with the pMP90 virulence plasmid showed high Col-0 floral dip transformation frequencies (0.76 to 1.57%). LMG201, which is genetically close to the Agrobacterium C58 strain, with the same virulence plasmid showed comparable or even higher transformation frequencies (1.22 to 2.28%), whereas the LBA4404 strain displayed reproducibly lower transformation frequencies (Agrobacterium chromosomal backgrounds had transformation frequencies between those of the C58C1 Rif(r) (pMP90) and LBA4404 reference strains. None of the strains could transform the C24 ecotype with a frequency higher than 0.1%. Strikingly, all Arabidopsis Col-0 floral dip transformation experiments showed a high transformation variability from plant to plant (even more than 50-fold) within and across the performed biological repeats for all analyzed Agrobacterium strains. Therefore, the physiology of the plant and, probably, the availability of competent flowers to be transformed determine, to a large extent, floral dip transformation frequencies.

  13. An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.

    Science.gov (United States)

    Shri, Manju; Rai, Arti; Verma, Pankaj Kumar; Misra, Prashant; Dubey, Sonali; Kumar, Smita; Verma, Sikha; Gautam, Neelam; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2013-04-01

    Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

  14. Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants.

    Science.gov (United States)

    Md Setamam, Nursuria; Jaafar Sidik, Norrizah; Abdul Rahman, Zainon; Che Mohd Zain, Che Radziah

    2014-06-30

    Capsicum annuum and Capsicum frutescens, also known as "chilies", belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month. In this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values Capsicum species. Both Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies.

  15. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

    NARCIS (Netherlands)

    Michielse, C.B.; Ram, A.F.J.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den

    2004-01-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins

  16. Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid.

    Science.gov (United States)

    Wibberg, Daniel; Blom, Jochen; Jaenicke, Sebastian; Kollin, Florian; Rupp, Oliver; Scharf, Birgit; Schneiker-Bekel, Susanne; Sczcepanowski, Rafael; Goesmann, Alexander; Setubal, Joao Carlos; Schmitt, Rüdiger; Pühler, Alfred; Schlüter, Andreas

    2011-08-20

    Agrobacterium sp. H13-3, formerly known as Rhizobium lupini H13-3, is a soil bacterium that was isolated from the rhizosphere of Lupinus luteus. The isolate has been established as a model system for studying novel features of flagellum structure, motility and chemotaxis within the family Rhizobiaceae. The complete genome sequence of Agrobacterium sp. H13-3 has been established and the genome structure and phylogenetic assignment of the organism was analysed. For de novo sequencing of the Agrobacterium sp. H13-3 genome, a combined strategy comprising 454-pyrosequencing on the Genome Sequencer FLX platform and PCR-based amplicon sequencing for gap closure was applied. The finished genome consists of three replicons and comprises 5,573,770 bases. Based on phylogenetic analyses, the isolate could be assigned to the genus Agrobacterium biovar I and represents a genomic species G1 strain within this biovariety. The highly conserved circular chromosome (2.82 Mb) of Agrobacterium sp. H13-3 mainly encodes housekeeping functions characteristic for an aerobic, heterotrophic bacterium. Agrobacterium sp. H13-3 is a motile bacterium driven by the rotation of several complex flagella. Its behaviour towards external stimuli is regulated by a large chemotaxis regulon and a total of 17 chemoreceptors. Comparable to the genome of Agrobacterium tumefaciens C58, Agrobacterium sp. H13-3 possesses a linear chromosome (2.15 Mb) that is related to its reference replicon and features chromosomal and plasmid-like properties. The accessory plasmid pAspH13-3a (0.6 Mb) is only distantly related to the plasmid pAtC58 of A. tumefaciens C58 and shows a mosaic structure. A tumor-inducing Ti-plasmid is missing in the sequenced strain H13-3 indicating that it is a non-virulent isolate. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Iodate and nitrate transformation by Agrobacterium/Rhizobium related strain DVZ35 isolated from contaminated Hanford groundwater.

    Science.gov (United States)

    Lee, Brady D; Ellis, Joshua T; Dodwell, Alex; Eisenhauer, Emalee E R; Saunders, Danielle L; Lee, M Hope

    2018-05-15

    Nitrate and radioiodine ( 129 I) contamination is widespread in groundwater underneath the Central Plateau of the Hanford Site. 129 I, a byproduct of nuclear fission, is of concern due to a 15.7 million year half-life, and toxicity. The Hanford 200 West Area contains plumes covering 4.3 km 2 with average 129 I concentrations of 3.5 pCi/L. Iodate accounts for 70.6% of the iodine present and organo-iodine and iodide make up 25.8% and 3.6%, respectively. Nitrate plumes encompassing the 129 I plumes have a surface area of 16 km 2 averaging 130 mg/L. A nitrate and iodate reducing bacterium closely related to Agrobacterium, strain DVZ35, was isolated from sediment incubated in a 129 I plume. Iodate removal efficiency was 36.3% in transition cultures, and 47.8% in anaerobic cultures. Nitrate (10 mM) was also reduced in the microcosm. When nitrate was spiked into the microcosms, iodate removal efficiency was 84.0% and 69.2% in transition and anaerobic cultures, respectively. Iodate reduction was lacking when nitrate was absent from the growth medium. These data indicate there is simultaneous reduction of nitrate and iodate by DVZ35, and iodate is reduced to iodide. Results provide the scientific basis for combined nitrogen and iodine cycling throughout the Hanford Site. Copyright © 2018. Published by Elsevier B.V.

  18. Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls

    Science.gov (United States)

    Kaitlin J. Palla; Paula M. Pijut

    2015-01-01

    An Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for white ash (Fraxinus americana) using hypocotyls as the initial explants. Hypocotyls isolated from mature embryos germinated on Murashige and Skoog (MS) medium supplemented with 22.2 µM 6-benzyladenine (BA) and 0.5 µM...

  19. Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid.

    Science.gov (United States)

    Ramírez-Bahena, Martha H; Vial, Ludovic; Lassalle, Florent; Diel, Benjamin; Chapulliot, David; Daubin, Vincent; Nesme, Xavier; Muller, Daniel

    2014-04-01

    Linear chromosomes are atypical in bacteria and likely a secondary trait derived from ancestral circular molecules. Within the Rhizobiaceae family, whose genome contains at least two chromosomes, a particularity of Agrobacterium fabrum (formerly A. tumefaciens) secondary chromosome (chromid) is to be linear and hairpin-ended thanks to the TelA protelomerase. Linear topology and telA distributions within this bacterial family was screened by pulse field gel electrophoresis and PCR. In A. rubi, A. larrymoorei, Rhizobium skierniewicense, A. viscosum, Agrobacterium sp. NCPPB 1650, and every genomospecies of the biovar 1/A. tumefaciens species complex (including R. pusense, A. radiobacter, A. fabrum, R. nepotum plus seven other unnamed genomospecies), linear chromid topologies were retrieved concomitantly with telA presence, whereas the remote species A. vitis, Allorhizobium undicola, Rhizobium rhizogenes and Ensifer meliloti harbored a circular chromid as well as no telA gene. Moreover, the telA phylogeny is congruent with that of recA used as a marker gene of the Agrobacterium phylogeny. Collectively, these findings strongly suggest that single acquisition of telA by an ancestor was the founding event of a large and diverse clade characterized by the presence of a linear chromid. This clade, characterized by unusual genome architecture, appears to be a relevant candidate to serve as a basis for a possible redefinition of the controversial Agrobacterium genus. In this respect, investigating telA in sequenced genomes allows to both ascertain the place of concerned strains into Agrobacterium spp. and their actual assignation to species/genomospecies in this genus. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

    Science.gov (United States)

    Tang, Wei; Lin, Jinxing; Newton, Ronald J

    2007-05-01

    Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

  1. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    Science.gov (United States)

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brac...

  2. Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

    Science.gov (United States)

    Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2015-01-01

    In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

  3. Minimum Requirements of Flagellation and Motility for Infection of Agrobacterium sp. Strain H13-3 by Flagellotropic Bacteriophage 7-7-1

    Science.gov (United States)

    Yen, Jiun Y.; Broadway, Katherine M.

    2012-01-01

    The flagellotropic phage 7-7-1 specifically adsorbs to Agrobacterium sp. strain H13-3 (formerly Rhizobium lupini H13-3) flagella for efficient host infection. The Agrobacterium sp. H13-3 flagellum is complex and consists of three flagellin proteins: the primary flagellin FlaA, which is essential for motility, and the secondary flagellins FlaB and FlaD, which have minor functions in motility. Using quantitative infectivity assays, we showed that absence of FlaD had no effect on phage infection, while absence of FlaB resulted in a 2.5-fold increase in infectivity. A flaA deletion strain, which produces straight and severely truncated flagella, experienced a significantly reduced infectivity, similar to that of a flaB flaD strain, which produces a low number of straight flagella. A strain lacking all three flagellin genes is phage resistant. In addition to flagellation, flagellar rotation is required for infection. A strain that is nonmotile due to an in-frame deletion in the gene encoding the motor component MotA is resistant to phage infection. We also generated two strains with point mutations in the motA gene resulting in replacement of the conserved charged residue Glu98, which is important for modulation of rotary speed. A change to the neutral Gln caused the flagellar motor to rotate at a constant high speed, allowing a 2.2-fold-enhanced infectivity. A change to the positively charged Lys caused a jiggly motility phenotype with very slow flagellar rotation, which significantly reduced the efficiency of infection. In conclusion, flagellar number and length, as well as speed of flagellar rotation, are important determinants for infection by phage 7-7-1. PMID:22865074

  4. Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos

    Directory of Open Access Journals (Sweden)

    Mauro Antonio Orlando Di

    2000-01-01

    Full Text Available Twenty-five Brazilian soybean cultivars were studied for susceptibility to four strains of Agrobacterium tumefaciens (C58, Ach5, Bo542 and A281 and for their ability to produce somatic embryos. Twelve plants of each cultivar were inoculated in a greenhouse at 4-6 weeks of age, using 12 inoculation sites per plant. The number of galls formed on plants were counted 8-10 weeks after inoculation. To study ability to produce somatic embryos, immature cotyledons, 4-6 mm in length, were plated onto N10 medium for induction of somatic embryogenesis, using four Petri dishes with 20 cotyledons for each cultivar. The embryogenic tissues were transferred onto new N10 medium six times at 15-day intervals and the number of somatic embryos per cultivar determined. Significant interaction between soybean cultivars and A. tumefaciens strains was observed; the most virulent strain was A281. The opine type apparently had no effect on strain virulence, and the most embryogenic cultivars were IAS-5, Cristalina, FT-Cometa, IAC-7 and OC-3.

  5. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat

    Directory of Open Access Journals (Sweden)

    Doherty Angela

    2005-09-01

    Full Text Available Abstract Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.

  6. An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

    Science.gov (United States)

    Han, Guomin; Shao, Qian; Li, Cuiping; Zhao, Kai; Jiang, Li; Fan, Jun; Jiang, Haiyang; Tao, Fang

    2018-05-01

    Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 10 6 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

  7. Attachment study of Agrobacterium tumefaciens to yam spp

    African Journals Online (AJOL)

    Charles S.Wortmann

    dodecandra) berries (Type 44) on different developmental stages of. Lymnaea ... flukicidal drugs and the use of molluscides to kill snail intermediate hosts are ... age groups of fasciola transmitting snails and assessing time-concentration.

  8. Attachment study of Agrobacterium tumefaciens to yam spp

    African Journals Online (AJOL)

    Charles S.Wortmann

    for seed yield per plant indicated the complexity of this trait and the ... chickpea with 142,170 hectares of production area and 1,047,440 quintals of .... Mean values for 10 morphological characters in 50 grass pea populations of Ethiopia,. 1998.

  9. Attachment study of Agrobacterium tumefaciens to yam spp

    African Journals Online (AJOL)

    Charles S.Wortmann

    of microcontinents. Key words/phrases: Accretion, cratonisation, isotope systems, subduction, ... N-S striking structures dominate Pan-African tectonics, although ... (a) Foliation trends, shear sense and brittle faults in the Gore-Gambella area; heavy .... intergrown sillimanite, quartz and plagioclase (Plate 1F), and large garnet.

  10. Agrobacterium tumefaciens-mediated transformation of biofuel plant ...

    African Journals Online (AJOL)

    Yomi

    2010-09-27

    Sep 27, 2010 ... several plant species including Arabidopsis (Chang et al.,. 1994; Clough and Bent, ... and Technology Talents Scheme of Yunnan Province. (Grant no. .... of shoots and roots from Jatropha curcas L. explants. J. Horticult. Sci.

  11. Attachment study of Agrobacterium tumefaciens to yam spp

    African Journals Online (AJOL)

    Charles S.Wortmann

    guttatus (Scrophulariaceae). Amer. J. Bot. 77:1505–1507. 13. Dole, J.A. (1992). Reproductive assurance mechanisms in three taxa of the Mimulus guttatus complex (Scrophulariaceae). Amer. J. Bot. 79:650–659. 14. Ellstrand, N.C. and Foster, K.W. (1983). Impact of population structure on the apparent out-crossing rate of ...

  12. Attachment study of Agrobacterium tumefaciens to yam spp

    African Journals Online (AJOL)

    Charles S.Wortmann

    high geothermal gradient areas near the acidic magma chamber. In the MER the main geothermal reservoirs are Pliocene basalts and ignimbrites (Tamiru Alemayehu, 1993;. Berhanu Gizaw, 1996; Tamiru Alemayehu and Vernier, 1997). Almost all thermal centres are found in close association with acidic volcanic centres.

  13. Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

    Science.gov (United States)

    Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B...

  14. Agrobacterium rhizogenes-induced cotton hairy root culture as an alternative tool for cotton functional genomics

    Science.gov (United States)

    Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In cer...

  15. Agrobacterium-mediated transformation as a tool for functional genomics in fungi

    NARCIS (Netherlands)

    Michielse, C.B.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

    2005-01-01

    In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and

  16. 77 FR 40880 - Agrobacterium radiobacter; Registration Review Proposed Decision; Notice of Availability

    Science.gov (United States)

    2012-07-11

    ...: Ann Sibold, Regulatory Action Leader, Biopesticides and Pollution Prevention Division (7511P), Office... pathogen Agrobacterium tumefaciens) when applied to seeds, roots and/or stems of nonbearing fruit, nut and.... Matthews, Director, Biopesticides and Pollution Prevention Division, Office of Pesticide Programs. [FR Doc...

  17. Stable Agrobacterium-mediated transformation of Norway spruce embryogenic tissues using somatic embryo explants

    Czech Academy of Sciences Publication Activity Database

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

    2011-01-01

    Roč. 57, č. 7 (2011), s. 277-280 ISSN 1212-4834 R&D Projects: GA MZe QH71290 Institutional research plan: CEZ:AV0Z50510513 Keywords : Agrobacterium tumefaciens * genetic engineering * GUS activity * Picea abies (L.) Karst Subject RIV: EB - Genetic s ; Molecular Biology

  18. Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda

    Science.gov (United States)

    Micah E. Stevens; Paula M. Pijut

    2014-01-01

    Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to...

  19. An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

    Science.gov (United States)

    Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

    2014-01-01

    Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. Copyright © 2014 Elsevier GmbH. All rights reserved.

  20. A host-specific biological control of grape crown gall by Agrobacterium vitis strain F2/5: its regulation and population dynamics.

    Science.gov (United States)

    Kaewnum, Supaporn; Zheng, Desen; Reid, Cheryl L; Johnson, Kameka L; Gee, Jodi C; Burr, Thomas J

    2013-05-01

    Nontumorigenic Agrobacterium vitis strain F2/5 is able to prevent crown gall caused by tumorigenic A. vitis on grape but not on other plant species such as tobacco. Mutations in a quorum-sensing transcription factor, aviR, and in caseinolytic protease (clp) component genes clpA and clpP1 resulted in reduced or loss of biological control. All mutants were complemented; however, restoration of biological control by complemented clpA and clpP1 mutants was dependent on the copy number of vector that was used as well as timing of application of the complemented mutants to grape wounds in relation to inoculation with pathogen. Mutations in other quorum-sensing and clp genes and in a gene associated with polyketide synthesis did not affect biological control. It was determined that, although F2/5 inhibits transformation by tumorigenic A. vitis strains on grape, it does not affect growth of the pathogen in wounded grape tissue over time.

  1. An Efficient Agrobacterium-Mediated Transformation System for Poplar

    Science.gov (United States)

    Movahedi, Ali; Zhang, Jiaxin; Amirian, Rasoul; Zhuge, Qiang

    2014-01-01

    Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis. PMID:24933641

  2. Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia

    Directory of Open Access Journals (Sweden)

    Kuzmanović N.

    2012-01-01

    Full Text Available In 2010, a serious outbreak of crown gall disease was observed on grapevines (Vitis vinifera L. cv. Cabernet Sauvignon in several commercial vineyards located in the Vojvodina province, Serbia. Bacteria were isolated from the young tumor tissue on nonselective YMA medium and five representative strains were selected for further identification. Tumorigenic (Ti plasmid was detected in all strains by PCR using primers designed to amplify the virC pathogenicity gene, producing a 414-bp PCR product. The strains were identified as Agrobacterium vitis using differential physiological and biochemical tests, and a multiplex PCR assay targeting 23S rRNA gene sequences. In the pathogenicity assay, all strains induced characteristic symptoms on inoculated tomato and grapevine plants. They were less virulent on tomato plants in comparison to the reference strains of A. tumefaciens and A. vitis. [Ministarstva nauke Republike Srbije, br. III46008: Development of integrated management of harmful organisms in plant production in order to overcome resistance and to improve food quality and safety

  3. Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction.

    Science.gov (United States)

    Xu, X Q; Li, L P; Pan, S Q

    2001-11-01

    Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell. An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium-plant interaction. In this paper, we report a feedback regulation pathway that controls the expression of katA in A. tumefaciens cells. We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection. This represents a new regulatory factor for catalase induction in bacteria. More importantly, a feedback regulation was observed when the katA-gfp expression was studied in different genetic backgrounds. We found that introduction of a wild-type katA gene encoding a functional catalase into A. tumefaciens cells could repress the katA-gfp expression over 60-fold. The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2. In addition, the katA-gfp expression of one bacterial cell could be repressed by other surrounding catalase-proficient bacterial cells. Furthermore, mutation at katA caused a 10-fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium. These results suggest that the endogenous H2O2 generated during A. tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria. Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA-gfp expression. The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A

  4. Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer

    NARCIS (Netherlands)

    Michielse, C.B.; Salim, K.; Ragas, P.; Ram, A.F.J.; Kudla, B.; Jarry, B.; Punt, P.J.; Hondel, C.A.M.J.J. van den

    2004-01-01

    Two transformation systems, based on the use of CaCl2/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS+ marker from Aspergillus nidulans, and

  5. Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

    DEFF Research Database (Denmark)

    Flodgaard, Lars; Dalgaard, Paw; Andersen, Jens Bo

    2005-01-01

    Bioluminescence is a common phenotype in marine bacteria, such As Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets......) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non...

  6. Optimization of Agrobacterium-Mediated Transformation in Soybean

    Science.gov (United States)

    Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan

    2017-01-01

    High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide

  7. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.

    Science.gov (United States)

    Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Pham, Thu Ha; Phan, Tuan-Nghia; Tran, Van-Tuan

    2016-12-01

    Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

  8. Optimization of genetic transformation with Agrobacterium rhizogenes

    International Nuclear Information System (INIS)

    Blanco, M.; Valverde, R.; Gomez, L.

    2002-01-01

    To optimize the genetic transformation efficiency using Agrobacterium rhizogenes, carrot sections inoculated with the Agrobacterium strain A4TC were co-cultivated with acetosyringone, phloroglucinol, and a mix of both. Acetosyringone is one of the phenolic compounds produced by plant tissues in response to wounding, which induces the transfer of T-DNA from the agrobacteria to the plant. Phloroglucinol is also a phenolic compound; however, it has a synergistic action with auxins by partially inhibiting cytokinin activity. The highest transformation efficiency (75%) was obtained with acetosyringone (100 mM) in combination with phloroglucinol (25 mg l - 1 ). In general, a 6-day co-cultivation, independently of treatments, induced the best transformation rate. Inclusion of 100 mg l - 1 kanamycin efficiently discriminated transformed roots from non-transgenic ones. This paper also presents a novel bacterial elimination method, by which Agrobacterium can be completely eliminated in 48 h with Cefotaxime at a dosage of 500 mg l - 1 . Author [es

  9. Differential roles of glucosinolates and camalexin at different stages of Agrobacterium-mediated transformation.

    Science.gov (United States)

    Shih, Po-Yuan; Chou, Shu-Jen; Müller, Caroline; Halkier, Barbara Ann; Deeken, Rosalia; Lai, Erh-Min

    2018-03-02

    Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation. © 2018 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

  10. Efficient and genotype-independent Agrobacterium--mediated tomato transformation.

    Science.gov (United States)

    Park, Sung Hun; Morris, Jay L; Park, Jung Eun; Hirschi, Kendal D; Smith, Roberta H

    2003-10-01

    An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.

  11. Agrobacterium-mediated gene transfer in plants and biosafety considerations.

    Science.gov (United States)

    Mehrotra, Shweta; Goyal, Vinod

    2012-12-01

    Agrobacterium, the natures' genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world's population.

  12. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

    Science.gov (United States)

    Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

    2015-08-01

    The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi. Copyright © 2015. Published by Elsevier Inc.

  13. Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid

    Science.gov (United States)

    Wetzel, Margaret E.; Kim, Kun-Soo; Miller, Marilyn; Olsen, Gary J.

    2014-01-01

    The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved. PMID:24363349

  14. mu-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina.

    OpenAIRE

    Kim, R Y; Gasser, R; Wistow, G J

    1992-01-01

    mu-Crystallin is the major component of the eye lens in several Australian marsupials. The complete sequence of kangaroo mu-crystallin has now been obtained by cDNA cloning. The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes. RNA analysis of kangaroo tissues shows that mu-cryst...

  15. Agrobacterium: nature's genetic engineer.

    Science.gov (United States)

    Nester, Eugene W

    2014-01-01

    Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun's old observations and also explain why Agrobacterium is nature's genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering.

  16. High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

    Science.gov (United States)

    Mehrotra, Meenakshi; Sanyal, Indraneel; Amla, D V

    2011-09-01

    To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

  17. Breeding of Coenzyme Q10 Produced Strain by Low-Energy Ion Implantation and Optimization of Coenzyme Q10 Fermentation

    International Nuclear Information System (INIS)

    Xu Dejun; Zheng Zhiming; Wang Peng; Wang Li; Yuan Hang; Yu Zengliang

    2008-01-01

    In order to increase the production efficiency of coenzyme Q 10 , the original strain Agrobacterium tumefaciens ATCC 4452 was mutated by means of Nitrogen ions implantation. A mutant strain, ATX 12, with high contents of coenzyme Q 10 was selected. Subsequently, the conditions such as carbohydrate concentration, nitrogen source concentration, inoculum's size, seed age, aeration and temperature which might affect the production of CoQ 10 were investigated in detail. Under optimal conditions, the maximum concentration of the intracellular CoQ 10 reached 200.3 mg/L after 80 h fed-batch fermentation, about 245% increasing in CoQ 10 production after ion implantation, compared to the original strain. (ion beam bioengineering)

  18. Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09.

    Science.gov (United States)

    Chen, Yun-mei; Xu, Hai-yang; Wang, Yang; Zhang, Jian-fa; Wang, Shi-ming

    2014-11-01

    Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. ZX09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreoscilla hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide (CO) difference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physiological changes included its elevated respiration rate and cellular invertase activity.

  19. Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K-powered ER/actin network.

    Science.gov (United States)

    Yang, Qinghua; Li, Xiaoyang; Tu, Haitao; Pan, Shen Q

    2017-03-14

    Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium -delivered factors are trafficked through the cytoplasm. In this study, we monitored the movement of Agrobacterium -delivered VirE2 inside plant cells by using a split-GFP approach in real time. Agrobacterium -delivered VirE2 trafficked via the endoplasmic reticulum (ER) and F-actin network inside plant cells. During this process, VirE2 was aggregated as filamentous structures and was present on the cytosolic side of the ER. VirE2 movement was powered by myosin XI-K. Thus, exogenously produced and delivered VirE2 protein can use the endogenous host ER/actin network for movement inside host cells. The A. tumefaciens pathogen hijacks the conserved host infrastructure for virulence trafficking. Well-conserved infrastructure may be useful for Agrobacterium to target a wide range of recipient cells and achieve a high efficiency of transformation.

  20. Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

    Science.gov (United States)

    Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

    This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

  1. [Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].

    Science.gov (United States)

    Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

    2014-01-01

    To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance.

  2. Agrobacterium T-DNA-encoded protein Atu6002 interferes with the host auxin response

    Science.gov (United States)

    Lacroix, Benoît; Gizatullina, Diana I.; Babst, Benjamin A.; Gifford, Andrew N.; Citovsky, Vitaly

    2013-01-01

    Summary Several genes in the Agrobacterium tumefaciens transferred (T) DNA encode proteins that are involved in developmental alterations leading to the formation of tumors in infected plants. We investigated the role of the protein encoded by the Atu6002 gene, the function of which is completely unknown. The Atu6002 expression occurs in Agrobacterium-induced tumors, and is also activated upon activation of plant cell division by growth hormones. Within the expressing plant cells, the Atu6002 protein is targeted to the plasma membrane. Interestingly, constitutive ectopic expression of Atu6002 in transgenic tobacco plants lead to a severe developmental phenotype characterized by stunted growth, shorter internodes, lanceolate leaves, increased branching, and modified flower morphology. These Atu6002-expressing plants also displayed impaired response to auxin. However, auxin cellular uptake and polar transport were not significantly inhibited in these plants, suggesting that Atu6002 interferes with auxin perception or signaling pathways. PMID:24128370

  3. Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

    Science.gov (United States)

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

    2015-01-01

    Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant.

  4. Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis.

    Science.gov (United States)

    Zhang, Hongtao; Setubal, Joao Carlos; Zhan, Xiaobei; Zheng, Zhiyong; Yu, Lijun; Wu, Jianrong; Chen, Dingqiang

    2011-06-01

    Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

  5. Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

    Science.gov (United States)

    Yadav, Reena; Mehrotra, Meenakshi; Singh, Aditya K; Niranjan, Abhishek; Singh, Rani; Sanyal, Indraneel; Lehri, Alok; Pande, Veena; Amla, D V

    2017-01-01

    Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 μg/g), chlorogenic acid (0.0978 ± 0.0046 μg/g), and quercetin (0.0971 ± 0.0046 μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

  6. 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...

  7. Advances in transforming kudzu (Pueraria phaseoloides and carrot (Daucus carota var. Danvers 126 roots with different Agrobacterium rhizogenes strains for increasing MA fungi growth

    Directory of Open Access Journals (Sweden)

    Marisol Medina Sierra

    2002-07-01

    Full Text Available Kudzú (P. phaseoloides and carrot (D. carota roots were transformed in this survey into different kinds of culture medium by using five different A. rhizogenes strains. These presented different behaviour both in carrot transformation by A. rhizogenes 15834, A.r.8196 and A.r.2659 strains as well as kudzu transformation by A.r.15834 and A.r.1724 strains. Transformed carrot root growth was increased in WM culture medium, whilst transformed kudzu root growth did not increase in either the same medium or in modified MS medium. Transformed carrot roots were used for G. intrarradices increase and sporulation; however, wild AMF strains, isolated from a mining area (the lower Cauca area of Antioquia, did not grow either in roots from this specie or those from kudzu, in spite of this plant having great affinity for wild AMF strains. The results represent an advance in the procedure for DNA isolation and keeping AMF collections, required for other research.

  8. A reliable in vitro fruiting system for armillaria mellea for evaluation of agrobacterium tumefaciens transformation vectors

    Science.gov (United States)

    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 has hindered research, and necessitated dependence on intermittently available wild-collected basidiospores. Here we describe a reliable, rep...

  9. Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla

    Science.gov (United States)

    Phomopsis seed decay (PSD) of soybean is caused primarily by the fungal pathogen Phomopsis longicolla. PSD impairs seed germination, reduces seedling vigor, and can substantially reduce stand establishment. In hot and humid conditions, PSD can cause significant yield losses. Few studies have explore...

  10. Identification of Juglans wild relatives resistant to crown gall caused by Agrobacterium tumefaciens

    Science.gov (United States)

    Wild species are a source of useful agronomic traits for crop plants including but not limited to pathogen resistance, drought tolerance, and salt tolerance (Aradhya and Kluepfel 2012). To exploit this natural diversity of disease resistance, we are conducting the first systematic exploration of th...

  11. Highly efficient transformation system for Malassezia furfur and Malassezia pachydermatis using Agrobacterium tumefaciens-mediated transformation

    NARCIS (Netherlands)

    Celis, A.M.; Vos, Aurin; Triana, S.; Medina, C.A.; Escobar Salazar, Natalia; Restrepo, S.; Wosten, Han; de Cock, Hans

    2017-01-01

    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,

  12. Deviating T-DNA transfer from Agrobacterium tumefaciens to plants

    DEFF Research Database (Denmark)

    van der Graaff, Eric; den Dulk-Ras, A; Hooykaas, P J

    1996-01-01

    -region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.......We analyzed 29 T-DNA inserts in transgenic Arabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data....... Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T...

  13. trans-Acting Virulence Functions of the Octopine Ti Plasmid from Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Kan, Jan van; Schilperoort, Rob

    1984-01-01

    All Ti plasmid-encoded virulence functions that were studied act in trans. An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized. Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called

  14. REGENERASI RUMPUT LAUT Kappaphycus alvarezii HASIL TRANSFORMASI GEN Sitrat Sintase MENGGUNAKAN Agrobacterium tumefaciens SECARA IN VITRO

    Directory of Open Access Journals (Sweden)

    Emma Suryati

    2016-02-01

    seleksi 85%, dan efisiensi regenerasi thalus non transgenik sebesar 95% pada media non selektif. Media recovery dengan penambahan pupuk PES memperlihatkan sintasan yang paling baik pada regenerasi thalus transgenik. Hasil analisis PCR memperlihatkan K. alvarezii transgenik putatif mengandung transgen PaCS di bawah kendali promoter 35S CaMV.

  15. Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

    Science.gov (United States)

    Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

    2017-02-15

    Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Improvements in the transformation of Arabidopsis thaliana C24 leaf-discs by Agrobacterium tumefaciens

    DEFF Research Database (Denmark)

    van der Graaff, Eric; Hooykaas, P J

    1996-01-01

    We report here an efficient Arabidopsis leafdisc transformation protocol yielding an average transformation frequency of 1.6 transgenic shoots per leaf explant 4 weeks after the bacterial infection period. Subsequent cultivation in vitro is such that a high percentage (85-90%) of the primary...... transformants produces seeds with an average seed yield of 100-300 seeds per plant. This improved transformation protocol yields mainly (70%) transformants segregating for a single T-DNA locus of which 68% actually contain one T-DNA insert. The objective is to generate a pool of independent transformants...

  17. Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.

    Science.gov (United States)

    Li, D D; Shi, W; Deng, X X

    2003-12-01

    Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

  18. Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum.

    Science.gov (United States)

    Kuzmanović, Nemanja; Puławska, Joanna; Prokić, Anđelka; Ivanović, Milan; Zlatković, Nevena; Jones, Jeffrey B; Obradović, Aleksa

    2015-09-01

    Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (Agrobacterium species. The major cellular fatty acids of the novel strains were 18:1 w7c (72.8-77.87%) and 16:0 (6.82-8.58%). Phenotypic features allowed their differentiation from closely related species. Polyphasic characterization showed that the three strains represent a novel species of the genus Agrobacterium, for which the name Agrobacterium arsenijevicii sp. nov. is proposed. The type strain of A. arsenijevicii is KFB 330(T) (= CFBP 8308(T) = LMG 28674(T)). Copyright © 2015 Elsevier GmbH. All rights reserved.

  19. Ecological dynamics and complex interactions of Agrobacterium megaplasmids.

    Science.gov (United States)

    Platt, Thomas G; Morton, Elise R; Barton, Ian S; Bever, James D; Fuqua, Clay

    2014-01-01

    As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it's Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together.

  20. Bacillus subtilis affects miRNAs and flavanoids production in Agrobacterium-Tobacco interaction.

    Science.gov (United States)

    Nazari, Fahimeh; Safaie, Naser; Soltani, Bahram Mohammad; Shams-Bakhsh, Masoud; Sharifi, Mohsen

    2017-09-01

    Agrobacterium tumefaciens is a very destructive plant pathogen. Selection of effective biological agents against this pathogen depends on more insight into molecular plant defence responses during the biocontrol agent-pathogen interaction. Auxin as a phytohormone is a key contributor in pathogenesis and plant defence and accumulation of auxin transport carriers are accompanied by increasing in flavonoid and miRNAs concentrations during plant interactions with bacteria. The aim of this research was molecular analysis of Bacillus subtilis (ATCC21332) biocontrol effect against A. tumefaciens (IBRC-M10701) pathogen interacting with Nicotiana tabacum plants. Tobacco plants were either treated with both or one of the challenging bacteria and the expression of miRNAs inside the plants were analysed through qRT-PCR. The results indicated that the bacterial treatments affect expression level of nta-miRNAs. In tobacco plants treated only with A. tumefaciens the expression of nta-miR393 was more than that was recorded for nta-miR167 (3.8 folds, P subtilis (2.1 folds, P subtilis alone, was similar to the amount recorded for the plants challenged with the both bacteria. This study suggests a relationship between the upregulation of nta-miR167, nta-miR393 and accumulation of flavanoid compounds. Overall, the expression of these miRNAs as well as flavonoid derivatives has the potential of being used as biomarkers for the interaction of B. subtilis and A. tumefaciens model system in N. tabacum. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. ORF Sequence: NC_003062 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available 6 [Agrobacterium tumefaciens str. C58] MRHGNSGRKLNRTASHRKAMFANMAASLITHEQIVTTLPKAKEIRPIVERLVTLGKRGDLHARRQAISQIKDQDAVRKLFDAIASRYATRNGGYLRIMKAGYRQGDNAALAVVEFVERDVDAKGAADKARVAAEAAAAEAA

  2. ORF Sequence: NC_003063 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available 1 [Agrobacterium tumefaciens str. C58] MIMGEGGEMGVGIFQIDMDWLHRWLRHRRGPGSGKTSGNRNLKPRLTPAAAQQDHRQMLCFMTLNAAVGVLIGICVGAALIFLDIGGLGARIGRAASPFVPVLLVVVPIASLFGAAAAASAILLMPYEKKFRDDDDDARGE

  3. A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean.

    Science.gov (United States)

    Li, Caifeng; Zhang, Haiyan; Wang, Xiurong; Liao, Hong

    2014-11-01

    Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean. An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

  4. ORF Alignment: NC_003063 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003063 gi|15890664 >1n8kA 9 361 1 333 5e-31 ... ref|NP_534796.1| xylitol dehydroge...nase [Agrobacterium tumefaciens str. C58] ... gb|AAL45112.1| xylitol dehydrogenase [Agrobacterium ... ... ... tumefaciens str. C58] gb|AAK89121.1| AGR_L_1091p ... [Agrobacterium tumefaciens str. C58] pir||G98199 xylitol... ... (strain C58, Cereon) pir||AB3087 xylitol dehydrogenase ... Atu4318 [imported] - Agrobacter

  5. ORF Alignment: NC_003305 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003305 gi|17938007 >1n8kA 9 361 1 333 5e-31 ... ref|NP_534796.1| xylitol dehydroge...nase [Agrobacterium tumefaciens str. C58] ... gb|AAL45112.1| xylitol dehydrogenase [Agrobacterium ... ... ... tumefaciens str. C58] gb|AAK89121.1| AGR_L_1091p ... [Agrobacterium tumefaciens str. C58] pir||G98199 xylitol... ... (strain C58, Cereon) pir||AB3087 xylitol dehydrogenase ... Atu4318 [imported] - Agrobacter

  6. Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

    Science.gov (United States)

    Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

    2014-01-01

    The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

  7. Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

    Directory of Open Access Journals (Sweden)

    Hiroaki Mano

    Full Text Available The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium. We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP. Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholinoethanesulfonic acid (MES buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max and pea (Pisum sativum. The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

  8. Direct visualization of Agrobacterium-delivered VirE2 in recipient cells

    Science.gov (United States)

    Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

    2014-01-01

    Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec−1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

  9. Canthaxanthin production with modified Mucor circinelloides strains.

    Science.gov (United States)

    Papp, Tamás; Csernetics, Arpád; Nagy, Gábor; Bencsik, Ottó; Iturriaga, Enrique A; Eslava, Arturo P; Vágvölgyi, Csaba

    2013-06-01

    Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.

  10. Factor affecting Agrobacterium -mediated transformation of rice ...

    African Journals Online (AJOL)

    Potato is a very important food crop and is adversely affected by fungus. Agrobacterium-mediated transformation can play an important role in the improvement of potato. The present study was conducted to optimize the different factors affecting Agrobacterium-mediated transformation of chitinase gene. Nodes were used as ...

  11. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation.

    Science.gov (United States)

    Kwon, Tackmin

    2016-09-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection.

  12. Bacteriophytochromes control conjugation in Agrobacterium fabrum.

    Science.gov (United States)

    Bai, Yingnan; Rottwinkel, Gregor; Feng, Juan; Liu, Yiyao; Lamparter, Tilman

    2016-08-01

    Bacterial conjugation, the transfer of single stranded plasmid DNA from donor to recipient cell, is mediated through the type IV secretion system. We performed conjugation assays using a transmissible artificial plasmid as reporter. With this assay, conjugation in Agrobacterium fabrum was modulated by the phytochromes Agp1 and Agp2, photoreceptors that are most sensitive in the red region of visible light. In conjugation studies with wild-type donor cells carrying a pBIN-GUSINT plasmid as reporter that lacked the Ti (tumor inducing) plasmid, no conjugation was observed. When either agp1(-) or agp2(-) knockout donor strains were used, plasmid DNA was delivered to the recipient, indicating that both phytochromes suppress conjugation in the wild type donor. In the recipient strains, the loss of Agp1 or Agp2 led to diminished conjugation. When wild type cells with Ti plasmid and pBIN-GUS reporter plasmid were used as donor, a high rate of conjugation was observed. The DNA transfer was down regulated by red or far-red light by a factor of 3.5. With agp1(-) or agp2(-) knockout donor cells, conjugation in the dark was about 10 times lower than with the wild type donor, and with the double knockout donor no conjugation was observed. These results imply that the phytochrome system has evolved to inhibit conjugation in the light. The decrease of conjugation under different temperature correlated with the decrease of phytochrome autophosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Is VIP1 important for Agrobacterium-mediated transformation?

    Science.gov (United States)

    Shi, Yong; Lee, Lan-Ying; Gelvin, Stanton B

    2014-09-01

    Agrobacterium genetically transforms plants by transferring and integrating T-(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus-tagged VirE2 or Venus-tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1-Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY-2 protoplasts, regardless of whether VirE2 was co-expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium-mediated transformation or VirE2 subcellular localization. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  14. Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes.

    Science.gov (United States)

    Aarrouf, J; Castro-Quezada, P; Mallard, S; Caromel, B; Lizzi, Y; Lefebvre, V

    2012-02-01

    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 with the two constructs pBIN-gus and pHKN29-gfp of A. rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN-gus or A4RS-pHKN29-gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29-gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29-gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene. We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P. capsici interaction.

  15. Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

    Science.gov (United States)

    Ibrahim, Evra Raunie

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

  16. Genetic Transformation of Metroxylon sagu (Rottb. Cultures via Agrobacterium-Mediated and Particle Bombardment

    Directory of Open Access Journals (Sweden)

    Evra Raunie Ibrahim

    2014-01-01

    Full Text Available Sago palm (Metroxylon sagu is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L. Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.

  17. Metabolic engineering of Agrobacterium sp. ATCC31749 for curdlan production from cellobiose.

    Science.gov (United States)

    Shin, Hyun-Dong; Liu, Long; Kim, Mi-Kyoung; Park, Yong-Il; Chen, Rachel

    2016-09-01

    Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing β-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe.

  18. Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

    Science.gov (United States)

    Lacroix, Benoît; Citovsky, Vitaly

    2015-11-20

    During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

  19. Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration[W

    Science.gov (United States)

    Vaghchhipawala, Zarir E.; Vasudevan, Balaji; Lee, Seonghee; Morsy, Mustafa R.; Mysore, Kirankumar S.

    2012-01-01

    Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)–mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate. PMID:23064322

  20. Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.

    Science.gov (United States)

    Paul, Anamika; Bakshi, Souvika; Sahoo, Debee Prasad; Kalita, Mohan Chandra; Sahoo, Lingaraj

    2012-04-01

    An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

  1. Agrobacterium-mediated transformation of lipomyces

    Science.gov (United States)

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

    2018-03-13

    This disclosure provides Agrobacterium-mediated transformation methods for the oil-producing (oleaginous) yeast Lipomyces sp., as well as yeast produced by the method. Such methods utilize Agrobacterium sp. cells that have a T-DNA binary plasmid, wherein the T-DNA binary plasmid comprises a first nucleic acid molecule encoding a first protein and a second nucleic acid molecule encoding a selective marker that permits growth of transformed Lipomyces sp. cells in selective culture media comprising an antibiotic.

  2. Purification and biochemical characterization of a highly thermostable bacteriocin isolated from Brevibacillus brevis strain GM100.

    Science.gov (United States)

    Ghadbane, Mouloud; Harzallah, Daoud; Laribi, Atef Ibn; Jaouadi, Bassem; Belhadj, Hani

    2013-01-01

    A bacteriocin-producing (11,000 AU mL(-1)) strain was isolated from the rhizosphere of healthy Algerian plants Ononis angustissima Lam., and identified as Brevibacillus brevis strain GM100. The bacteriocin, called Bac-GM100, was purified to homogeneity from the culture supernatant, and, based on MALDI-TOF/MS analysis, was a monomer protein with a molecular mass of 4375.66 Da. The 21 N-terminal residues of Bac-GM100 displayed 65% homology with thurincin H from Bacillus thuringiensis. Bac-GM100 was extremely heat-stable (20 min at 120 °C), and was stable within a pH range of 3-10. It proved sensitive to various proteases, which demonstrated its protein nature. It was also found to display a bactericidal mode of action against gram-negative (Salmonella enteric ATCC 43972, Pseudomonas aeruginosa ATCC 49189, and Agrobacterium tumefaciens C58) and gram-positive (Enterococcus faecalis ENSAIA 631 and Staphylococcus aureus ATCC 6538) bacteria, and a fungistatic mode of action against the pathogenic fungus Candida tropicalis R2 CIP 203.

  3. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    Science.gov (United States)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

  4. Agrobacterium deltaense sp. nov., an endophytic bacteria isolated from nodule of Sesbania cannabina.

    Science.gov (United States)

    Yan, Jun; Li, Yan; Han, Xiao Zeng; Chen, Wen Feng; Zou, Wen Xiu; Xie, Zhihong; Li, Meng

    2017-09-01

    A Gram-negative, non-spore-forming, aerobic rods, strain YIC4121 T , was isolated from root nodule of Sesbania cannabina grown in Dongying (Yellow River Delta), Shandong Province, PR China. Based on phylogenetic analysis of 16 S rRNA gene sequences, strain YIC4121 T was assigned to the genus Agrobacterium with 99.7, 99.3, 99.0, 98.8 and 98.7% sequence similarities to Agrobacterium radiobacter LMG140 T , A. pusense NRCPB10 T , A. arsenijevicii KFB 330 T , A. nepotum 39/7 T and A. larrymoorei ATCC51759 T . Analysis of the concatenated housekeeping genes (recA-atpD-glnII), showed lower sequence similarities (≤94.6%) between strain YIC4121 T and other recognized Agrobacterium species. Strain YIC4121 T shared whole-genome average nucleotide identities (ANI) 87.94, 91.27 and 77.42%, with A. pusense NRCPB10 T , A. radiobacter LMG140 T and A. larrymoorei ATCC51759 T . The predominant cellular fatty acids were C 19:0 cyclo ω8c, summed feature 2 (C 12:0 aldehyde/unknown 10.9525), summed feature 8 (C 18:1 ω7c/C 18:1 ω6c), C 16:0 3 OH and C 16:0 . The G + C content of strain YIC4121 T was 59.80 mol%. Tween 80, lactulose, citric acid, α-ketoglutaric acid, glycyl-L-glutamic acid and 2, 3-butanediol could not be utilized as carbon source, distinguishing strain YIC4121 T from the other related species. Based on the distinctly genetic and phenotypic dissimilarity, a novel species Agrobacterium deltaense sp. nov. is proposed with YIC4121 T (=HAMBI 3654 T  = LMG 29283 T ) as the type strain.

  5. AGROBACTERIUM-MEDIATED TRANSFORMATION OF COMPOSITAE PLANTS. I. CONSTRUCTION OF TRANSGENIC PLANTS AND «HAIRY» ROOTS WITH NEW PROPERTIES

    Directory of Open Access Journals (Sweden)

    N. A.Matvieieva

    2013-02-01

    Full Text Available The review explores some of the recent advances and the author's own researchs concerning biotechnological approaches for Agrobacterium tumefaciens- and A. rhizogenes-mediated transformation of Compositae family plants. This paper reviews the results of genetic transformation of Compositae plants, including edible (Cichorium intybus, Lactuca sativa, oil (Helianthus annuus, decorative (Gerbera hybrida, medical (Bidens pilosa, Artemisia annua, Artemisia vulgaris, Calendula officinalis, Withania somnifera etc. plant species. Some Compositae genetic engineering areas are considered including creation of plants, resistant to pests, diseases and herbicides, to the effect of abiotic stress factors as well as plants with altered phenotype. The article also presents the data on the development of biotechnology for Compositae plants Cynara cardunculus, Arnica montana, Cichorium intybus, Artemisia annua "hairy" roots construction.

  6. Agrobacterium VirB10, an ATP energy sensor required for type IV secretion.

    Science.gov (United States)

    Cascales, Eric; Christie, Peter J

    2004-12-07

    Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa. Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate [T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] of the Agrobacterium tumefaciens VirB/D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel. Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility. VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation. Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9. We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 "bridge" between inner and outer membrane subassemblies of the VirB/D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope.

  7. Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line.

    Science.gov (United States)

    Cho, Myeong-Je; Wu, Emily; Kwan, Jackie; Yu, Maryanne; Banh, Jenny; Linn, Wutt; Anand, Ajith; Li, Zhi; TeRonde, Susan; Register, James C; Jones, Todd J; Zhao, Zuo-Yu

    2014-10-01

    An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

  8. Patogenecidad de agrobacterium tumefaciens en algunas especies de plantas de flóres de exportación

    OpenAIRE

    Ovalle Germán; Benincore Germán; Arbeláez Germán

    1984-01-01

    Tres aislamientos de tumefaelens Agrobaeterlum de rosa de las plantas afectadas con agalla de la corona en la Sabana de Bogotá se han caracterizado e identificado por pruebas de laboratorio y mediante la inoculación en plantas de tomate, zanahoria y remolacha. Patogenicidad fue probado en el campo de rosas, claveles, limonium, chrysanthermum y plantas gipsophila que son especies importantes en el país para la producción de flores de exportación. Su patogenia se ha probado también en las plant...

  9. Patogenecidad de agrobacterium tumefaciens en algunas especies de plantas de flóres de exportación

    Directory of Open Access Journals (Sweden)

    Ovalle Germán

    1984-12-01

    Full Text Available Tres aislamientos de tumefaelens Agrobaeterlum de rosa de las plantas afectadas con agalla de la corona en la Sabana de Bogotá se han caracterizado e identificado por pruebas de laboratorio y mediante la inoculación en plantas de tomate, zanahoria y remolacha. Patogenicidad fue probado en el campo de rosas, claveles, limonium, chrysanthermum y plantas gipsophila que son especies importantes en el país para la producción de flores de exportación. Su patogenia se ha probado también en las plantas de eucaliptus, remolacha, tomate y geranio, anfitriones común de las bacterias, estas pruebas se llevaron a cabo en 45 plantas de día y el material de propagación. Los aislamientos fueron altamente patógena de plantas de rosa y eucallptus, moderadamente patógenas de clavel y de hielo y estadísticas presentados pathogeneclty muy bajo en chrysanthemun. No hubo infección en gypsophila. Las plantas de tomate, la remolacha y el geranio presente una alta susceptibilidad a la bacteria y pueden ser de gran utilidad para el diagnóstico.Three isolates of Agrobaeterlum tumefaelens from rose plants affected with crown gall in the Savanna of Bogota have been characterized and identified by laboratory test and by inoculation in tomato plants, carrot and beet root disks. Pathogenecity was tested in the field on rose, carnation, statice, chrysanthermum and gipsophila plants which are important species in the country for the production of export flowers. Their pathogenecity was also tested on eucaliptus, beet, tomato and geranium plants, common hosts of the bacteria; these tests were carried out on 45 days old plants and propagation material. The isolates were highly pathogenic on rose and eucallptus plants, moderately pathogenic on carnation and stat ice and presented very low pathogeneclty on chrysanthemun. There was no Infection on gypsophila. Tomato, beet and geranIum plants present high susceptibility to the bacteria and can be of great usefuiness for the diagnosis of the disease.

  10. Estudios orientados a la transformación de papa criolla (Solanum Phureja mediada por Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Diana Angélica Carvajal Bernal

    2004-07-01

    explantes en medio de regeneración con adición de cefatoxina (250 mg/l. En estas condiciones, se obtuvieron regenerantes potencialmente transformados, y mediante la aplicación de la técnica de PCR se determinó que ninguno era transgénico. El presente estudio se desarrolló con financiación de la Universidad Nacional de Colombia y CEVIPAPA (CV-03-005-02.

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

    KAUST Repository

    Ali, Shawkat; Bakkeren, Guus

    2014-01-01

    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

  12. SacB-SacR gene cassette as the negative selection marker to suppress Agrobacterium overgrowth in Agrobacterium-mediated plant transformation

    Science.gov (United States)

    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. 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 ...

  13. Inhibitory and Toxic Effects of Volatiles Emitted by Strains of Pseudomonas and Serratia on Growth and Survival of Selected Microorganisms, Caenorhabditis elegans, and Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Alexandra A. Popova

    2014-01-01

    Full Text Available In previous research, volatile organic compounds (VOCs emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans. VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds—ketones (2-nonanone, 2-heptanone, 2-undecanone and dimethyl disulfide—were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.

  14. Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

    Science.gov (United States)

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

    2014-06-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)-CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

  15. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

    Science.gov (United States)

    Cardoza, V.; Stewart, C. N.

    2003-01-01

    An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

  16. Optimization of Agrobacterium -mediated transformation parameters ...

    African Journals Online (AJOL)

    Agrobacterium-mediated transformation factors for sweet potato embryogenic calli were optimized using -glucuronidase (GUS) as a reporter. The binary vector pTCK303 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Transformation parameters were optimized including bacterial ...

  17. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

    Science.gov (United States)

    2014-01-01

    Background Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous

  18. Agrobacterium: nature’s genetic engineer

    Science.gov (United States)

    Nester, Eugene W.

    2015-01-01

    Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun’s old observations and also explain why Agrobacterium is nature’s genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442

  19. Degradation of dibenzofuran via multiple dioxygenation by a newly isolated Agrobacterium sp. PH-08.

    Science.gov (United States)

    Le, T T; Murugesan, K; Nam, I-H; Jeon, J-R; Chang, Y-S

    2014-03-01

    To demonstrate the biodegradation of dibenzofuran (DF) and its structural analogs by a newly isolated Agrobacterium sp. PH-08. To assess the biodegradation potential of newly isolated Agrobacterium sp. PH-08, various substrates were evaluated as sole carbon sources in growth and biotransformation experiments. ESI LC-MS/MS analysis revealed the presence of angular degrading by-products as well as lateral dioxygenation metabolites in the upper pathway. The metabolites in the lower pathway also were detected. In addition, the cometabolically degraded daughter compounds of DF-related compounds such as BP and dibenzothiophene (DBT) in dual substrate degradation were observed. Strain PH-08 exhibited the evidence of meta-cleavage pathway as confirmed by the activity and gene expression of catechol-2,3-dioxygenase. Newly isolated bacterial strain, Agrobacterium sp. PH-08, grew well with and degraded DF via both angular and lateral dioxygenation as demonstrated by metabolites identified through ESI LC-MS/MS and GC-MS analyses. The other heterocyclic pollutants were also cometabolically degraded. Few reports have described the complete degradation of DF by a cometabolic lateral pathway. Our study demonstrates the novel results that the newly isolated strain utilized the DF as a sole carbon source and mineralized it via multiple dioxygenation. © 2013 The Society for Applied Microbiology.

  20. Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.

    Science.gov (United States)

    Ceasar, S Antony; Ignacimuthu, S

    2011-09-01

    A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.

  1. Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

    Directory of Open Access Journals (Sweden)

    Julien Lang

    2014-10-01

    Full Text Available By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour, a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D of 0.6 µM greater than that for nopaline (KD of 3.7 µM. Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to

  2. Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

    Science.gov (United States)

    Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

    2014-10-01

    By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche

  3. Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana.

    Science.gov (United States)

    Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi

    2014-04-19

    Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

  4. 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.

  5. Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants.

    Science.gov (United States)

    Barton, Ian S; Fuqua, Clay; Platt, Thomas G

    2018-01-01

    Many important pathogens maintain significant populations in highly disparate disease and non-disease environments. The consequences of this environmental heterogeneity in shaping the ecological and evolutionary dynamics of these facultative pathogens are incompletely understood. Agrobacterium tumefaciens, the causative agent for crown gall disease of plants has proven a productive model for many aspects of interactions between pathogens and their hosts and with other microbes. In this review, we highlight how this past work provides valuable context for the use of this system to examine how heterogeneity and transitions between disease and non-disease environments influence the ecology and evolution of facultative pathogens. We focus on several features common among facultative pathogens, such as the physiological remodelling required to colonize hosts from environmental reservoirs and the consequences of competition with host and non-host associated microbiota. In addition, we discuss how the life history of facultative pathogens likely often results in ecological tradeoffs associated with performance in disease and non-disease environments. These pathogens may therefore have different competitive dynamics in disease and non-disease environments and are subject to shifting selective pressures that can result in pathoadaptation or the within-host spread of avirulent phenotypes. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  6. Agrobacterium-mediated transformation of the wild orchid Cattleya maxima Lindl

    Directory of Open Access Journals (Sweden)

    Augusta Yadira Cueva-Agila

    2018-03-01

    Full Text Available Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80 % than untransformed controls (45 - 57 %, and this contrast was maximized in hormone-free, Murashige and Skoog (MS medium (80 % of the transformed plants compared to 57 % of the untransformed ones. This finding supports the notion that SERK plays an important role in Orchid embryogenesis

  7. A cytometry microparticle platform approach for screening tobacco microRNA changes after agrobacterium delivery

    Energy Technology Data Exchange (ETDEWEB)

    Powell, Joshua D.; Chen, Qiang; Mason, Hugh S.

    2016-08-01

    Abstract Key message nta-miR-398 is significantly up-regulated while nta-miR-428d is significantly down-regulated in tobacco after agroinfiltration AbstractMicroRNAs are a class of non-coding regulatory RNAs that can modulate development as well as alter innate antiviral defenses in plants. In this study we explored host changes at the microRNA level within tobacco (Nicotiana benthamiana) after expression of a recombinant anti-Ebola GP1 antibody through Agrobacterium tumefaciens agroinfiltration delivery. A multiplex nanoparticle-based cytometry assay tracked the host expression changes of 53 tobacco microRNAs. Our results revealed that the most abundant microRNAs in actively growing leaves corresponded to nanoparticle probes specific to nta-mir-6149 and nta-miR-168b. After agroinfiltration, probes targeting nta-mir-398 and nta-mir-482d were significantly altered in their respective expression levels and were further verified through RT-qPCR analysis. To our knowledge this study is the first to profile microRNA expression in tobacco after agroinfiltration using a multiplex nanoparticle approach.

  8. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop.

    Science.gov (United States)

    Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve; Kreuze, Jan F

    2015-05-05

    Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world's arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

  9. Characterization of a new pathovar of Agrobacterium vitis causing banana leaf blight in China.

    Science.gov (United States)

    Huang, Siliang; Long, Mengling; Fu, Gang; Lin, Shanhai; Qin, Liping; Hu, Chunjin; Cen, Zhenlu; Lu, Jie; Li, Qiqin

    2015-01-01

    A new banana leaf blight was found in Nanning city, China, during a 7-year survey (2003-2009) of the bacterial diseases on banana plants. Eight bacterial strains were isolated from affected banana leaves, and identified as an intraspecific taxon of Agrobacterium vitis based on their 16S rDNA sequence similarities with those of 37 randomly selected bacterial strains registered in GenBank database. The representative strain Ag-1 was virulent on banana leaves and shared similar growth and biochemical reactions with the reference strain IAM14140 of A. vitis. The strains causing banana leaf blight were denominated as A. vitis pv. musae. The traditional A. vitis strains virulent to grapevines were proposed to be revised as A. vitis pv. vitis. This is the first record of a new type of A. vitis causing banana leaf blight in China. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

    Science.gov (United States)

    Juan, J X; Yu, X H; Jiang, X M; Gao, Z; Zhang, Y; Li, W; Duan, Y D; Yang, G

    2015-01-30

    ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

  11. A simple and stable method of tagging Agrobacterium fabrum C58 for environmental monitoring

    Directory of Open Access Journals (Sweden)

    Meriam BOURI

    2017-05-01

    Full Text Available Agrobacterium fabrum is one of the eleven Agrobacterium spp. complex species that has been observed to carry a Ti plasmid and induce crown gall, a disease causing significant damage to economically important perennial agricultural crops. Members of this species complex, including A. fabrum, are morphologically indistinguishable from one another on culture media and are known to grow together in soil and within host galls. Consequently, the tracking of this species in its natural environment requires a cautious approach to tagging strains without altering any of their ecologically important traits. A gentamicin resistant cassette (aacC1 was inserted, by homologous recombination, into a non-coding region of the A. fabrum C58 chromosome between the genes atu1182 and atu1183. The resultant strain did not show any significant in vitro growth differences compared to the wild-type strain, and the marker was stable in rich medium, both with and without selective pressure. The mutant/marked strain was indistinguishable from the parental strain for ability to induce galls, grow in bulk soil and colonize the rhizosphere of tomato plants. Easy, precise, safe and stable tagging of the A. fabrum C58 genome facilitates environmental population surveys by either simple selection or direct detection by PCR. This methodology provides understanding of the ecology of this species complex as an integral part of managing the soil microbiota for improved crown gall management.

  12. Extreme resistance to two Brazilian strains of Potato virus Y (PVY in transgenic potato, cv. Achat, expressing the PVYº coat protein Resistência extrema a duas estirpes do Potato virus Y (PVY de batata transgênica, cv. Achat, expressando o gene da capa protéica do PVY O

    Directory of Open Access Journals (Sweden)

    Eduardo Romano

    2001-07-01

    Full Text Available The coat protein (CP gene of the potato virus Y strain "o" (PVY O was introduced into potato, cultivar Achat, via Agrobacterium tumefaciens-mediated transformation. Sixty three putative transgenic lines were challenged against the Brazilian strains PVY-OBR and PVY-NBR. An extremely resistant phenotype, against the two strains, was observed in one line, denominated 1P. No symptoms or positive ELISA results were observed in 16 challenged plants from this line. Another clone, named as 63P, showed a lower level of resistance. Southern blot analysis showed five copies of the CP gene in the extremely resistant line and at least three copies in the other resistant line. The stability of the integrated transgenes in the extreme resistant line was examined during several in vitro multiplications over a period of three years, with no modification in the Southern pattern was observed. The stability of the transgenes, the absence of primary infections and the relatively broad spectrum of resistance suggest that the extremely resistant line obtained in this work can be useful for agricultural purposes.O gene da capa protéica (CP do Potato virus Y estirpe "o", foi introduzido em batata cultivar Achat, via Agrobacterium tumefaciens. Sessenta e três linhas possivelmente transgênicas foram desafiadas com as estirpes brasileiras PVY-OBR e PVY-NBR. Uma linha apresentou extrema resistência às duas estirpes inoculadas, e foi denominado clone 1P. Não foram observados sintomas sistêmicos de infecção e as plantas foram negativas em Elisa. Outra linha, denominada clone 63P, mostrou algum nível de resistência. Análises por Southern blot indicaram a presença de pelo menos cinco cópias do gen CP no clone 1P e pelo menos três cópias no clone 63P. A estabilidade do gene introduzido no clone 1P foi avaliada durante três anos, após várias multiplicações in vitro. Não foram observadas mudanças no padrão do Southern blot. A estabilidade do transgene, na

  13. A highly efficient method for Agrobacterium mediated transformation ...

    African Journals Online (AJOL)

    An Agrobacterium mediated transformation method was developed for the Thai rice variety, Pathumthani 1 (PT1), and the Indian rice variety, Pokkali (PKL). Various aspects of the transformation method, including callus induction, callus age, Agrobacterium concentration and co-cultivation period were examined, in order to ...

  14. Host genes involved in Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Soltani, Jalal

    2009-01-01

    Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the

  15. Agrobacterium -induced hypersensitive necrotic reaction in plant cells

    African Journals Online (AJOL)

    High necrosis and poor survival rate of target plant tissues are some of the major factors that affect the efficiency of Agrobacterium-mediated T-DNA transfer into plant cells. These factors may be the result of, or linked to, hypersensitive defense reaction in plants to Agrobacterium infection, which may involve the recognition ...

  16. Do leaf surface characteristics affect Agrobacterium infection in tea

    Indian Academy of Sciences (India)

    The host range specificity of Agrobacterium with five tea cultivars and an unrelated species (Artemisia parviflora) having extreme surface characteristics was evaluated in the present study. The degree of Agrobacterium infection in the five cultivars of tea was affected by leaf wetness, micro-morphology and surface chemistry.

  17. Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

    Science.gov (United States)

    Raji, Jennifer A; Frame, Bronwyn; Little, Daniel; Santoso, Tri Joko; Wang, Kan

    2018-01-01

    Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

  18. AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION OF SORGHUM USING TISSUE CULTURE-BASED AND POLLEN-MEDIATED APPROACHES

    Directory of Open Access Journals (Sweden)

    Elkonin L.A.

    2012-08-01

    Full Text Available Genetic transformation is a powerful tool for genetic improvement of arable crops. Genetic engineering approaches are especially important for modification of starch and protein contents, vitamin and micronutrient concentration, improvement of nutritive value of protein fractions, and increase tolerance to environmental stresses. Application of transgenic technologies for genetic improvement of sorghum, a highly productive heat tolerant and drought resistant crop, is extremely important since climate aridization in many regions all over the globe hampers sustainable production of traditional cereals, such as wheat, maize and barley. However, sorghum, in spite of great number of investigations, is one of the most recalcitrant crop species to genetic modification. The most frequently reported problems are a low frequency of transformation and silencing of transgenes. Using the A. tumefaciens strain AGL0/p35SGIB with the bar and gus-intron genes under the nos and CaMV35S promoters, respectively, we studied different methods of Agrobacterium-mediated genetic transformation of the grain sorghum: in vitro culture-based techniques, by inoculation of immature embryos or embryo-derived calli, and pollen-mediated approach, by inoculation of flowering panicles. Four lines of grain sorghum – Milo-10, [9E] Milo-10 (CMS-line, KVV-114, and KVV-45 – were used. In both approaches, for activation of vir-genes agrobacterial cell suspension was grown in the AB or modified AB media with acetosyringone at room temperature. In vitro culture approach was effective for obtaining transgenic plants in the lines Milo-10 and KVV-45, which were able to produce embryogenic callus from immature embryos after their co-cultivation with agrobacterial cell suspension. Callus cultures tolerant to glufosinate ammonium (GA and capable to plant regeneration were obtained. The frequency of immature embryos producing PCR-positive transgenic plants varied in different experiments

  19. Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

    Science.gov (United States)

    Govender, Nisha; Wong, Mui-Yun

    2017-04-01

    A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

  20. Agrobacterium-mediated genetic transformation of Coffea arabica (L. is greatly enhanced by using established embryogenic callus cultures

    Directory of Open Access Journals (Sweden)

    Lashermes Philippe

    2011-05-01

    Full Text Available Abstract Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%. At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our

  1. Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

    Science.gov (United States)

    2011-01-01

    Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium

  2. Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

    OpenAIRE

    Hao, Guixia; Burr, Thomas J.

    2006-01-01

    Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both...

  3. High-throughput Agrobacterium-mediated barley transformation

    Directory of Open Access Journals (Sweden)

    Snape John W

    2008-09-01

    Full Text Available Abstract Background Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low ( Results A robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos with Agrobacterium strain AGL1, carrying vectors from the pBract series that contain the hpt gene (conferring hygromycin resistance as a selectable marker. Results of large scale experiments utilising the luc (firefly luciferase gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene. Conclusion This protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.

  4. Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Tang, Lixia; Lutje Spelberg, Jeffrey H.; Fraaije, Marco W.; Janssen, DB

    2003-01-01

    Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic

  5. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

    Science.gov (United States)

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-07-27

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Promoter deletions are essential for transformation of lettuce by the T-cyt gene: the phenotypes of transgenic plants

    NARCIS (Netherlands)

    Curtis, H.E.C.; Jordi, W.; Davelaar, E.; Power, J.B.; Laat, de A.M.M.; Davey, M.R.

    1999-01-01

    The Agrobacterium T- cyt gene was transferred into lettuce, Latuca sativa‘Saladin’ using a genotype-independent transformation procedure employing a supervirulent Agrobacterium tumefaciens strain carrying the binary vector pMOG23. Kanamycin-resistant shoots were initiated from inoculated explants

  7. Agrobacterium salinitolerans sp. nov., a saline-alkaline-tolerant bacterium isolated from root nodule of Sesbania cannabina.

    Science.gov (United States)

    Yan, Jun; Li, Yan; Yan, Hui; Chen, Wen Feng; Zhang, Xiaoxia; Wang, En Tao; Han, Xiao Zeng; Xie, Zhi Hong

    2017-06-01

    Two Gram-staining-negative, aerobic bacteria (YIC 5082T and YIC4104) isolated from root nodules of Sesbania cannabina grown in a high-salt and alkaline environment were identified as a group in the genus Agrobacterium because they shared 100 and 99.7 % sequence similarities of 16S rRNA and recA+atpD genes, respectively. These two strains showed 99.2/100 % and 93.9/95.4 % 16S rRNA and recA+atpD gene sequence similarities to Agrobacterium radiobacter LMG140T and Agrobacterium. pusense NRCPB10T, respectively. The average nucleotide identities (ANI) of genome sequences were 89.95 % or lower between YIC 5082T and the species of the genus Agrobacterium examined. Moreover, these two test strains formed a unique nifH lineage deeply separated from other rhizobia. Although the nodC gene was not detected in YIC 5082T and YIC4104, they could form effective root nodules on S. cannabina plants. The main cellular fatty acids in YIC 5082T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C19 : 0cyclo ω8c, summed feature 2 (C12 : 0 aldehyde/unknown equivalent chain length 10.9525) and C16 : 0. The DNA G+C content of YIC 5082T was 59.3 mol%. The failure to utilize d-sorbitol as a carbon source distinguished YIC 5082T from the type strains of related species. YIC 5082T could grow in presence of 5.0 % (w/v) NaCl and at a pH of up to 10.0. Based on results regarding the genetic and phenotypic properties of YIC 5082T and YIC4104 the name Agrobacterium salinitolerans sp. nov. is proposed and YIC 5082T (=HAMBI 3646T=LMG 29287T) is designed as the type strain.

  8. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis.

    Science.gov (United States)

    Rana, Mohammad M; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

    2016-07-15

    Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L(-1) sucrose, 0.1 g·L(-1) l-glutamine and 5 g·L(-1) polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L(-1) sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

  9. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis

    Directory of Open Access Journals (Sweden)

    Mohammad M. Rana

    2016-07-01

    Full Text Available Tea (Camellia sinensis L. is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS basal salts medium supplemented with 30 g·L−1 sucrose, 0.1 g·L−1 l-glutamine and 5 g·L−1 polyvinylpolypyrrolidone (PVPP as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L−1 sucrose. Additionally, the reporter genes β-glucuronidase (gusA and cyan fluorescent protein (cfp were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

  10. Traversing the Cell: Agrobacterium T-DNA’s Journey to the Host Genome

    Science.gov (United States)

    Gelvin, Stanton B.

    2012-01-01

    The genus Agrobacterium is unique in its ability to conduct interkingdom genetic exchange. Virulent Agrobacterium strains transfer single-strand forms of T-DNA (T-strands) and several Virulence effector proteins through a bacterial type IV secretion system into plant host cells. T-strands must traverse the plant wall and plasma membrane, traffic through the cytoplasm, enter the nucleus, and ultimately target host chromatin for stable integration. Because any DNA sequence placed between T-DNA “borders” can be transferred to plants and integrated into the plant genome, the transfer and intracellular trafficking processes must be mediated by bacterial and host proteins that form complexes with T-strands. This review summarizes current knowledge of proteins that interact with T-strands in the plant cell, and discusses several models of T-complex (T-strand and associated proteins) trafficking. A detailed understanding of how these macromolecular complexes enter the host cell and traverse the plant cytoplasm will require development of novel technologies to follow molecules from their bacterial site of synthesis into the plant cell, and how these transferred molecules interact with host proteins and sub-cellular structures within the host cytoplasm and nucleus. PMID:22645590

  11. Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

    Science.gov (United States)

    Stephan, B I; Alvarez Crespo, M C; Kemppainen, M J; Pardo, A G

    2017-05-01

    Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

  12. 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 ...

  13. 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...

  14. 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.

  15. 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.

  16. 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...

  17. Hypericin and hyperforin production in St. John’s wort in vitro culture: Influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens

    Czech Academy of Sciences Publication Activity Database

    Pavlík, M.; Vacek, Jan; Klejdus, B.; Kubáň, V.

    2007-01-01

    Roč. 55, č. 15 (2007), s. 6147-6153 ISSN 0021-8561 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : hypericin * hyperforin * saccharose Subject RIV: BO - Biophysics Impact factor: 2.532, year: 2007

  18. Characterization of Agrobacterium species by capillary isoelectric focusing

    Czech Academy of Sciences Publication Activity Database

    Süle, S.; Horká, Marie; Matoušková, H.; Kubesová, Anna; Šalplachta, Jiří; Horký, J.

    2012-01-01

    Roč. 132, č. 1 (2012), s. 81-89 ISSN 0929-1873 R&D Projects: GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : capillary isoelectric focusing * Agrobacterium spp. * identification Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.610, year: 2012

  19. Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

    1998-01-01

    The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

  20. agrobacterium-mediated transformation of common bean abstract

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    confirmées par la culture histochimique pour activité GUS étaient obtenues dans les plantules ... plant tissues but not in bacterial cells and can be ... fluorescent protein (GFP) techniques (Zambre et ..... affecting Agrobacterium-mediated trans-.

  1. Isolation and characterization of curdlan produced by Agrobacterium HX1126 using α-lactose as substrate.

    Science.gov (United States)

    Liu, Yongmei; Gu, Qiuya; Ofosu, Fred Kwame; Yu, Xiaobin

    2015-11-01

    A strain Agrobacterium HX1126 was isolated from soil sample near the canal in Wuxi. α-lactose was used as the sole carbon source for the production of an exopolysaccharide which was named PLHX. The highest production of PLHX (21.4g/L) was obtained under nitrogen depletion. PLHX composed mainly of glucose, with lower amounts of galactose and aminogalactose. The structure of the product was confirmed by NMR and FTIR and was identified as curdlan. This exopolysaccharide formed a gel when 30g/L was put in boiling water for 10min, with an achieved gel strength of 831g/cm(2). Moreover, a hypothesis for higher gel strength production is proposed. The gel forming property makes this exopolysaccaride a good potential application in the food, pharmaceutical and cosmetic industries. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Cell wall biochemical alterations during Agrobacterium-mediated expression of haemagglutinin-based influenza virus-like vaccine particles in tobacco.

    Science.gov (United States)

    Le Mauff, François; Loutelier-Bourhis, Corinne; Bardor, Muriel; Berard, Caroline; Doucet, Alain; D'Aoust, Marc-André; Vezina, Louis-Philippe; Driouich, Azeddine; Couture, Manon M-J; Lerouge, Patrice

    2017-03-01

    Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

    Science.gov (United States)

    Pecenková, Tamara; Janda, Martin; Ortmannová, Jitka; Hajná, Vladimíra; Stehlíková, Zuzana; Žárský, Viktor

    2017-09-01

    Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated. Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. maculicola (Psm) and P. syringae pv. tomato DC3000 (Pst), as well as Agrobacterium tumefaciens (Atu) and Escherichia coli (Eco). Root hair lengths were measured after 24 and 48h of direct exposure to each bacterial strain. Several Arabidopsis mutants with impaired responses to pathogens, impaired ethylene perception and defects in the exocyst vesicle tethering complex that is involved in secretion were also analysed. Arabidopsis seedling roots infected with Psm or Pst responded similarly to when infected with plant growth-promoting rhizobacteria; root hair growth was stimulated and primary root growth was inhibited. Other plant- and soil-adapted bacteria induced similar root hair responses. The most compromised root hair growth stimulation response was found for the knockout mutants exo70A1 and ein2. The single immune pathways dependent on salicylic acid, jasmonic acid and PAD4 are not directly involved in root hair growth stimulation; however, in the mutual cross-talk with ethylene, they indirectly modify the extent of the stimulation of root hair growth. The Flg22 peptide does not initiate root hair stimulation as intact bacteria do, but pretreatment with Flg22 prior to Psm inoculation abolished root hair growth stimulation in an FLS2 receptor kinase-dependent manner. These early response phenomena are not associated with changes in auxin levels, as monitored with the pDR5::GUS auxin reporter. Early stimulation of root hair growth is an effect of an unidentified component of living plant pathogenic bacteria. The root

  4. Agrobacterium-Mediated Transformation of Leaf Base Segments.

    Science.gov (United States)

    Gasparis, Sebastian

    2017-01-01

    Agrobacterium-mediated transformation has become a routine method of genetic engineering of cereals, gradually replacing the biolistic protocols. Simple integration patterns of transgenic loci, decent transformation efficiency, and technical simplicity are the main advantages offered by this method. Here we present a detailed protocol for the production of transgenic oat plants by Agrobacterium-mediated transformation of leaf base segments. The use of leaf explants as target tissues for transformation and in vitro regeneration of transgenic plants may be a good alternative for genotypes which are not susceptible to regeneration from immature or mature embryos. We also describe the biochemical and molecular analysis procedures of the transgenic plants including a GUS histochemical assay, and Southern blot, both of which are optimized for application in oat.

  5. Cannabis sativa L. genetically transformed root based culture via Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Farnoush Berahmand

    2016-09-01

    Full Text Available It is an increased interest in the therapeutic potential of Cannabis sativa L. (marijuana for treatment of multiple sclerosis and HIV neuropathy. Because of limitation in cultivation of this plant, an efficient hairy root induction system for Cannabis sativa L. was developed in the present study. Agrobacterium rhizogenes mediated transformation performed by two different co-cultivation mediums and four different bacterial strains including A4, ATCC15834, MSU440, and A13 (MAFF-02-10266. Genomic DNA from putative transgenic hairy root lines and the control root was extracted using a modified CTAB protocol. Molecular analysis of transformed root lines was confirmed by polymerase chain reaction using specific primers of the rolB gene. The transformation frequency by Murashige and Skoog co-cultivation medium resulting in hairy root induction frequencies of 42.3%, 46.3%, 68.6% and 39.3% by A4, ATCC15834, MSU440, and A13 strains, respectively. There was no significant difference between MS or ½ MS co-cultivation mediums.  This study established a reliable protocol for induction of hairy roots of C. sativa. The best A. rhisogenes strain was MSU440. It was observed no significant difference between MS and ½ MS co-cultivation mediums on transformation frequency.

  6. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

    Science.gov (United States)

    Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

    2017-02-01

    CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  7. Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae

    OpenAIRE

    Liu, C.-M.; McLean, P. A.; Sookdeo, C. C.; Cannon, F. C.

    1991-01-01

    Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). All organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not a...

  8. 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...

  9. Identification of substituent groups and related genes involved in salecan biosynthesis in Agrobacterium sp. ZX09.

    Science.gov (United States)

    Xu, Linxiang; Cheng, Rui; Li, Jing; Wang, Yang; Zhu, Bin; Ma, Shihong; Zhang, Weiming; Dong, Wei; Wang, Shiming; Zhang, Jianfa

    2017-01-01

    Salecan, a soluble β-1,3-D-glucan produced by a salt-tolerant strain Agrobacterium sp. ZX09, has been the subject of considerable interest in recent years because of its multiple bioactivities and unusual rheological properties in solution. In this study, both succinyl and pyruvyl substituent groups on salecan were identified by an enzymatic hydrolysis following nuclear magnetic resonance (NMR), HPLC, and MS analysis. The putative succinyltransferase gene (sleA) and pyruvyltransferase gene (sleV) were determined and cloned. Disruption of the sleA gene resulted in the absence of succinyl substituent groups on salecan. This defect could be complemented by expressing the sleA cloned in a plasmid. Thus, the sleA and sleV genes located in a 19.6-kb gene cluster may be involved in salecan biosynthesis. Despite the lack of succinyl substituents, the molecular mass of salecan generated by the sleA mutant did not substantially differ from that generated by the wild-type strain. Loss of succinyl substituents on salecan changed its rheological characteristics, especially a decrease in intrinsic viscosity.

  10. Callogenesis in root explants of four species of the family Solanaceae after inducing by Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Zahra Shakeran

    2015-09-01

    Full Text Available Studying explants affected by Agrobacterium rhizogenes shows that in addition to possible formation of hairy roots, it is likely that callogenesis can be induced in these tissues. The T-DNA region of A. rhizogenes codes enzymes that participate in biosynthesis of plants growth hormones. These hormones also affect callogenesis, hence, the formation of various calluses with different morphological properties are possible. It is very likely that the level of biosynthetic growth hormone, the plasmid carried by each bacteria strain, the position of T-DNA, and the level of gene expression contribute to this morphologic variation. In this study, the root explants of four species of the family Solanaceae namely Atropa belladonna, Datura metel, D. stramonium and Hyoscyamus niger were induced by using different strains of A. rhizogenes (A4, A7, AR15834, AR318, AR9402 and AR9543. Some of these explants entered callus phase and formed various calluses with different colors and shapes. Moreover, in some callus samples hairy roots were also appeared. These variations were probably caused by variations in the levels and ratios of auxin and cytokinine hormons after the induction. As shown in previous studies, the amount of secondary metabolites is reduced due to undifferentiated tissue produced in the callogenesis process.

  11. Biolistic- and Agrobacterium-mediated transformation protocols for wheat.

    Science.gov (United States)

    Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László

    2012-01-01

    After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.

  12. Effect of gamma irradiation on Agrobacterium-mediated genetic transformation of Japanese lawngrass (Zoysia japonica Steud.)

    International Nuclear Information System (INIS)

    Zhang Lei; Anhui Agricultural Univ., Hefei; Hu Fanrong; Zhang Linlin; Wang Xueyan; Wu Dianxing; Ma Chuanxi

    2004-01-01

    The effects of gamma irradiation on Agrobacterium-mediated genetic transformation were investigated in the current paper, using embryonic calli derived from the mature seeds of Japanese lawngrass (Zoysia japonica Steud.). The result indicated that the GUS transient expression rates were enhanced with the increasing doses when treated by doses lower than 4 Gy, however it would be decreased when treated by doses higher than 4 Gy. Based on the survival rate and GUS transient expression rate, 2 Gy is the optimal dose for Agrobacterium-mediated genetic transformation. Further observation found that 36 hours reculture after gamma irradiation is the most appropriate for agrobacterium infection. (authors)

  13. Study on attachment of agrobacterium tumefanciens with citrus reticulata var. tankan hayata embryogenic cells

    International Nuclear Information System (INIS)

    Wang Shengbin; Yu Rangcai; Zou Weiquan; Huang Ziran

    2002-01-01

    32 P-NaH 2 PO 4 in LB medium had no negative effect on the growth of Agrobacterium tumefanciens when below the concentration of 1.169 x 10 5 Bq/ml, indicating that Agrobacterium tumefanciens could be radiolabeled with 32 P-NaH 2 PO 4 . The kinetic experiment showed that attachment of agrobacterium tumefanciens to citrus embryogenic cells was a dynamic binding. The number of bacteria attached to embryogenic cells was correlated with bacterial population and was not affected by Aceto-syringone (As) or Tween-20 in the co-cultivation medium

  14. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) shoot apex with a fungal phytase gene improves phosphorus acquisition.

    Science.gov (United States)

    Ma, Zhiying; Liu, Jianfeng; Wang, Xingfen

    2013-01-01

    Cotton is an important world economic crop plant. It is considered that cotton is recalcitrant to in vitro proliferation. Somatic embryogenesis and plant regeneration has been successful by using hypocotyl, whereas it is highly genotype dependent. Here, a genotype-independent cotton regeneration protocol from shoot apices is presented. Shoot apices from 3- to 5-day-old seedlings of cotton are infected with an Agrobacterium strain, EHA105, carrying the binary vector pC-KSA contained phytase gene (phyA) and the marker gene neomycin phosphotransferase (NPTII), and directly regenerated as shoots in vitro. Rooted shoots can be obtained within 6-8 weeks. Plants that survived by leaf painting kanamycin (kan) were -further analyzed by DNA and RNA blottings. The transgenic plants with increased the phosphorus (P) acquisition efficiency were obtained following the transformation method.

  15. Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.

    Science.gov (United States)

    Sun, Wenyi; Yang, Xiaobing; Wang, Xueying; Lin, Xinping; Wang, Yanan; Zhang, Sufang; Luan, Yushi; Zhao, Zongbao K

    2017-07-01

    To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

  16. Artemisia tilesii Ledeb hairy roots establishment using Agrobacterium rhizogenes-mediated transformation.

    Science.gov (United States)

    Matvieieva, N A; Shakhovsky, A M; Belokurova, V B; Drobot, K O

    2016-05-18

    An efficient and rapid protocol for the establishment of Artemisia tilesii "hairy" root culture is reported. Leaf explants of aseptically growing plants were cocultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5-6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator Emistim on the stimulation of A. tilesii "hairy" root growth were studied. Maximum stimulating effect both for the control and for transgenic roots was observed in case of root cultivation on the media supplemented with IBA-up to 7.95- and 9.1-fold biomass increase, respectively. Cultivation on the medium with 10 μl/L Emistime has also led to the control roots growth stimulation (up to 2.75-fold). Emistime at 5 μl/L concentration led to 5.46-fold mass increase in only one "hairy" root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.

  17. An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

    Science.gov (United States)

    Anand, Ajith; Bass, Steven H; Wu, Emily; Wang, Ning; McBride, Kevin E; Annaluru, Narayana; Miller, Michael; Hua, Mo; Jones, Todd J

    2018-05-01

    A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.

  18. Agrobacterium-assisted selenium nanoparticles: molecular aspect of antifungal activity

    Science.gov (United States)

    Kumar, Anil; Bera, Smritilekha; Singh, Man; Mondal, Dhananjoy

    2018-03-01

    Selenium nanoparticles (SeNPs) were synthesized through the bioreduction of sodium selenite (Na2SeO3) using gram-negative agrobacterium (AGBT) species. Subsequently, their physicochemical properties (pH, viscosity and surface tension) and medicinal activities as anti-dermatophyte against soil keratinophilic fungi at the molecular level were assessed. UV-visible and FTIR spectroscopic data of the biologically synthesized SeNPs were then recorded for confirming the presence of native biological materials adhered to nanoparticles, which are inherently required to enhance the stability and solubility through inhibition of the nanoparticle’s natural aggregation and agglomeration. The λ max value between 290-300 nm in the absorption spectra of the biogenic materials in different concentrations of the Na2SeO3 corroborated the presence of SeNPs in the solution. The interaction of SeNPs in solution state was further studied through the determination of pH, viscosity and surface tension values of agrobacterium-derived SeNPs in different solvents. The pH value of SeNPs dispersed in water is reported as above 7.0 and the average viscosity, and surface tensions of the SeNPs are appeared as near to the water. The particle size distribution was further determined by DLS and the highest % of particle size of the synthesized SeNPs is found in between 200-300 nm. The anti-dermatophyte activity and molecular interaction with fungi DNA molecules were assessed providing the highest anti-dermatophyte activity at 0.1 M concentration and it is observed that the quantities and qualities of fungi DNA were affected by SeNPs. Considering all the outcomes of the studies together, our findings suggest that agrobacterium-mediated synthesis of SeNPs is dependent on bacterial metabolisms but not on the concentration of Na2SeO3 and are promising selenium-derived species with potential application in the prevention of fungal infection through denaturation of fungi DNA.

  19. Genetic transformation of switchgrass.

    Science.gov (United States)

    Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

    2009-01-01

    Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

  20. Maize (Zea mays L.).

    Science.gov (United States)

    Frame, Bronwyn; Warnberg, Katey; Main, Marcy; Wang, Kan

    2015-01-01

    Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

  1. Molecular Characterization of the Genes pcaG and pcaH, Encoding Protocatechuate 3,4-Dioxygenase, Which Are Essential for Vanillin Catabolism in Pseudomonas sp. Strain HR199

    Science.gov (United States)

    Overhage, Jörg; Kresse, Andreas U.; Priefert, Horst; Sommer, Horst; Krammer, Gerhard; Rabenhorst, Jürgen; Steinbüchel, Alexander

    1999-01-01

    Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional β subunit of the protocatechuate 3,4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis,cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway

  2. Expressão eficiente do gene reporter beta-glucuronidase nos tecidos vasculares de batata (Solanum tuberosum L. utilizando de um promotor específico (BRA3 de Agrobacterium rhizogenes Efficient expression of beta-glucuronidase reporter gene in vascular tissue of potato (Solanum tuberosum L. utilizing a specific promoter (BRA3 from Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Antonio Carlos Torres

    2003-06-01

    Full Text Available Promotores tecido-específico controlam a transcrição de genes em diferentes tecidos vegetais bem como em diferentes estádios de desenvolvimento da planta, levando à indução de distintos níveis de atividade transiente e/ou estável do gene. Tais promotores podem ser empregados para a expressão seletiva de genes de interesse. O promotor rol A de Agrobacterium rhizogenes, por exemplo, é floema-específico, sugerindo que possa ser empregado em estratégias de defesa de plantas que são infectadas por vírus com replicação restrita ao floema. A expressão do gene marcador da ß-glucuronidase (gus dirigido pelo promotor rol A (pBRA3 foi observada em plantas transgênicas de batata (cvs. Macaca e Baronesa. Entrenós e secções de folhas foram submetidos ao cocultivo com A. tumefaciens. A atividade do gene gus avaliada em brotações resistentes à canamicina não se restringiu ao floema (alto nível de expressão do gene, mas também se manifestou no xilema dos caules. As expressões transiente e estável são, no entanto, tecido-específicas, localizadas sobretudo no sistema vascular de entrenós e ausente em raízes e folhas. As plantas gus positivas foram micropropagadas, plantadas em casa de vegetação e avaliadas por PCR, utilizando-se 'primers' específicos para o gene npt II. Nenhuma alteração fenotípica foi observada em plantas transgênicas, em relação às não transformadas.Tissue-especific promoters allow the modulation of gene transcription in different tissue types as well as in different stages of plant development, leading different levels of transient and stable activity of the gene product. These promoters have been employed for selective gene expression. The Agrobacterium rhizogenes rol A gene promoter (BRA3 controls phloem-specific expression indicating that this promoter might have an important role in plant defense strategies against virus which replicated only in the phloem. The expression of

  3. 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.

  4. Effective elimination of chimeric tissue in transgenics for the stable genetic transformation of lesquerella fendleri

    Science.gov (United States)

    In order to improve the potential of Lesquerella fendleri as a valuable industrial oilseed crop, a stable genetic transformation system was developed. Genetic transformation was performed by inoculating leaf segments with an Agrobacterium tumefaciens strain AGL1 carrying binary vector pCAMBIA 1301.1...

  5. Effective Agrobacterium–mediated transformation of pineapple with ...

    African Journals Online (AJOL)

    user7

    Agrobacterium tumefaciens strain LBA4404 harboring a plant expression vector pUHA1 containing a ... hybridization of this self-incompatible monocot species, only a few new hybrids ... CYP1A1 is a member of the P450 gene family isolated.

  6. 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

  7. Genetic transformation of garlic ( Allium sativum L.) with tobacco ...

    African Journals Online (AJOL)

    Garlic yield and quality have decreased due to white rot disease caused by Sclerotium cepivorum Berk. A transformation protocol to introduce tobacco chitinase and glucanase genes into garlic embryogenic calli using Agrobacterium tumefaciens has been established. LBA4404 strain having pC2301CHGLU plasmid with ...

  8. In planta transformation of sorghum (Sorghum bicolor (L.) Moench)

    Indian Academy of Sciences (India)

    An in planta transformation protocol for sorghum (Sorghum bicolor (L.) Moench) using shoot apical meristem of germinating seedlings is reported in this study. Agrobacterium tumefaciens strain, LBA4404 with pCAMBIA1303 vector and construct pCAMBIA1303TPS1 were individually used for transformation. Since, the ...

  9. Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

    Science.gov (United States)

    Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D; Xia, Lan-Qin

    2016-01-01

    Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium -mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

  10. Generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation

    Directory of Open Access Journals (Sweden)

    Wang Genping

    2016-09-01

    Full Text Available Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154 and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154 were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of ‘clean’ GM wheat containing only the foreign genes of agronomic importance.

  11. Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi.

    Science.gov (United States)

    Lin, Xinping; Liu, Sasa; Bao, Ruiqi; Gao, Ning; Zhang, Sufang; Zhu, Rongqian; Zhao, Zongbao Kent

    2017-11-01

    Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.

  12. Genetic control and regulatory mechanisms of succinoglycan and curdlan biosynthesis in genus Agrobacterium.

    Science.gov (United States)

    Wu, Dan; Li, Ang; Ma, Fang; Yang, Jixian; Xie, Yutong

    2016-07-01

    Agrobacterium is a genus of gram-negative bacteria that can produce several typical exopolysaccharides with commercial uses in the food and pharmaceutical fields. In particular, succinoglycan and curdlan, due to their good quality in high yield, have been employed on an industrial scale comparatively early. Exopolysaccharide biosynthesis is a multiple-step process controlled by different functional genes, and various environmental factors cause changes in exopolysaccharide biosynthesis through regulatory mechanisms. In this mini-review, we focus on the genetic control and regulatory mechanisms of succinoglycan and curdlan produced by Agrobacterium. Some key functional genes and regulatory mechanisms for exopolysaccharide biosynthesis are described, possessing a high potential for application in metabolic engineering to modify exopolysaccharide production and physicochemical properties. This review may contribute to the understanding of exopolysaccharide biosynthesis and exopolysaccharide modification by metabolic engineering methods in Agrobacterium.

  13. Transcriptome Analysis of Maize Immature Embryos Reveals the Roles of Cysteine in Improving Agrobacterium Infection Efficiency

    Science.gov (United States)

    Liu, Yan; Zhang, Zhiqiang; Fu, Junjie; Wang, Guoying; Wang, Jianhua; Liu, Yunjun

    2017-01-01

    Maize Agrobacterium-mediated transformation efficiency has been greatly improved in recent years. Antioxidants, such as, cysteine, can significantly improve maize transformation frequency through improving the Agrobacterium infection efficiency. However, the mechanism underlying the transformation improvement after cysteine exposure has not been elucidated. In this study, we showed that the addition of cysteine to the co-cultivation medium significantly increased the Agrobacterium infection efficiency of hybrid HiII and inbred line Z31 maize embryos. Reactive oxygen species contents were higher in embryos treated with cysteine than that without cysteine. We further investigated the mechanism behind cysteine-related infection efficiency increase using transcriptome analysis. The results showed that the cysteine treatment up-regulated 939 genes and down-regulated 549 genes in both Z31 and HiII. Additionally, more differentially expressed genes were found in HiII embryos than those in Z31 embryos, suggesting that HiII was more sensitive to the cysteine treatment than Z31. GO analysis showed that the up-regulated genes were mainly involved in the oxidation reduction process. The up-regulation of these genes could help maize embryos to cope with the oxidative stress stimulated by Agrobacterium infection. The down-regulated genes were mainly involved in the cell wall and membrane metabolism, such as, aquaporin and expansin genes. Decreased expression of these cell wall integrity genes could loosen the cell wall, thereby improving the entry of Agrobacterium into plant cells. This study offers insight into the role of cysteine in improving Agrobacterium-mediated transformation of maize immature embryos. PMID:29089955

  14. Agrobacterium rhizogenes-Mediated Transformation – a Non-GMO Platform For Developing Compact Ornamentals

    DEFF Research Database (Denmark)

    Lütken, Henrik Vlk; Hegelund, Josefine Nymark; Lauridsen, Uffe Bjerre

    of these compounds are potentially harmful to both the environment and human health. A new non-GMO molecular breeding strategy, as opposed to both the application of chemical growth retardants and conventional molecular breeding is Agrobacterium rhizogenes-mediated transformation. In this method, the soil borne...... for transformations, plants produced via this approach are not considered as GMOs in the European Union and Japan. We have developed an optimised Agrobacterium rhizogenes-mediated transformation platform useful for a wide range of ornamentals. Kalanchoë was the starting point and the effect of the rol-genes has now...

  15. Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

    Science.gov (United States)

    Jones, Richard W

    2016-01-01

    When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced. Published by Elsevier B.V.

  16. Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

    Science.gov (United States)

    Jia, Yuying; Yao, Xingdong; Zhao, Mingzhe; Zhao, Qiang; Du, Yanli; Yu, Cuimei; Xie, Futi

    2015-08-07

    The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

  17. Agrobacterium-mediated transformation of the recalcitrant Vanda Kasem's Delight orchid with higher efficiency.

    Science.gov (United States)

    Gnasekaran, Pavallekoodi; Antony, Jessica Jeyanthi James; Uddain, Jasim; Subramaniam, Sreeramanan

    2014-01-01

    The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600 nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 μM acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

  18. An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Pratheesh, P T; Vineetha, M; Kurup, G Muraleedhara

    2014-06-01

    Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 μM acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae.

  19. A technique to study Meloidogyne arenaria resistance in Agrobacterium rhizogenes-transformed peanut

    Science.gov (United States)

    A reliable peanut root transformation system would be useful to study the functions of genes involved in root biology and disease resistance. The objective of this study was to establish an effective protocol to produce composite plants mediated by Agrobacterium rhizogenes transformation. More tha...

  20. Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process

    Directory of Open Access Journals (Sweden)

    Yuying Jia

    2015-08-01

    Full Text Available The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman, which showed a relatively weak susceptibility. Gibberellin (GA levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA. Higher zeatin riboside (ZR content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA content, polyphenol oxidase (PPO and peroxidase (POD activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

  1. Optimization of Agrobacterium-mediated transient expression of heterologous genes in spinach

    DEFF Research Database (Denmark)

    Cao, Dang Viet; Pamplona, Reniel S.; Kim, Jiwon

    2017-01-01

    The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this st...

  2. Agrobacterium-Mediated Transformation of Bread and Durum Wheat Using Freshly Isolated Immature Embryos

    Science.gov (United States)

    Wu, Huixia; Doherty, Angela; Jones, Huw D.

    Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

  3. Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

    Science.gov (United States)

    Mhatre, Minal

    2013-01-01

    Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

  4. Agrobacterium-mediated transformation: state of the art and future prospect

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Great progress has been made in recent years in studies on the mechanism of Agrobacterium-mediated transformation and its application. Many details of the key molecular events within the bacterial cells involved in T-DNA transfer have been elucidated, and it is notable that some plant factors which were elusive before are purified and characterized. Vast kinds of species, which were either recalcitrant to or not included in the host range of Agrobacterium, can now be transformed by this bacterium, and they include the very important cereal species, gymnosperms, yeast and many filamentous fungi. The simple in vivo transformation of tissue in intact plants and the "agrolistic" methods to transform recalcitrant plants are the two novel technical achievements. Combined with other powerful techniques such as bacterial artificial chromosome, very large DNA fragment can be transformed into the plant genome by Agrobacterium. Further studies will elucidate more plant-encoded factors involved in T-DNA transformation and there is a need to develop more powerful Agrobacterium-based transformation systems to meet different needs in basic research and crop improvement practice.

  5. In Vitro Callogenesis and Agrobacterium-Mediated Transformation of Globe Artichoke

    NARCIS (Netherlands)

    Menin, B.; Moglia, A.; Comino, C.; Lanteri, S.; Herpen, van T.W.J.M.; Beekwilder, M.J.

    2012-01-01

    Micropropagation techniques have been widely applied in globe artichoke (C. cardunculus L. var. scolymus), however, efficient protocols for the establishment of in vitro callogenesis and organogenesis, a pre-requisite for Agrobacterium-mediated genetic transformation, have not been set up so far. We

  6. Improvement of Faba Bean Yield Using Rhizobium/Agrobacterium Inoculant in Low-Fertility Sandy Soil

    Directory of Open Access Journals (Sweden)

    Sameh H. Youseif

    2017-01-01

    Full Text Available Soil fertility is one of the major limiting factors for crop’s productivity in Egypt and the world in general. Biological nitrogen fixation (BNF has a great importance as a non-polluting and a cost-effective way to improve soil fertility through supplying N to different agricultural systems. Faba bean (Vicia faba L. is one of the most efficient nitrogen-fixing legumes that can meet all of their N needs through BNF. Therefore, understanding the impact of rhizobial inoculation and contrasting soil rhizobia on nodulation and N2 fixation in faba bean is crucial to optimize the crop yield, particularly under low fertility soil conditions. This study investigated the symbiotic effectiveness of 17 Rhizobium/Agrobacterium strains previously isolated from different Egyptian governorates in improving the nodulation and N2 fixation in faba bean cv. Giza 843 under controlled greenhouse conditions. Five strains that had a high nitrogen-fixing capacity under greenhouse conditions were subsequently tested in field trials as faba bean inoculants at Ismaillia Governorate in northeast Egypt in comparison with the chemical N-fertilization treatment (96 kg N·ha−1. A starter N-dose (48 kg N·ha−1 was applied in combination with different Rhizobium inoculants. The field experiments were established at sites without a background of inoculation under low fertility sandy soil conditions over two successive winter growing seasons, 2012/2013 and 2013/2014. Under greenhouse conditions, inoculated plants produced significantly higher nodules dry weight, plant biomass, and shoot N-uptake than non-inoculated ones. In the first season (2012/2013, inoculation of field-grown faba bean showed significant improvements in seed yield (3.73–4.36 ton·ha−1 and seed N-yield (138–153 Kg N·ha−1, which were higher than the uninoculated control (48 kg N·ha−1 that produced 2.97 Kg·ha−1 and 95 kg N·ha−1, respectively. Similarly, in the second season (2013

  7. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    Directory of Open Access Journals (Sweden)

    Benoît Lacroix

    Full Text Available VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  8. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    Science.gov (United States)

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  9. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    Jong, René M. de; Rozeboom, Henriëtte J.; Kalk, Kor H.; Tang, Lixia; Janssen, Dick B.; Dijkstra, Bauke W.

    2002-01-01

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  10. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    de Jong, RM; Rozeboom, HJ; Kalk, KH; Tang, Lixia; Janssen, DB; Dijkstra, BW

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  11. Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields

    Directory of Open Access Journals (Sweden)

    Nieto Joaquín J

    2010-07-01

    Full Text Available Abstract Background Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T. Results The most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the β-1,2-cyclic glucan from R

  12. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium

    DEFF Research Database (Denmark)

    Trieu, A.T.; Burleigh, S.H.; Kardailsky, I.V.

    2000-01-01

    Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium....... The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method....... Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T-2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M...

  13. [Methods for the detection of Agrobacterium from plant, soil and water samples].

    Science.gov (United States)

    Alippi, Adriana M; López, Ana C; Balatti, Pedro A

    2011-01-01

    The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly difficult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identification techniques by using a set of semi-selective and differential culture media in combination with a specific PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specific PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

  14. Isothermal Amplification and Lateral-Flow Assay for Detecting Crown-Gall-Causing Agrobacterium spp.

    Science.gov (United States)

    Fuller, Skylar L; Savory, Elizabeth A; Weisberg, Alexandra J; Buser, Jessica Z; Gordon, Michael I; Putnam, Melodie L; Chang, Jeff H

    2017-09-01

    Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

  15. [Proteomic analysis of curdlan-producing Agrobacterium sp. ATCC 31749 in response to dissolved oxygen].

    Science.gov (United States)

    Dai, Xiaomeng; Yang, Libo; Zheng, Zhiyong; Chen, Haiqin; Zhan, Xiaobei

    2015-08-04

    Curdlan is produced by Agrobacterium sp. ATCC 31749 under nitrogen limiting condition. The biosynthesis of crudlan is a typical aerobic bioprocess, and the production of curdlan would be severely restricted under micro-aerobic and anoxic conditions. Proteomic analysis of Agrobacterium sp. was conducted to investigate the effect of dissolved oxygen on the crucial enzymes involved in curdlan biosynthesis. Two-dimensional gel electrophoresis was performed to separate and visualize the differential expression of the intracellular proteins extracted from Agrobacterium sp. ATCC 31749 cultured under various dissolved oxygen levels (75%, 50%, 25% and 5%). In addition, a comparative proteomic analysis of the intracellular proteins expression level under various dissolved oxygen levels was done. Significant differently expressed proteins were identified by MALDI-TOF/TOF. Finally, we identified 15 differently expressed proteins involved in polysaccharide synthesis, fatty acid synthesis, amino acid synthesis pathway. Among these proteins, phosphoglucomutase and orotidine 5-phosphate decarboxylase were the key metabolic enzymes directing curdlan biosynthesis. Oxygen could affect the expression of the proteins taking charge of curdlan synthesis significantly.

  16. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    Science.gov (United States)

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  18. Agrobacterium rhizogenes transformed soybean roots differ in their nodulation and nitrogen fixation response to genistein and salt stress.

    Science.gov (United States)

    Dolatabadian, Aria; Modarres Sanavy, Seyed Ali Mohammad; Ghanati, Faezeh; Gresshoff, Peter M

    2013-07-01

    We evaluated response differences of normal and transformed (so-called 'hairy') roots of soybean (Glycine max L. (Merr.), cv L17) to the Nod-factor inducing isoflavone genistein and salinity by quantifying growth, nodulation, nitrogen fixation and biochemical changes. Composite soybean plants were generated using Agrobacterium rhizogenes-mediated transformation of non-nodulating mutant nod139 (GmNFR5α minus) with complementing A. rhizogenes K599 carrying the wild-type GmNFR5α gene under control of the constitutive CaMV 35S promoter. We used genetic complementation for nodulation ability as only nodulated roots were scored. After hairy root emergence, primary roots were removed and composite plants were inoculated with Bradyrhizobium japonicum (strain CB1809) pre-induced with 10 μM genistein and watered with NaCl (0, 25, 50 and 100 mM). There were significant differences between hairy roots and natural roots in their responses to salt stress and genistein application. In addition, there were noticeable nodulation and nitrogen fixation differences. Composite plants had better growth, more root volume and chlorophyll as well as more nodules and higher nitrogenase activity (acetylene reduction) compared with natural roots. Decreased lipid peroxidation, proline accumulation and catalase/peroxidase activities were found in 'hairy' roots under salinity stress. Genistein significantly increased nodulation and nitrogen fixation and improved roots and shoot growth. Although genistein alleviated lipid peroxidation under salinity stress, it had no significant effect on the activity of antioxidant enzymes. In general, composite plants were more competitive in growth, nodulation and nitrogen fixation than normal non-transgenic even under salinity stress conditions.

  19. Production, structural characterization and gel forming property of a new exopolysaccharide produced by Agrobacterium HX1126 using glycerol or d-mannitol as substrate.

    Science.gov (United States)

    Liu, Yongmei; Gu, Qiuya; Ofosu, Fred Kwame; Yu, Xiaobin

    2016-01-20

    A strain Agrobacterium HX1126 was isolated from soil sample near the canal in Wuxi. Glycerol was used as carbon source for the production of a new exopolysaccharide which was named PGHX. PGHX composed mainly of galactose, with lower amounts of arabinose and aminogalactose. It was found that this strain could use d-mannitol as carbon source to produce PGHX too. A method for the preparation of crude PGHX was proposed and the crude PGHX can be formed in a gel formation when 30 g/L was put into the boiling water for 10 min, with an achieved gel strength of 957 g/cm(2). The concentration of proteins in the crude product was considered to be an important parameter which directly influence the gel forming property. The highest production of PGHX (24.9 g/L) was obtained under the nitrogen depletion condition. The structure of the product was confirmed by NMR and FTIR. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Agrobacterium-mediated inoculation of chrysanthemum (Chrysanthemum morifolium) plants with chrysanthemum stunt viroid.

    Science.gov (United States)

    Nabeshima, Tomoyuki; Doi, Motoaki; Hosokawa, Munetaka

    2016-08-01

    Agroinfiltration was tested as a method of inoculation of chrysanthemum plants with chrysanthemum stunt viroid (CSVd). Binary vectors harboring dimeric CSVd sequences in sense and antisense orientations were constructed, and Agrobacterium transfected with these binary vectors was infiltrated into chrysanthemum leaves. Northern blotting and reverse transcription polymerase chain reaction analysis showed that local infection was established within 7 days and systemic infection within 20 days. CSVd polarities showed no difference in infectivity. This study showed that agroinfiltration of chrysanthemum plants is an easy, rapid, and cost-effective method for CSVd inoculation. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    Science.gov (United States)

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  2. The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection.

    Science.gov (United States)

    Bhattacharya, Amita; Sood, Priyanka; Citovsky, Vitaly

    2010-09-01

    Phenolics are aromatic benzene ring compounds with one or more hydroxyl groups produced by plants mainly for protection against stress. The functions of phenolic compounds in plant physiology and interactions with biotic and abiotic environments are difficult to overestimate. Phenolics play important roles in plant development, particularly in lignin and pigment biosynthesis. They also provide structural integrity and scaffolding support to plants. Importantly, phenolic phytoalexins, secreted by wounded or otherwise perturbed plants, repel or kill many microorganisms, and some pathogens can counteract or nullify these defences or even subvert them to their own advantage. In this review, we discuss the roles of phenolics in the interactions of plants with Agrobacterium and Rhizobium.

  3. Pepper, Sweet (Capsicum annuum)

    NARCIS (Netherlands)

    Heidmann, I.; Boutilier, K.A.

    2015-01-01

    Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium ( Agrobacterium tumefaciens ). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper

  4. 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 ...

  5. Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche

    Directory of Open Access Journals (Sweden)

    Xue Han

    2013-01-01

    Full Text Available Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L 6-benzylaminopurine and (0.08 mg/L naphthaleneacetic acid, we have achieved the highest frequency (90% for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0 and an infection time (20–30 min. According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30% than older leaves (10%.

  6. Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability*

    Science.gov (United States)

    Song, Zhang-yue; Tian, Jing-luan; Fu, Wei-zhe; Li, Lin; Lu, Ling-hong; Zhou, Lian; Shan, Zhi-hui; Tang, Gui-xiang; Shou, Hui-xia

    2013-01-01

    The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system. In this study, twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection, regeneration capacity, and stable transgenic efficiency. Three genotypes, Yuechun 04-5, Yuechun 03-3, and Tianlong 1, showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system. For the Tianlong 1, the average stable transformation efficiency is 4.59%, higher than that of control genotypes (Jack and Williams 82), which is enough for further genomic research and genetic engineering. While polymerase chain reaction (PCR), LibertyLink strips, and β-glucuronidase (GUS) staining assays were used to detect the insertion and expression of the transgene, leaves painted with 135 mg/L Basta could efficiently identify the transformants. PMID:23549846

  7. Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

    Science.gov (United States)

    Ding, Baoqing; Yuan, Yao-Wu

    2016-04-01

    The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

  8. High-Throughput Agrobacterium-mediated Transformation of Medicago Truncatula in Comparison to Two Expression Vectors

    International Nuclear Information System (INIS)

    Sultana, T.; Deeba, F.; Naqvi, S. M. S.

    2016-01-01

    Legumes have been turbulent to efficient Agrobacterium-mediated transformation for a long time. The selection of Medicago truncatula as a model legume plant for molecular analysis resulted in the development of efficient Agrobacterium-mediated transformation protocols. In current study, M. truncatula transformed plants expressing OsRGLP1 were obtained through GATEWAY technology using pGOsRGLP1 (pH7WG2.0=OsRGLP1). The transformation efficiency of this vector was compared with expression vector from pCAMBIA series over-expressing same gene (pCOsRGLP1). A lower number of explants generated hygromycin resistant plantlet for instance, 18.3 with pGOsRGLP1 vector as compared to 35.5 percent with pCOsRGLP1 vector. Transformation efficiency of PCR positive plants generated was 9.4 percent for pGOsRGLP1 while 21.6 percent for pCOsRGLP1. Furthermore 24.4 percent of explants generated antibiotic resistant plantlet on 20 mgl/sup -1/ of hygromycin which was higher than on 15 mgl/sup -1/ of hygromycin such as 12.2 percent. T/sub 1/ progeny analysis indicated that the transgene was inherited in Mendelian manner. The functionally active status of transgene was monitored by high level of Superoxide dismutase (SOD) activity in transformed progeny. (author)

  9. An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

    Directory of Open Access Journals (Sweden)

    Fatemeh Haddadi

    2015-02-01

    Full Text Available An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100% of shoot formation and the highest mean number of shoots (24 produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86% in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

  10. Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

    Science.gov (United States)

    Endo, Masaki; Kumagai, Masahiko; Motoyama, Ritsuko; Sasaki-Yamagata, Harumi; Mori-Hosokawa, Satomi; Hamada, Masao; Kanamori, Hiroyuki; Nagamura, Yoshiaki; Katayose, Yuichi; Itoh, Takeshi; Toki, Seiichi

    2015-01-01

    Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  11. Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

    Science.gov (United States)

    Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

    2011-04-01

    Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  12. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  13. Scale-Up of Agrobacterium rhizogenes-Mediated Hairy Root Cultures of Rauwolfia serpentina: A Persuasive Approach for Stable Reserpine Production.

    Science.gov (United States)

    Mehrotra, Shakti; Srivastava, Vikas; Goel, Manoj K; Kukreja, Arun K

    2016-01-01

    Roots of Rauwolfia serpentina, also known as "Sarpagandha" possess high pharmaceutical value due to the presence of reserpine and other medicinally important terpene indole alkaloids. Ever increasing commercial demand of R. serpentina roots is the major reason behind the unsystematic harvesting and fast decline of the species from its natural environment. Considering Agrobacterium rhizogenes-mediated hairy root cultures as an alternative source for the production of plant-based secondary metabolites, the present optimized protocol offers a commercially feasible method for the production of reserpine, the most potent alkaloid from R. serpentina roots. This end-to-end protocol presents the establishment of hairy root culture from the leaf explants of R. serpentina through the infection of A. rhizogenes strain A4 in liquid B5 culture medium and its up-scaling in a 5 L bench top, mechanically agitated bioreactor. The transformed nature of roots was confirmed through PCR-based rol A gene amplification in genomic DNA of putative hairy roots. The extraction and quantification of reserpine in bioreactor grown roots has been done using monolithic reverse phase high-performance liquid chromatography (HPLC).

  14. Jatropha (Jatropha curcas L.).

    Science.gov (United States)

    Maravi, Devendra Kumar; Mazumdar, Purabi; Alam, Shamsher; Goud, Vaibhav V; Sahoo, Lingaraj

    2015-01-01

    The seed oil of Jatropha (Jatropha curcas L.) as a source of biodiesel fuel is gaining worldwide importance. Commercial-scale exploration of Jatropha has not succeeded due to low and unstable seed yield in semiarid lands unsuitable for the food production and infestation to diseases. Genetic engineering is promising to improve various agronomic traits in Jatropha and to understand the molecular functions of key Jatropha genes for molecular breeding. We describe a protocol routinely followed in our laboratory for stable and efficient Agrobacterium tumefaciens-mediated transformation of Jatropha using cotyledonary leaf as explants. The 4-day-old explants are infected with Agrobacterium tumefaciens strain EHA105 harboring pBI121 plant binary vector, which contains nptII as plant selectable marker and gus as reporter. The putative transformed plants are selected on kanamycin, and stable integration of transgene(s) is confirmed by histochemical GUS assay, polymerase chain reaction, and Southern hybridization.

  15. Production of Phytophthora infestans-resistant potato (Solanum tuberosum) utilising Ensifer adhaerens OV14

    DEFF Research Database (Denmark)

    Wendt, Toni; Doohan, Fiona; Mullins, Ewen

    2012-01-01

    their gene transfer efficiencies. In this study we set out to identify alternative bacteria species that could (a) utilize vir genes for genetic transformation and (b) substitute for A. tumefaciens in existing transformation protocols, without a prerequisite for protocol modifications. To this end we...... isolated a collection (n=751) of plant-associated bacteria from the rhizosphere of commercially grown crops. Based on various screens, including plant transformation with the open-source vector pCAMBIA5105, we identified a strain of the bacterium Ensifer adhaerens with the capacity to transform both......Based on the use of Agrobacterium tumefaciens-mediated transformation commodity crop improvement through genetic engineering is the fastest adopted crop technology in the world (James 2010). However, the complexity of the Agrobacterium patent landscape remains a challenge for non-patent holders who...

  16. Histology of somatic embryos of eurycoma longifolia (simaroubaceae): relevance in agrobacterium rhizogenes-mediated transformation

    International Nuclear Information System (INIS)

    Balakrishnan, B.; Rabiah, S.S.; Keng, C.L.

    2014-01-01

    Histological analysis conducted on somatic embryos of Eurycoma longifolia shows the developmental structures that are remarkably similar to seeds found in the wild. The primary components of a growing somatic embryo are its shoot and root apical meristems indicated by dense layers of rapidly growing cells. The increased understanding of In vitro culture systems and anatomical changes provide information into cellular processes that govern genetic transformation of E. longifolia with Agrobacterium rhizogenes. The presence of meristematic regions on cultured somatic embryos suggests that they are suitable for genetic transformation as genetic elements could be transported to these regions where growth and differentiation are centered. This allows the successful integration and expression of transferred DNA in the host organism, leading the way for an efficient A. rhizogenes-mediated transformation protocol. (author)

  17. The effect of media composition on EDTA degradation by Agrobacterium sp

    International Nuclear Information System (INIS)

    Palumbo, A.V.; Lee, S.Y.; Boerman, P.

    1994-01-01

    EDTA degradation by an Agrobacterium sp. has been examined by quantifying 14 C-labeled CO 2 produced from iron-[2- 14 C] EDTA and by measured loss of nonlabeled EDTA by HPLC. Fe-EDTA degradation resulted in a rise in pH, nitrate concentration, and ammonia concentration. Addition of glycerol resulted in suppression of Fe-EDTA degradation and in a decrease in pH and NH 4 + concentration in the media. Addition of peptone or yeast extract did not affect degradation. Some of the components (e.g., biotin) of the media are not necessary for growth and biodegradation. Although cobalt-EDTA cannot be degraded, ferrous iron can be added to displace the cobalt. The purpose was to relate the results to remediation of radionuclide-contaminated soils. 12 refs., 4 figs., 2 tabs

  18. A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis

    Directory of Open Access Journals (Sweden)

    Polyana Kelly Martins

    2015-06-01

    Full Text Available The production and use of sugarcane in Brazil is very important for bioenergy production and is recognized as one of the most efficient in the world. In our laboratory, Setaria viridis is being tested as a model plant for sugarcane. S. viridis has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements that make it suitable for use as a model system. We report a highly efficient protocol for Agrobacterium-mediated genetic transformation of S. viridis. The optimization of several steps in tissue culture allowed the rapid regeneration of plants and increased the rate of transformation up to 29%. This protocol could become a powerful tool for functional genomics in sugarcane.

  19. Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis

    Directory of Open Access Journals (Sweden)

    Ruffing Anne M

    2012-02-01

    Full Text Available Abstract Background The ability to synthesize exopolysaccharides (EPS is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery. Results We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis. Conclusions This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

  20. An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol

    Directory of Open Access Journals (Sweden)

    Ülker Bekir

    2006-10-01

    Full Text Available Abstract Background The Agrobacterium vacuum (Bechtold et al 1993 and floral-dip (Clough and Bent 1998 are very efficient methods for generating transgenic Arabidopsis plants. These methods allow plant transformation without the need for tissue culture. Large volumes of bacterial cultures grown in liquid media are necessary for both of these transformation methods. This limits the number of transformations that can be done at a given time due to the need for expensive large shakers and limited space on them. Additionally, the bacterial colonies derived from solid media necessary for starting these liquid cultures often fail to grow in such large volumes. Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown. Results To avoid problems associated with large bacterial liquid cultures, we investigated whether bacteria grown on plates are also suitable for plant transformation. We demonstrate here that bacteria grown on plates can be used with similar efficiency for transforming plants even after one week of storage at 4°C. This makes it much easier to synchronize Agrobacterium and plants for transformation. DNA gel blot analysis was carried out on the T1 plants surviving the herbicide selection and demonstrated that the surviving plants are indeed transgenic. Conclusion The simplified method works as efficiently as the previously reported protocols and significantly reduces the workload, cost and time. Additionally, the protocol reduces the risk of large scale contaminations involving GMOs. Most importantly, many more independent transformations per day can be performed using this modified protocol.

  1. HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes.

    Science.gov (United States)

    Zalewski, Wojciech; Orczyk, Wacław; Gasparis, Sebastian; Nadolska-Orczyk, Anna

    2012-11-07

    CKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium. The mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T(1) lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T(0) plants and T(1) lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes. The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T(1) lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively

  2. DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

    Science.gov (United States)

    Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori

    2016-04-02

    Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high

  3. AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.

    Science.gov (United States)

    Tsuboyama, Shoko; Kodama, Yutaka

    2014-01-01

    The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

  4. NCBI nr-aa BLAST: CBRC-TSYR-01-0087 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TSYR-01-0087 ref|NP_356664.2| ice nucleation-like protein [Agrobacterium tumef...aciens str. C58] gb|AAK89449.2| ice nucleation-like protein [Agrobacterium tumefaciens str. C58] NP_356664.2 7e-17 20% ...

  5. NCBI nr-aa BLAST: CBRC-TSYR-01-1411 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TSYR-01-1411 ref|NP_356664.2| ice nucleation-like protein [Agrobacterium tumef...aciens str. C58] gb|AAK89449.2| ice nucleation-like protein [Agrobacterium tumefaciens str. C58] NP_356664.2 9e-39 19% ...

  6. ORF Alignment: NC_003305 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003305 gi|17937619 >1kmoA 2 661 49 702 6e-54 ... ref|NP_534408.1| exogenous ferric... siderophore receptor [Agrobacterium tumefaciens ... str. C58] gb|AAL44724.1| exogenous ferric sidero...phore ... receptor [Agrobacterium tumefaciens str. C58] ... pir||AF3038 exogenous ferric sider

  7. ORF Alignment: NC_003063 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003063 gi|15891046 >1kmoA 2 661 49 702 6e-54 ... ref|NP_534408.1| exogenous ferric... siderophore receptor [Agrobacterium tumefaciens ... str. C58] gb|AAL44724.1| exogenous ferric sidero...phore ... receptor [Agrobacterium tumefaciens str. C58] ... pir||AF3038 exogenous ferric sider

  8. ORF Alignment: NC_003063 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003063 gi|15890948 >1kmoA 11 661 74 716 3e-76 ... ref|NP_534507.1| hydroxamate-type ferris...iderophore receptor [Agrobacterium ... tumefaciens str. C58] gb|AAL44823.1| hydroxamate-type ... ferris...iderophore receptor [Agrobacterium tumefaciens ... str. C58] pir||AI3050 hydroxamate-type ferris

  9. ORF Alignment: NC_003305 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003305 gi|17937718 >1kmoA 11 661 74 716 3e-76 ... ref|NP_534507.1| hydroxamate-type ferris...iderophore receptor [Agrobacterium ... tumefaciens str. C58] gb|AAL44823.1| hydroxamate-type ... ferris...iderophore receptor [Agrobacterium tumefaciens ... str. C58] pir||AI3050 hydroxamate-type ferris

  10. Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum

    Czech Academy of Sciences Publication Activity Database

    Konieczny, R.; Obert, B.; Bleho, J.; Novák, Ondřej; Heym, C.; Tuleja, M.; Mueller, J.; Strnad, Miroslav; Menzel, D.; Šamaj, J.

    2011-01-01

    Roč. 168, č. 7 (2011), s. 722-729 ISSN 0176-1617 R&D Projects: GA ČR GA301/08/1649; GA MŠk ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511 Keywords : Agrobacterium * Callus * Common ice plant * Cytokinin content * GFP Subject RIV: EF - Botanics Impact factor: 2.791, year: 2011

  11. Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars.

    Science.gov (United States)

    Liu, Yan-Rong; Cen, Hui-Fang; Yan, Jian-Ping; Zhang, Yun-Wei; Zhang, Wan-Jun

    2015-07-01

    Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

  12. Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

    Science.gov (United States)

    Rolloos, Martijn; Hooykaas, Paul J J; van der Zaal, Bert J

    2015-02-09

    Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

  13. Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation.

    Science.gov (United States)

    Nahampun, Hartinio N; López-Arredondo, Damar; Xu, Xing; Herrera-Estrella, Luis; Wang, Kan

    2016-05-01

    Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco and Arabidopsis allowed plants to grow in media with Phi as the sole phosphorous source. The phosphite oxidoreductase (PTXD) enzyme catalyzes the conversion of Phi into phosphate (Pi) that can then be metabolized by plants and utilized as their essential phosphorous source. Here we assess an alternative selectable marker based on a bacterial ptxD gene for Agrobacterium-mediated maize transformation. We compared the transformation frequencies of maize using either the ptxD/Phi selection system or a standard herbicide bar/bialaphos selection system. Two maize genotypes, a transformation amenable hybrid Hi II and an inbred B104, were tested. Transgene presence, insertion copy numbers, and ptxD transcript levels were analyzed and compared. This work demonstrates that the ptxD/Phi selection system can be used for Agrobacterium-mediated maize transformation of both type I and type II callus culture and achieve a comparable frequency as that of the herbicide bar/bialaphos selection system.

  14. When plant virology met Agrobacterium: the rise of the deconstructed clones.

    Science.gov (United States)

    Peyret, Hadrien; Lomonossoff, George P

    2015-10-01

    In the early days of molecular farming, Agrobacterium-mediated stable genetic transformation and the use of plant virus-based vectors were considered separate and competing technologies with complementary strengths and weaknesses. The demonstration that 'agroinfection' was the most efficient way of delivering virus-based vectors to their target plants blurred the distinction between the two technologies and permitted the development of 'deconstructed' vectors based on a number of plant viruses. The tobamoviruses, potexviruses, tobraviruses, geminiviruses and comoviruses have all been shown to be particularly well suited to the development of such vectors in dicotyledonous plants, while the development of equivalent vectors for use in monocotyledonous plants has lagged behind. Deconstructed viral vectors have proved extremely effective at the rapid, high-level production of a number of pharmaceutical proteins, some of which are currently undergoing clinical evaluation. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Agrobacterium mediated transformation of brassica juncea (l.) czern with chitinase gene conferring resistance against fungal infections

    International Nuclear Information System (INIS)

    Ahmad, B.; Ambreen, S.; Khan, I.

    2015-01-01

    Brassica juncea (Czern and Coss., L.) is an important oilseed crop. Since it is attacked by several bacterial and fungal diseases, therefore, we developed an easy and simple protocol for the regeneration and transformation of B. juncea variety RAYA ANMOL to give rise to transgenic plants conferring resistance against various fungal diseases. The transformation was carried out using Agrobacterium with Chitinase gene. This gene was isolated from Streptomyces griseus HUT6037. We used two types of explants for transformation i.e. hypocotyls and cotyledons. Only hypocotyls explants showed good results regarding callus initiation. Different hormonal concentrations were applied i.e. BAP 2, 4 and 6 mgL-1 and NAA 0.1, 0.2 and 0.3 mgL-1. However, high transformation efficiency was observed by supplementing the medium with combination of 2 mgL-1 BAP and 0.2 mgL-1 for initiation of callus. Similarly 10 mgL-1 kanamycin and 200 mgL-1 cefotaxime also proved successful for the selection of transformed callus. In order to confirm the presence of transgenic callus Polymerase chain reaction was performed using specific primers for Chitinase gene. (author)

  16. Simultaneous denitrification and phosphorus removal by Agrobacterium sp. LAD9 under varying oxygen concentration.

    Science.gov (United States)

    Ma, Tao; Chen, Qian; Gui, Mengyao; Li, Can; Ni, Jinren

    2016-04-01

    Although efficient aerobic denitrification has received increasing attention, few studies have been made on simultaneous denitrification and phosphorus removal (SDPR) under aerobic condition. In this study, SDPR by an efficient aerobic denitrifier, Agrobacterium sp. LAD9, was firstly demonstrated. High nitrate and phosphorus removal rates of 7.50 and 1.02 mg L(-1) h(-1) were achieved in wide range of O2 concentration from 5.92 to 20.02 mg L(-1). The N2O production would be inhibited as O2 concentration exceeded 11.06 mg L(-1), while the phosphorus removal efficiency would be generally improved with increasing O2 concentration. (15)N mass spectrometry revealed that nitrogen removal accorded with the typical aerobic denitrification pathway, while (31)P nuclear magnetic resonance spectroscopy ((31)P NMR) indicated the fate of phosphorus to cells, extracellular polymeric substances (EPS), and polyphosphate (poly-P) of the denitrifier. EPS acted as a reservoir of phosphorus and the transformation of poly-P was dynamic and depended on initial orthophosphate (ortho-P) content. The aerobic SDPR would greatly simplify the conventional wastewater treatment processes which required separated considerations of nitrogen and phosphorus removal.

  17. Key Amino Acids in the Bacterial (6-4) Photolyase PhrB from Agrobacterium fabrum.

    Science.gov (United States)

    Graf, Dominik; Wesslowski, Janine; Ma, Hongju; Scheerer, Patrick; Krauß, Norbert; Oberpichler, Inga; Zhang, Fan; Lamparter, Tilman

    2015-01-01

    Photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation. PhrB from Agrobacterium fabrum represents a new group of prokaryotic (6-4) photolyases which contain an iron-sulfur cluster and a DMRL chromophore. We performed site-directed mutagenesis in order to assess the role of particular amino acid residues in photorepair and photoreduction, during which the FAD chromophore converts from the oxidized to the enzymatically active, reduced form. Our study showed that Trp342 and Trp390 serve as electron transmitters. In the H366A mutant repair activity was lost, which points to a significant role of His366 in the protonation of the lesion, as discussed for the homolog in eukaryotic (6-4) photolyases. Mutants on cysteines that coordinate the Fe-S cluster of PhrB were either insoluble or not expressed. The same result was found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines was mutated and for expression in minimal medium with limited Fe concentrations. We therefore assume that the Fe-S cluster is required for protein stability. We further mutated conserved tyrosines that are located between the DNA lesion and the Fe-S cluster. Mutagenesis results showed that Tyr424 was essential for lesion binding and repair, and Tyr430 was required for efficient repair. The results point to an important function of highly conserved tyrosines in prokaryotic (6-4) photolyases.

  18. Key Amino Acids in the Bacterial (6-4 Photolyase PhrB from Agrobacterium fabrum.

    Directory of Open Access Journals (Sweden)

    Dominik Graf

    Full Text Available Photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation. PhrB from Agrobacterium fabrum represents a new group of prokaryotic (6-4 photolyases which contain an iron-sulfur cluster and a DMRL chromophore. We performed site-directed mutagenesis in order to assess the role of particular amino acid residues in photorepair and photoreduction, during which the FAD chromophore converts from the oxidized to the enzymatically active, reduced form. Our study showed that Trp342 and Trp390 serve as electron transmitters. In the H366A mutant repair activity was lost, which points to a significant role of His366 in the protonation of the lesion, as discussed for the homolog in eukaryotic (6-4 photolyases. Mutants on cysteines that coordinate the Fe-S cluster of PhrB were either insoluble or not expressed. The same result was found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines was mutated and for expression in minimal medium with limited Fe concentrations. We therefore assume that the Fe-S cluster is required for protein stability. We further mutated conserved tyrosines that are located between the DNA lesion and the Fe-S cluster. Mutagenesis results showed that Tyr424 was essential for lesion binding and repair, and Tyr430 was required for efficient repair. The results point to an important function of highly conserved tyrosines in prokaryotic (6-4 photolyases.

  19. Agrobacterium rhizogenes-mediated transformation of Arachis hypogaea: an efficient tool for functional study of genes

    Directory of Open Access Journals (Sweden)

    Shuai Liu

    2016-09-01

    Full Text Available We have developed a technique for efficient transformation of hairy roots of Arachis hypogaea L. using Agrobacterium rhizogenes K599, and have validated this approach for the investigation of gene function. As a model transgene, AhAREB1, a drought-resistance gene from peanut, was fused to green fluorescent protein, and four parameters that might influence the transformation efficiency were tested. The optimal procedure involved the use of petioles with four expanded leaves as explants, infection by K599 at optical density (OD600 of 0.6 for 15 min and co-cultivation for 2 d, giving transformation efficiencies of up to 91%. Hairy roots from transgenic peanut plants overexpressing AhAREB1 were unaffected by treatment with polyethylene glycol (PEG, demonstrating increased drought tolerance, whereas control roots showed clear signs of plasmolysis. Transgenic roots accumulated less superoxide anion (O2− than control roots under drought conditions. Additionally, transgenic roots displayed upregulation of four stress-response genes encoding WRKY transcription factor (WRKY33, MYB transcription factor (MYB92, abscisic acid receptor (PYL5 and dehydrin 2 (DHN2.

  20. Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes.

    Science.gov (United States)

    Lacroix, Benoît; Citovsky, Vitaly

    2018-03-03

    Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research.

  1. Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.

    Science.gov (United States)

    Wu, Dongliang; Navet, Natasha; Liu, Yingchao; Uchida, Janice; Tian, Miaoying

    2016-09-06

    As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility. In this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion. This system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

  2. Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina.

    Science.gov (United States)

    Caro, Luis A; Santecchia, Natalia; Marinangeli, Pablo A; Curvetto, Néstor R; Hernández, Luis F

    2003-12-01

    The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.

  3. The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers.

    Science.gov (United States)

    Nagano, Soshichiro; Scheerer, Patrick; Zubow, Kristina; Michael, Norbert; Inomata, Katsuhiko; Lamparter, Tilman; Krauß, Norbert

    2016-09-23

    Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    Science.gov (United States)

    Zheng, Desen; Burr, Thomas J

    2016-02-01

    Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

  5. The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers*

    Science.gov (United States)

    Nagano, Soshichiro; Scheerer, Patrick; Zubow, Kristina; Michael, Norbert; Inomata, Katsuhiko; Lamparter, Tilman; Krauß, Norbert

    2016-01-01

    Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion. PMID:27466363

  6. Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

    DEFF Research Database (Denmark)

    Buch, C.; Sigh, J.; Nielsen, J.

    2003-01-01

    Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One...... strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains...

  7. Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud].

    Science.gov (United States)

    An, Xia; Wang, Bo; Liu, Lijun; Jiang, Hui; Chen, Jie; Ye, Shengtuo; Chen, Leiyu; Guo, Pingan; Huang, Xing; Peng, Dingxiang

    2014-05-01

    In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l(-1) was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD600 of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l(-1) in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25%. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.

  8. Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua Methods for the detection of Agrobacterium from plant, soil and water samples

    Directory of Open Access Journals (Sweden)

    Adriana M Alippi

    2011-12-01

    Full Text Available El género Agrobacterium incluye especies ftopatógenas que inducen la formación de agallas en el cuello o la proliferación de raíces en cabellera en más de 600 especies de dicotiledóneas, y especies no patógenas cuyo hábitat natural es el suelo. Como no es posible erradicar a las especies patógenas y habida cuenta de que más del 80 % de las infecciones puede provenir de viveros, es importante evitar la diseminación de la enfermedad. Por ello, el objetivo de este trabajo ha sido desarrollar técnicas sensibles y precisas que, aisladamente o combinadas, permitan detectar la presencia de especies y biovares de Agrobacterium a partir de muestras de material vegetal, suelo y agua. Se comprobó que con la estrategia combinada de realizar aislamientos en los medios semiselectivos D1, D1-M y YEM-RCT; PCR multiplex sobre el gen 23S ADNr; PCR específca sobre los genes virC1 y virC2 y bioensayos en plántulas de pimiento cv. California Wonder y en hojas cortadas de kalanchoe, se reduce la posibilidad de obtener falsos negativos y/o falsos positivos. Por lo expuesto, esta combinación de técnicas constituye una herramienta adecuada para el diagnóstico de cepas patógenas de Agrobacterium a partir de distintos tipos de muestras.The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly diffcult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identifcation techniques by using a set of semi-selective and differential culture media in combination with a specifc PCR to amplify a partial sequence derived

  9. The Stringent Response Induced by Phosphate Limitation Promotes Purine Salvage in Agrobacterium fabrum.

    Science.gov (United States)

    Sivapragasam, Smitha; Deochand, Dinesh K; Meariman, Jacob K; Grove, Anne

    2017-10-31

    Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds is enhanced by acidic pH and limiting inorganic phosphate. Nutrient starvation may also trigger the stringent response, and purine salvage is among the pathways expected to be favored under such conditions. We show here that phosphate limitation induces the stringent response, as evidenced by production of (p)ppGpp, and that the xdhCSML operon encoding the purine salvage enzyme xanthine dehydrogenase is upregulated ∼15-fold. The xdhCSML operon is under control of the TetR family transcription factor XdhR; direct binding of ppGpp to XdhR attenuates DNA binding, and the enhanced xdhCSML expression correlates with increased cellular levels of (p)ppGpp. Xanthine dehydrogenase may also divert purines away from salvage pathways to form urate, the ligand for the transcription factor PecS, which in the plant pathogen Dickeya dadantii is a key regulator of virulence gene expression. However, urate levels remain low under conditions that produce increased levels of xdhCSML expression, and neither acidic pH nor limiting phosphate results in induction of genes under control of PecS. Instead, expression of such genes is induced only by externally supplemented urate. Taken together, our data indicate that purine salvage is favored during the stringent response induced by phosphate starvation, suggesting that control of this pathway may constitute a novel approach to modulating virulence. Because bacterial purine catabolism appears to be unaffected, as evidenced by the absence of urate accumulation, we further propose that the PecS regulon is induced by only host-derived urate.

  10. Comparison of agrobacterium mediated wheat and barley transformation with nucleoside diphosphate kinase 2 (NDPK2) gene

    International Nuclear Information System (INIS)

    Waheed, U.; Shah, M.M.; Smedley, M.; Harwood, W.

    2016-01-01

    An efficient and reliable transformation system is imperative for improvement of important crop species like barley and wheat. Wheat transformation is complex due to larger genome size and polyploidy while barley has a limitation of genotypic dependency. The objective of current study was to compare the relative transformation efficiency of wheat and barley using specific expression vector pBRACT 214-NDPK2 constructed through gateway cloning carrying Nucleoside Diphosphate Kinase 2 (NDPK2) gene. The vector was used to compare the transformation response in both crops using immature embryos through Agrobacterium mediated transformation. Both wheat and barley showed different responses towards callus induction and regeneration. Immature embryos of 1.5 to 2 mm in diameter was found optimum for wheat callus induction while 1 to 1.5 mm for barley. Both embryogenic and non-embryogenic calli were found in wheat with significantly greater tendency for embryogenecity in barley. The overall regeneration response was found different for all transformed wheat and barley cultivars. Wheat cultivars showed good response initially that drastically slowed down in later stages with the exception of Fielder that reached to the green shoots with good roots. The barley transformed lines showed good regeneration response as compared to wheat. PCR analysis of putative transformants using genomic DNA showed a maximum of 27% transformation efficiency in barely. No true transformation response was obtained in all cultivars of wheat used in this study. The protocol developed for wheat and barley transformation will greatly be helpful in crop improvement programme through genetic engineering especially in diploid relatives of cereals. (author)

  11. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  12. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins.RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes.SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  13. Agrobacterium rhizogenes mediated transformation of Rhodiola sp. – an approach to enhance the level of bioactive compounds

    DEFF Research Database (Denmark)

    Møller Hansen, Martin; Lauridsen, Uffe Bjerre; Hegelund, Josefine Nymark

    Agrobacterium rhizogenes mediated transformation of Rhodiola sp. – an approach to enhance the level of bioactive compounds. Martin Møller Hansen1, Uffe Bjerre Lauridsen2, Josefine Nymark Hegelund3, Renate Müller4, Jihong Liu Clarke5, Henrik Lütken6 University of Copenhagen, Faculty of Science...... to wild type roots. The purpose of this study is to obtain HRs containing rol-genes from Rhodiola sp. for future sustainable production in bioreactors. Materials and Methods Whole stems of R. rosea and two accessions of R. pachyclados were sterilized with ethanol and NaOCl. The stems were then cut...

  14. Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

    Science.gov (United States)

    Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

    2010-01-01

    Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

  15. A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

    Science.gov (United States)

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

  16. Establishment of an efficient virus-induced gene silencing (VIGS) assay in Arabidopsis by Agrobacterium-mediated rubbing infection.

    Science.gov (United States)

    Manhães, Ana Marcia E de A; de Oliveira, Marcos V V; Shan, Libo

    2015-01-01

    Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.

  17. Optimización de la transformación genética con Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Mónica Blanco

    2003-01-01

    Full Text Available Para optimizar la eficiencia en la transformación genética con Agrobacterium rhizogenes, secciones de zanahoria fueron inoculadas con la cepa de Agrobacterium A4TC y co-cultivadas con acetosiringona, floroglucinol y una combinación de ambos. La acetosiringona es uno de los compuestos fenólicos que liberan los tejidos vegetales como respuesta a heridas, el cual induce la transferencia del T-ADN de la agrobacteria a la planta. El floroglucinol es también un compuesto fenólico; sin embargo, ejerce un efecto sinergístico con las auxinas, inhibiendo parcialmente la actividad de las citocininas. La mayor eficiencia en la transformación (75% se obtuvo con el uso de acetosiringona (100 mM en combinación con floroglucinol (25 mg l-1. En general, 6 días de co-cultivo, independientemente de los tratamientos, fue el tiempo que indujo el mayor porcentaje de transformación. La inclusión de 100 mg l-1 de kanamicina resultó un mecanismo eficiente para discriminar entre raíces transformadas y no transformadas. En adición, este trabajo presenta un novedoso sistema con el cual, una vez realizada la transformación, la bacteria puede ser eliminada completamente en 48 h con Cefotaxime en dosis de 500 mg l-1.

  18. Nitrite-mediated hydrolysis of epoxides catalyzed by halohydrin dehalogenase from Agrobacterium radiobacter AD1 : A new tool for the kinetic resolution of epoxides

    NARCIS (Netherlands)

    Hasnaoui, Ghania; Lutje Spelberg, Jeffrey H.; de Vries, Erik; Tang, Lixia; Hauer, Bernhard; Janssen, Dick B.

    2005-01-01

    Halohydrin dehalogenase obtained from Agrobacterium radiobacter AD1, has been tested for the nitrite-mediated ring opening of epoxides. This reaction mainly leads to the formation of unstable hydroxynitrite ester intermediates, which can be further hydrolyzed to the corresponding diols. This

  19. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1996-02-01

    Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

  20. Susceptibility of Some Stone and Pome Fruit Rootstocks to Crown Gall

    Directory of Open Access Journals (Sweden)

    A. Rhouma

    2005-12-01

    Full Text Available The susceptibility of different fruit rootstocks to crown gall disease was investigated in greenhouse and field experiments with numerous strains of Agrobacterium tumefaciens over three years. Plants were inoculated in the roots and shoots for pot experiments. Field experiments were performed in a naturally contaminated nursery plot. The genotypes Prunus dulcis and P. persica showed a high level of susceptibility to A. tumefaciens. Among the stone rootstocks, bitter almond was highly susceptible in all experiments. Apricot and Cadaman rootstocks displayed low susceptibility but larger galls, showing that there was no relation between rootstock susceptibility and gall size. Among pome rootstocks, quince BA29 was resistant to the disease, while MM106 was susceptible in potted trials; however, in the field essays, pome rootstocks did not become galled, possibly because the strains had selected for and adapted to stone rootstocks.

  1. Implementación de un protocolo para la producción de raíces pilosas (hairy roots de uña de gato (Uncaria tomentosa mediante transformación con Agrobacterium rhizogenes Implementation of a protocol for the production of hairy roots of cat’s claw (Uncaria tomentosa by Agrobacterium rhizogenes mediated transformation

    Directory of Open Access Journals (Sweden)

    Giovanni Garro Monge

    2012-11-01

    compounds is concentrated. Because of this,the production of hairy roots becomes an alternative technique for scale production of the metabolites of interest. In this research, a protocol was implemented using agroinfection of U. tomentosa shoot tips with wild strains of Agrobacterium rizhogenes (AR1500 and A4RS and maintenance of hairy roots obtained. In collaboration with the Laboratory of Molecular Biology of the PIPRA program (UC Davis, we determined the efficacy of this agroinfection protocol and the use of other molecular tools which showed satisfactory results in testing agroinfection under the methodologies established.

  2. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

  3. Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

  4. Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium -mediated system

    Institute of Scientific and Technical Information of China (English)

    翟文学; 李晓兵; 田文忠; 周永力; 潘学彪; 曹守云; 赵显峰; 赵彬; 章琦; 朱立煌

    2000-01-01

    A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

  5. Improvement of Coenzyme Q10 Production: Mutagenesis Induced by High Hydrostatic Pressure Treatment and Optimization of Fermentation Conditions

    Directory of Open Access Journals (Sweden)

    Yahong Yuan

    2012-01-01

    Full Text Available Coenzyme Q10 (CoQ10, ubiquinone, a potent antioxidative dietary supplement, was produced by submerged fermentation using Agrobacterium tumefaciens instead of chemical synthesis or solvent extraction. Agrobacterium tumefaciens 1.2554 was subjected to mutagenesis using a series of treatments including high hydrostatic pressure (HHP treatment, UV irradiation, and diethyl sulfate (DES treatment to obtain mutant strains showing higher CoQ10 production than wild-type strains. A mutant strain PK38 with four genetic markers was isolated: the specific CoQ10 content of the mutant strain increased by 52.83% compared with the original strain. Effects of carbon and nitrogen sources on CoQ10 production with PK38 were studied. Sucrose at concentration of 30 g/l was tested as the best carbon source, and yeast extract at concentration of 30 g/l supplemented with 10 g/l of ammonium sulfate was identified to be the most favorable for CoQ10 production using PK38. Fed-batch culture strategy was then used for increasing production of CoQ10 in 5-l fermentor. Using the exponential feeding fed-batch culture of sucrose, cell growth and CoQ10 formation were significantly improved. With this strategy, the final cell biomass, CoQ10 production, and specific CoQ10 production increased by 126.11, 173.12, and 22.76%, respectively, compared to those of batch culture.

  6. Phases of crown-gall transformation susceptible to hydroxyurea

    Directory of Open Access Journals (Sweden)

    Aldona Rennert

    2014-01-01

    Full Text Available With the use of bacterial strains, both sensitive and resistant to hydroxyurea the action of this inhibitor on tumour formation on the leaves of Kalanchoe daigremontiana infected with Agrobacterium tumefaciens was tested for five days after inoculation. The results are in agreement with the opinion that the anti-tumour effect of hydroxyurea applied in the induction phase (between 18 and 60 h after inoculation is the result of its direct action on plant cells, whereas inhibition of tumour formation by the inhibitor in the inoculation period depends on its action on the pathogenic bacteria.

  7. NARINGENIN ENHANCED EFFICIENCY OF GUS ACTIVITY IN Passiflora mollissima (H.B.K. Bailey

    Directory of Open Access Journals (Sweden)

    G.O. Cancino

    2004-06-01

    Full Text Available The flavonoid naringenin has been investigated as a possible vir gene inducer in Agrobacterium-mediated transformation in Passiflora mollissima, P. giberti and Nicotiana tabacum cv. Xanthi. The transformation efficiency percentage of explants showing blue GUS expression and the extent of staining following inoculation with Agrobacterium tumefaciens strains EHA 105 and 1065, carrying gus and nptII genes was enhanced with the supplementation of the co-cultivation medium with naringenin. Supplementation of medium with 100µM (strain EHA 105 and 300 µM (strain 1065 naringenin was most effective at enhancing mean (±s.e.m., n=3 GUS activity in leaf explants (20.3 ± 2.4%, strain EHA; 105; 6.0 ± 0.57%, strain 1065 and nodal segments (16.7 ± 2.4% strain EHA 105; 8.3 ± 0.57% strain 1065 of P. mollissima. In P. giberti and N. tabacum maximum GUS activity was obtained in leaf and root explants with 100µM naringenin for both strains analysed. Additionally, when naringenin was added to Luria Bertani (LB medium, both bacterial growth via optical density and colony forming units were higher when compared to control. This is the first report of the use of naringenin to enhance gene transfer from Agrobacterium to plants. These findings suggest that naringenin can be used as an alternative to acetosyringone for vir gene induction in Agrobacterium. This approach may be especially useful in plants that are generally recalcitrant to Agrobacterium-mediatedtransformation.

  8. Description of recovery method used for curdlan produced by Agrobacterium sp. IFO 13140 and its relation to the morphology and physicochemical and technological properties of the polysaccharide.

    Science.gov (United States)

    Mangolim, Camila Sampaio; Silva, Thamara Thaiane da; Fenelon, Vanderson Carvalho; Koga, Luciana Numata; Ferreira, Sabrina Barbosa de Souza; Bruschi, Marcos Luciano; Matioli, Graciette

    2017-01-01

    Curdlan is a linear polysaccharide considered a dietary fiber and with gelation properties. This study evaluated the structure, morphology and the physicochemical and technological properties of curdlan produced by Agrobacterium sp. IFO 13140 recovered by pre-gelation and precipitation methods. Commercial curdlan submitted or otherwise to the pre-gelation process was also evaluated. The data obtained from structural analysis revealed a similarity between the curdlan produced by Agrobacterium sp. IFO 13140 (recovered by both methods) and the commercial curdlans. The results showed that the curdlans evaluated differed significantly in terms of dispersibility and gelation, and only the pre-gelled ones had significant potential for food application, because this method influence on the size of the particles and in the presence of NaCl. In terms of technological properties, the curdlan produced by Agrobacterium sp. IFO 13140 (pre-gelation method) had a greater water and oil holding capacity (64% and 98% greater, respectively) and a greater thickening capacity than the pre-gelled commercial curdlan. The pre-gelled commercial curdlan displayed a greater gelling capacity at 95°C than the others. When applied to food, only the pre-gelled curdlans improved the texture parameters of yogurts and reduced syneresis. The curdlan gels, which are rigid and stable in structure, demonstrated potential for improving the texture of food products, with potential industrial use.

  9. A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants.

    Science.gov (United States)

    Hahn, Simone; Giritch, Anatoli; Bartels, Doreen; Bortesi, Luisa; Gleba, Yuri

    2015-06-01

    Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant-based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T-DNA of Agrobacterium encodes viral replicons capable of cell-to-cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume- and cost-sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high-volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large-scale production of many other cost-sensitive proteins. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Community of endophytic fungi from the medicinal and edible plant ...

    African Journals Online (AJOL)

    Received: 26 September 2016. Revised accepted: 12 ... against Bacillus subtilis, Staphylococcus aureus, Agrobacterium tumefaciens, Escherichia coli and. Pseudomonas .... macroscopic traits of the colonies (color, colony diameter, aspect ...

  11. Metabolic Engineering Strategies for the Optimization of Medicinal and Aromatic Plants : Expectations and Realities

    NARCIS (Netherlands)

    Kayser, O.; Baricevic, D; Novak, J; Pank, F

    2010-01-01

    In recent years classic genetic and molecular biology strategies (Bioballistics, Agrobacterium tumefaciens transformation, recombinant enzymes) for production of natural compounds or even breeding of medicinal and aromatic plants have expanded and improved productivity of plant-derived fine

  12. Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants.

    Science.gov (United States)

    Bettini, Priscilla P; Marvasi, Massimiliano; Fani, Fabiola; Lazzara, Luigi; Cosi, Elena; Melani, Lorenzo; Mauro, Maria Luisa

    2016-10-01

    Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b 6 /f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (F v /F m , rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. Crystallization, X-ray diffraction analysis and preliminary structure determination of the polygalacturonase PehA from Agrobacterium vitis

    International Nuclear Information System (INIS)

    Vordtriede, Paul B.; Yoder, Marilyn D.

    2008-01-01

    The acidic polygalacturonase PehA from A. vitis has been crystallized. A molecular-replacement solution indicated a right-handed parallel β-helix fold. Polygalacturonases are pectate-degrading enzymes that belong to glycoside hydrolase family 28 and hydrolyze the α-1,4 glycosidic bond between neighboring galacturonasyl residues of the homogalacturonan substrate. The acidic polygalacturonase PehA from Agrobacterium vitis was overexpressed in Escherichia coli, where it accumulated in the periplasmic fraction. It was purified to homogeneity via a two-step chromatography procedure and crystallized using the hanging-drop vapour-diffusion technique. PehA crystals belonged to space group P2 1 , with unit-cell parameters a = 52.387, b = 62.738, c = 149.165 Å, β = 89.98°. Crystals diffracted to 1.59 Å resolution and contained two molecules per asymmetric unit. An initial structure determination by molecular replacement indicated a right-handed parallel β-helix fold

  14. The use of phosphomannose isomerase selection system for Agrobacterium-mediated transformation of tobacco and flax aimed for phytoremediation.

    Science.gov (United States)

    Hilgert, Jitka; Sura-De Jong, Martina; Fišer, Jiří; Tupá, Kateřina; Vrbová, Miroslava; Griga, Miroslav; Macek, Tomáš; Žiarovská, Jana

    2017-05-04

    A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

  15. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    Science.gov (United States)

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2016-01-01

    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

  16. Characterisation and microstructure of reduced-fat chicken patties made with a novel polymer from Agrobacterium radiobacter k84.

    Science.gov (United States)

    Calliari, Caroline Maria; de Souza, Evandro Leite; Castro-Goméz, Raúl Jorge Hernan; Honório, Vanessa Gonçalves; Magnani, Marciane

    2015-04-15

    Chicken patties elaborated with a novel polymer from Agrobacterium radiobacter k84 (ARB) were characterised during 60days of frozen storage. After cooking, formulations without ARB (F0), with ARB 5 g/100 g (F5) and ARB 10 g/100 g (F10) presented 4.23%, 2.83% and 0.11% fat, respectively. No differences were observed to water holding capacity, cooking yield and shear force among formulations. Microstructural analysis showed formation of meat emulsion for F5 and gel for F10. Colour and chicken flavour decreased with increase of ARB; no difference was found for tenderness among the formulations. Overall acceptance showed higher scores for F0 when compared to F5 and F10. Lipid oxidation was not a limiting factor for stability of patties; all formulations presented suitable microbiological quality over the assessed period. These results suggest ARB as a promising fat substitute, capable of maintain the quality aspects of chicken patties, although a negative impact in colour has been found. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. An improved method for Agrobacterium rhizogenes-mediated transformation of tomato suitable for the study of arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Ho-Plágaro, Tania; Huertas, Raúl; Tamayo-Navarrete, María I; Ocampo, Juan A; García-Garrido, José M

    2018-01-01

    Solanum lycopersicum , an economically important crop grown worldwide, has been used as a model for the study of arbuscular mycorrhizal (AM) symbiosis in non-legume plants for several years and several cDNA array hybridization studies have revealed specific transcriptomic profiles of mycorrhizal tomato roots. However, a method to easily screen candidate genes which could play an important role during tomato mycorrhization is required. We have developed an optimized procedure for composite tomato plant obtaining achieved through Agrobacterium rhizogenes -mediated transformation. This protocol involves the unusual in vitro culture of composite plants between two filter papers placed on the culture media. In addition, we show that DsRed is an appropriate molecular marker for the precise selection of cotransformed tomato hairy roots . S. lycopersicum composite plant hairy roots appear to be colonized by the AM fungus Rhizophagus irregularis in a manner similar to that of normal roots, and a modified construct useful for localizing the expression of promoters putatively associated with mycorrhization was developed and tested. In this study, we present an easy, fast and low-cost procedure to study AM symbiosis in tomato roots.

  18. Agrobacterium-mediated transformation and direct shoot regeneration in Iranian tomato (Solanum lycopersicum L.) cultivar Falat- CH

    International Nuclear Information System (INIS)

    Kauser, N.; Khan, S.

    2016-01-01

    Falat CH is an important commercial tomato cultivar being used in Iran. In this article an optimized protocol with increased transformation and regeneration rate for this tomato variety is reported. Several explants including cotyledon, leaf and hypocotyl were evaluated for direct shoot formation and the effect of various combinations of BAP, Zeatin, IAA and IBA were studied. It is the first report on two cytokinins BAP and Zeatin in various combinations to evaluate the synergetic effect of cytokinins on direct shoot regeneration. The synergetic combination of 1.5mg/l BAP, 0.5 mg/l Zeatin and 0.2 mg/l IAA was considered as the best treatment which resulted in higher plant regeneration rates from all of the explants over previous reported methods. Using the best regeneration treatment obtained, the HBsAg gene was transferred into the tomato explants using Agrobacterium mediated transformation technique Percent of the putative transgenic plants regenerated was 68%. PCR of putative transformed plants showed that 87.1% of regenerated plants amplified nptII and HBsAg gene when specifically designed primers were used giving a final transformation rate of 34.85%. (author)

  19. Effect of nitrogen source concentration on curdlan production by Agrobacterium sp. ATCC 31749 grown on prairie cordgrass hydrolysates.

    Science.gov (United States)

    West, Thomas P

    2016-01-01

    The effect of nitrogen source concentration on the production of the polysaccharide curdlan by the bacterium Agrobacterium sp. ATCC 31749 from hydrolysates of prairie cordgrass was examined. The highest curdlan concentrations were produced by ATCC 31749 when grown on a medium containing a solids-only hydrolysate and the nitrogen source ammonium phosphate (2.2 mM) or on a medium containing a complete hydrolysate and 3.3 mM ammonium phosphate. The latter medium sustained a higher level of bacterial curdlan production than the former medium after 144 hr. Biomass production by ATCC 31749 was highest after 144 hr when grown on a medium containing a solids-only hydrolysate and 2.2 or 8.7 mM ammonium phosphate. On the medium containing the complete hydrolysate, biomass production by ATCC 31749 was highest after 144 hr when 3.3 mM ammonium phosphate was present. Bacterial biomass production after 144 hr was greater on the complete hydrolysate medium compared to the solids-only hydrolysate medium. Curdlan yield produced by ATCC 31749 after 144 hr from the complete hydrolysate medium containing 3.3 mM ammonium phosphate was higher than from the solids-only hydrolysate medium containing 2.2 mM ammonium phosphate.

  20. ORF Alignment: NC_003304 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003304 gi|17935259 >1tjlA 17 141 10 134 5e-37 ... ref|NP_532049.1| dnaK deletion s...uppressor protein [Agrobacterium tumefaciens str. ... C58] gb|AAL42365.1| dnaK deletion suppressor pr...otein ... [Agrobacterium tumefaciens str. C58] pir||AG2743 dnaK ... deletion suppressor protei

  1. ORF Alignment: NC_003062 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003062 gi|15888685 >1tjlA 17 141 10 134 5e-37 ... ref|NP_532049.1| dnaK deletion s...uppressor protein [Agrobacterium tumefaciens str. ... C58] gb|AAL42365.1| dnaK deletion suppressor pr...otein ... [Agrobacterium tumefaciens str. C58] pir||AG2743 dnaK ... deletion suppressor protei

  2. Genetic transformation of extremophilic fungi Acidea extrema and Acidothrix acidophila

    Czech Academy of Sciences Publication Activity Database

    Hršelová, Hana; Hujslová, Martina; Gryndler, Milan

    2015-01-01

    Roč. 60, č. 4 (2015), s. 365-371 ISSN 0015-5632 R&D Projects: GA ČR(CZ) GAP504/11/0484 Institutional support: RVO:61388971 Keywords : TUMEFACIENS-MEDIATED TRANSFORMATION * AGROBACTERIUM-TUMEFACIENS * FILAMENTOUS FUNGI Subject RIV: EE - Microbiology, Virology Impact factor: 1.335, year: 2015

  3. Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics

    Directory of Open Access Journals (Sweden)

    Liu Wei

    2009-06-01

    Full Text Available Abstract Background Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. Superroot of Lotus corniculatus is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic L. corniculatus plants from Superroot-derived leaves through A. tumefaciens-mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification. Results In the present study, we developed an A. rhizogenes-mediated transformation of Superroot-derived L. corniculatus for gene function investigation, combining the efficient A. rhizogenes-mediated transformation and the rapid regeneration system of Superroot. The transformation system using A. rhizogenes K599 harbouring pGFPGUSPlus was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD600 = 0.6, and co-cultivation on medium (pH 5.4 at 22°C for 2 days enhanced the transformation frequency significantly. As proof of concept, Superroot-derived L. corniculatus was transformed with a gene from wheat encoding an Na+/H+ antiporter (TaNHX2 using the described system. Transgenic Superroot plants were obtained and had increased salt tolerance, as expected from the expression of TaNHX2. Conclusion A rapid and efficient tool for gene function investigation in L. corniculatus was developed, combining the simplicity and high efficiency of the Superroot regeneration system and the availability of A. rhizogenes-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could

  4. Strain-Modulated Epitaxy

    National Research Council Canada - National Science Library

    Brown, April

    1999-01-01

    Strain-Modulated Epitaxy (SME) is a novel approach, invented at Georgia Tech, to utilize subsurface stressors to control strain and therefore material properties and growth kinetics in the material above the stressors...

  5. Hamstring strain - aftercare

    Science.gov (United States)

    Pulled hamstring muscle; Sprain - hamstring ... There are 3 levels of hamstring strains: Grade 1 -- mild muscle strain or pull Grade 2 -- partial muscle tear Grade 3 -- complete muscle tear Recovery time depends ...

  6. [Processes of plant colonization by Methylobacterium strains and some bacterial properties ].

    Science.gov (United States)

    Romanovskaia, V A; Stoliar, S M; Malashenko, Iu R; Dodatko, T N

    2001-01-01

    The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere. Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicum cells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M. extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens). Neither epiphytic and soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, and M. fujisawaense catalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.

  7. Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF

    Science.gov (United States)

    García-Cano, Elena; Magori, Shimpei; Sun, Qi; Ding, Zehong; Lazarowitz, Sondra G.; Citovsky, Vitaly

    2015-01-01

    Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis. PMID:26571494

  8. Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

    Directory of Open Access Journals (Sweden)

    Elena García-Cano

    Full Text Available Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.

  9. Genome and proteome analysis of 7-7-1, a flagellotropic phage infecting Agrobacterium sp H13-3

    Directory of Open Access Journals (Sweden)

    Kropinski Andrew M

    2012-05-01

    Full Text Available Abstract Background The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage. Methods The sequence of the 7-7-1 genome was determined by pyro(454sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS. Results Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100 and large (orf112 subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7, putative tail fibre (orf102, portal (orf113, major capsid (orf115 and tail sheath (orf126 proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114.

  10. Quorum sensing molecules production by nosocomial and soil isolates Acinetobacter baumannii.

    Science.gov (United States)

    Erdönmez, Demet; Rad, Abbas Yousefi; Aksöz, Nilüfer

    2017-12-01

    Acinetobacter species remain alive in hospitals on various surfaces, both dry and moist, forming an important source of hospital infections. These bacteria are naturally resistant to many antibiotic classes. Although the role of the quorum sensing system in regulating the virulence factors of Acinetobacter species has not been fully elucidated, it has been reported that they play a role in bacterial biofilm formation. The biofilm formation helps them to survive under unfavorable growth conditions and antimicrobial treatments. It is based on the accumulation of bacterial communication signal molecules in the area. In this study, we compared the bacterial signal molecules of 50 nosocomial Acinetobacter baumannii strain and 20 A. baumannii strain isolated from soil. The signal molecules were detected by the biosensor bacteria (Chromobacterium violaceum 026, Agrobacterium tumefaciens A136, and Agrobacterium tumefaciens NTL1) and their separation was determined by thin-layer chromatography. As a result, it has been found that soil-borne isolates can produce 3-oxo-C8-AHL and C8-AHL, whereas nosocomial-derived isolates can produce long-chain signals such as C10-AHL, C12-AHL, C14-AHL and C16-AHL. According to these results, it is possible to understand that these signal molecules are found in the infection caused by A. baumannii. The inhibition of this signaling molecules in a communication could use to prevent multiple antibiotic resistance of these bacteria.

  11. A strain gauge

    DEFF Research Database (Denmark)

    2016-01-01

    The invention relates to a strain gauge of a carrier layer and a meandering measurement grid positioned on the carrier layer, wherein the strain gauge comprises two reinforcement members positioned on the carrier layer at opposite ends of the measurement grid in the axial direction....... The reinforcement members are each placed within a certain axial distance to the measurement grid with the axial distance being equal to or smaller than a factor times the grid spacing. The invention further relates to a multi-axial strain gauge such as a bi-axial strain gauge or a strain gauge rosette where each...... of the strain gauges comprises reinforcement members. The invention further relates to a method for manufacturing a strain gauge as mentioned above....

  12. Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

    Science.gov (United States)

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2017-08-01

    Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM -1 ) and recombinant protein productivity per unit area-time (g m -2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m -2 than at a low plant density of 100 plants m -2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

    Science.gov (United States)

    Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

    2011-07-01

    Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

  14. TRANSIENT EXPRESSION OF β-GLUCURONIDASE IN ...

    African Journals Online (AJOL)

    ACSS

    2014-08-28

    Aug 28, 2014 ... production des cultivars de résistance adaptée aux charançons de la patate douce. Mots Clés: Agrobacterium tumefaciens, β-glucuronidase, Ipomoea batatas. INTRODUCTION. Plant transformation through Agrobacterium species is the most commonly used system since the transferred DNA has minimal ...

  15. ORF Alignment: NC_003305 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003305 gi|17938192 >1v7zA 16 254 17 254 3e-36 ... ref|NP_534981.1| creatinine amid...ohydrolase [Agrobacterium tumefaciens str. C58] ... gb|AAL45297.1| creatinine amidohydrolase [Agrobac... C58] pir||AC3110 ... creatinine amidohydrolase [imported] - Agrobacterium

  16. ORF Alignment: NC_003063 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003063 gi|15890482 >1v7zA 16 254 17 254 3e-36 ... ref|NP_534981.1| creatinine amid...ohydrolase [Agrobacterium tumefaciens str. C58] ... gb|AAL45297.1| creatinine amidohydrolase [Agrobac... C58] pir||AC3110 ... creatinine amidohydrolase [imported] - Agrobacterium

  17. Isolation and characterization of the heavy metal resistant bacteria CCNWRS33-2 isolated from root nodule of Lespedeza cuneata in gold mine tailings in China

    International Nuclear Information System (INIS)

    Wei Gehong; Fan Lianmei; Zhu Wenfei; Fu Yunyun; Yu Jianfu; Tang Ming

    2009-01-01

    A total of 108 strains of bacteria were isolated from root nodules of wild legumes growing in gold mine tailings in northwest of China and were tested for heavy metal resistance. The results showed that the bacterial strain CCNWRS33-2 isolated from Lespedeza cuneata was highly resistant to copper, cadmium, lead and zinc. The strain had a relatively high mean specific growth rate under each heavy metal stress test and exhibited a high degree of bioaccumulation ability. The partial sequence of the copper resistance gene copA was amplified from the strain and a sequence comparison with our Cu-resistant PCR fragment showed a high homology with Cu-resistant genes from other bacteria. Phylogenetic analysis based on the 16S rRNA gene sequence showed that CCNWRS33-2 belongs to the Rhizobium-Agrobacterium branch and it had 98.9% similarity to Agrobactrium tumefaciens LMG196

  18. Isolation and characterization of the heavy metal resistant bacteria CCNWRS33-2 isolated from root nodule of Lespedeza cuneata in gold mine tailings in China

    Energy Technology Data Exchange (ETDEWEB)

    Wei Gehong [College of Life Science, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A and F University, Yangling Shaanxi 712100 (China)], E-mail: weigehong@yahoo.com.cn; Fan Lianmei; Zhu Wenfei; Fu Yunyun; Yu Jianfu; Tang Ming [College of Life Science, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A and F University, Yangling Shaanxi 712100 (China)

    2009-02-15

    A total of 108 strains of bacteria were isolated from root nodules of wild legumes growing in gold mine tailings in northwest of China and were tested for heavy metal resistance. The results showed that the bacterial strain CCNWRS33-2 isolated from Lespedeza cuneata was highly resistant to copper, cadmium, lead and zinc. The strain had a relatively high mean specific growth rate under each heavy metal stress test and exhibited a high degree of bioaccumulation ability. The partial sequence of the copper resistance gene copA was amplified from the strain and a sequence comparison with our Cu-resistant PCR fragment showed a high homology with Cu-resistant genes from other bacteria. Phylogenetic analysis based on the 16S rRNA gene sequence showed that CCNWRS33-2 belongs to the Rhizobium-Agrobacterium branch and it had 98.9% similarity to Agrobactrium tumefaciens LMG196.

  19. Three dimensional strained semiconductors

    Science.gov (United States)

    Voss, Lars; Conway, Adam; Nikolic, Rebecca J.; Leao, Cedric Rocha; Shao, Qinghui

    2016-11-08

    In one embodiment, an apparatus includes a three dimensional structure comprising a semiconductor material, and at least one thin film in contact with at least one exterior surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the three dimensional structure. In another embodiment, a method includes forming a three dimensional structure comprising a semiconductor material, and depositing at least one thin film on at least one surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the structure.

  20. Transformation of Rhodiola rosea with rol-genes from Agrobacterium rhizogenes to enhance the production of bioactive compounds and analyses of key genes in their pathways

    DEFF Research Database (Denmark)

    Stofberg, Jimmi; Antypa, Natalia-Meropi; Müller, Renate

    Agrobacterium rhizogenes leads to development of hairy roots (HRs) at the infection site. Transformed HRs have for several plant resulted in a higher contents of secondary metabolites compared to untransformed wild types. The method comprises clonal propagation of in vitro and in vivo material followed by A...