Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

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Tae-Kyun Oh

2014-09-01

Full Text Available A full-length phytase gene (phy of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR, and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5, an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F, the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

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Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

2014-01-01

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs. PMID:25192284

The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L. is equivalent to obinutuzumab produced in CHO cells.

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Jin Won Lee

Full Text Available Plants have attracted attention as bio-drug production platforms because of their economical and safety benefits. The preliminary efficacy of ZMapp, a cocktail of antibodies produced in N. benthamiana (Nicotiana benthamiana L., suggested plants may serve as a platform for antibody production. However, because the amino acid sequences of the Fab fragment are diverse and differences in post-transcriptional processes between animals and plants remain to be elucidated, it is necessary to confirm functional equivalence of plant-produced antibodies to the original antibody. In this study, Obinutuzumab, a third generation anti-CD20 antibody, was produced in N. benthamiana leaves (plant-obinutuzumab and compared to the original antibody produced in glyco-engineered Chinese hamster ovary (CHO cells (CHO-obinutuzumab. Two forms (with or without an HDEL tag were generated and antibody yields were compared. The HDEL-tagged form was more highly expressed than the non-HDEL-tagged form which was cleaved in the N-terminus. To determine the equivalence in functions of the Fab region between the two forms, we compared the CD20 binding affinities and direct binding induced cell death of a CD20-positive B cells. Both forms showed similar CD20 binding affinities and direct cell death of B cell. The results suggested that plant-obinutuzumab was equivalent to CHO-obinutuzumab in CD20 binding, cell aggregation, and direct cell death via binding. Therefore, our findings suggest that Obinutuzumab is a promising biosimilar candidate that can be produced efficiently in plants.

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

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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.

Changes induced by the Pepper mild mottle tobamovirus on the chloroplast proteome of Nicotiana benthamiana.

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Pineda, M; Sajnani, C; Barón, M

2010-01-01

We have analyzed the chloroplast proteome of Nicotiana benthamiana using two-dimensional gel electrophoresis and mass spectrometry followed by a database search. In order to improve the resolution of the two-dimensional electrophoresis gels, we have made separate maps for the low and the high pH range. At least 200 spots were detected. We identified 72 polypeptides, some being isoforms of different multiprotein families. In addition, changes in this chloroplast proteome induced by the infection with the Spanish strain of the Pepper mild mottle virus were investigated. Viral infection induced the down-regulation of several chloroplastidic proteins involved in both the photosynthetic electron-transport chain and the Benson-Calvin cycle.

Transient expression of hemagglutinin antigen from low pathogenic avian influenza A (H7N7 in Nicotiana benthamiana.

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Selvaraju Kanagarajan

Full Text Available The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His tag, and an endoplasmic retention signal (SEKDEL. The construct was cloned into a Cowpea mosaic virus (CPMV-based vector (pEAQ-HT and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19 containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0, as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi. Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of

Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

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Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

2014-01-01

The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

Plum pox virus accumulates mutations in different genome parts during a long-term maintenance in Prunus host plants and passage in Nicotiana benthamiana.

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Vozárová, Z; Kamencayová, M; Glasa, M; Subr, Z

2013-01-01

Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural conditions in Middle Europe. Comparison of complete genome sequences obtained from subisolates of a PPV-M isolate maintained experimentally over a 6-year period in different Prunus host species and passaged in Nicotiana benthamiana was performed with the aim to highlight the mutations potentially connected with the virus-host adaptation. The results showed that the lowest number of non-silent mutations was accumulated in PPV-M maintained in peach (original host species), approximately two times higher diversity was recorded in plum, apricot and N. benthamiana, indicating the genetic determination of the PPV host preference. The sequence variability of Prunus subisolates was distributed more or less evenly along the PPV genome and no amino acid motif could be outlined as responsible for the host adaptation. In N. benthamiana the mutations were accumulated notably in the P1 and P3 genes indicating their non-essentiality in the infection of this experimental host plant.

SGS3 Cooperates with RDR6 in Triggering Geminivirus-Induced Gene Silencing and in Suppressing Geminivirus Infection in Nicotiana Benthamiana

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Fangfang Li

2017-09-01

Full Text Available RNA silencing has an important role in defending against virus infection in plants. Plants with the deficiency of RNA silencing components often show enhanced susceptibility to viral infections. RNA-dependent RNA polymerase (RDRs mediated-antiviral defense has a pivotal role in resistance to many plant viruses. In RDR6-mediated defense against viral infection, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3, was also found to fight against some viruses in Arabidopsis. In this study, we showed that SGS3 from Nicotiana benthamiana (NbSGS3 is required for sense-RNA induced post-transcriptional gene silencing (S-PTGS and initiating sense-RNA-triggered systemic silencing. Further, the deficiency of NbSGS3 inhibited geminivirus-induced endogenous gene silencing (GIEGS and promoted geminivirus infection. During TRV-mediated NbSGS3 or N. benthamiana RDR6 (NbRDR6 silencing process, we found that their expression can be effectively fine-tuned. Plants with the knock-down of both NbSGS3 and NbRDR6 almost totally blocked GIEGS, and were more susceptible to geminivirus infection. These data suggest that NbSGS3 cooperates with NbRDR6 against GIEGS and geminivirus infection in N. benthamiana, which provides valuable information for breeding geminivirus-resistant plants.

Effects of simultaneously elevated temperature and CO2 levels on Nicotiana benthamiana and its infection by different positive-sense RNA viruses are cumulative and virus type-specific.

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Del Toro, Francisco J; Rakhshandehroo, Farshad; Larruy, Beatriz; Aguilar, Emmanuel; Tenllado, Francisco; Canto, Tomás

2017-11-01

We have studied how simultaneously elevated temperature and CO 2 levels [climate change-related conditions (CCC) of 30°C, 970 parts-per-million (ppm) of CO 2 vs. standard conditions (SC) of 25°C, ~ 405ppm CO 2 ] affect physiochemical properties of Nicotiana benthamiana leaves, and also its infection by several positive-sense RNA viruses. In previous works we had studied effects of elevated temperature, CO 2 levels separately. Under CCC, leaves of healthy plants almost doubled their area relative to SC but contained less protein/unit-of-area, similarly to what we had found under conditions of elevated CO 2 alone. CCC also affected the sizes/numbers of different foliar cell types differently. Under CCC, infection outcomes in titers and symptoms were virus type-specific, broadly similar to those observed under elevated temperature alone. Under either condition, infections did not significantly alter the protein content of leaf discs. Therefore, effects of elevated temperature and CO 2 combined on properties of the pathosystems studied were overall cumulative. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

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Donghui Zhang

Full Text Available In the remodeling pathway for the synthesis of phosphatidylcholine (PC, acyl-CoA-dependent lysophosphatidylcholine (lysoPC acyltransferase (LPCAT catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2. Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△ disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA and α-linolenic acid (18:3n3, ALA into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3, while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana.

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Angela Chaparro-Garcia

2011-01-01

Full Text Available The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae, such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs, contributes to P. infestans resistance.We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs.We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.

Differentially expressed genes in healthy and plum pox virus-infected Nicotiana benthamiana plants.

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Vozárová, Z; Žilová, M; Šubr, Z

2015-12-01

Viruses use both material and energy sources of their hosts and redirect the production of disposable compounds in order to make viral replication more efficient. Metabolism of infected organisms is modified by these enhanced requirements as well by their own defense response. Resulting complex story consists of many regulation events on various gene expression levels. Elucidating these processes may contribute to the knowledge on virus-host interactions and to evolving new antiviral strategies. In our work we applied a subtractive cloning technique to compare the transcriptomes of healthy and plum pox virus (PPV)-infected Nicotiana benthamiana plants. Several genes were found to be induced or repressed by the PPV infection. The induced genes were mainly related to general stress response or photosynthesis, several repressed genes could be connected with growth defects evoked by the infection. Interestingly, some genes usually up-regulated by fungal or bacterial infection were found repressed in PPV-infected plants. Potential involvement of particular differently expressed genes in the process of PPV infection is discussed.

Identification of mRNAs that move over long distances using an RNA-Seq analysis of Arabidopsis/Nicotiana benthamiana heterografts.

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Notaguchi, Michitaka; Higashiyama, Tetsuya; Suzuki, Takamasa

2015-02-01

Phloem is a conductive tissue that allocates nutrients from mature source leaves to sinks such as young developing tissues. Phloem also delivers proteins and RNA species, such as small RNAs and mRNAs. Intensive studies on plant systemic signaling revealed the essential roles of proteins and RNA species. However, many of their functions are still largely unknown, with the roles of transported mRNAs being particularly poorly understood. A major difficulty is the absence of an accurate and comprehensive list of mobile transcripts. In this study, we used a hetero-graft system with Nicotiana benthamiana as the recipient scion and Arabidopsis as the donor stock, to identify transcripts that moved long distances across the graft union. We identified 138 Arabidopsis transcripts as mobile mRNAs, which we collectively termed the mRNA mobilome. Reverse transcription-PCR, quantitative real-time PCR and droplet digital PCR analyses confirmed the mobility. The transcripts included potential signaling factors and, unexpectedly, more general factors. In our investigations, we found no preferred transcript length, no previously known sequence motifs in promoter or transcript sequences and no similarities between the level of the transcripts and that in the source leaves. Grafting experiments regarding the function of ERECTA, an identified transcript, showed that no function of the transcript mobilized. To our knowledge, this is the first report identifying transcripts that move over long distances using a hetero-graft system between different plant taxa. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

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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.

Tracking the potyviral P1 protein in Nicotiana benthamiana plants during plum pox virus infection.

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Vozárová, Z; Glasa, M; Šubr, Z W

The P1 protein is derived from the N terminus of potyvirus-coded polyprotein. In addition to the proteolytic activity essential for its maturation, it probably participates in suppression of host defense and/or in virus replication. Clear validation of the P1 in vivo function(s), however, is not yet available. We applied an infectious cDNA clone of plum pox virus (PPV), where the P1 was N-fused with a hexahistidine tag, to trace this protein in Nicotiana benthamiana plants during the PPV infection. Immunoblot analysis with the anti-his antibody showed a diffuse band corresponding to the molecular weight about 70-80 kDa (about twice larger than expected) in the root samples from early stage of infection. This signal culminated on the sixth day post inoculation, later it rapidly disappeared. Sample denaturation by boiling in SDS before centrifugal clarification was essential, indicating strong affinity of P1-his to some plant compound sedimenting with the tissue and cell debris.

Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants.

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Kovalskaya, Natalia; Foster-Frey, Juli; Donovan, David M; Bauchan, Gary; Hammond, Rosemarie W

2016-01-01

The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells.

Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

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Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-04-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

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Schwab Wilfried

2011-03-01

Full Text Available Abstract Background Plant lipoxygenases (LOXs have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV based vector system. Results A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml-1 cell cultures which can oxygenate linoleic acid resulting in high yields (18 μmol ml-1 cell cultures of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 μmol mg-1 crude protein. The yield of C9-aldehydes from linoleic acid was 64%. Conclusion The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E,12(Z-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent.

Production of the main celiac disease autoantigen by transient expression in Nicotiana benthamiana

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Vanesa Soledad Marin Viegas

2015-12-01

Full Text Available Celiac Disease (CD is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2, is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion and vacuolar (C-terminal KISIA fusion TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9 to 16 fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB were induced by the ELP, with a consequent 2 fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant

Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

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Zhu, Feng; Xi, De-Hui; Yuan, Shu; Xu, Fei; Zhang, Da-Wei; Lin, Hong-Hui

2014-06-01

Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

Production of complex multiantennary N-glycans in Nicotiana benthamiana plants.

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Nagels, Bieke; Van Damme, Els J M; Pabst, Martin; Callewaert, Nico; Weterings, Koen

2011-03-01

In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions.

Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway.

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Andersen-Ranberg, Johan; Kongstad, Kenneth Thermann; Nafisi, Majse; Staerk, Dan; Okkels, Finn Thyge; Mortensen, Uffe Hasbro; Lindberg Møller, Birger; Frandsen, Rasmus John Normand; Kannangara, Rubini

2017-10-05

Carminic acid is a C-glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane-bound enzymes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana.

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Haihong Jia

Full Text Available The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS, reduced enzyme activities, elevated malondialdehyde (MDA content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS.

Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

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Pêra, Francisco F P G; Mutepfa, David L R; Khan, Ayesha M; Els, Johann H; Mbewana, Sandiswa; van Dijk, Alberdina A A; Rybicki, Edward P; Hitzeroth, Inga I

2015-12-02

Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher

Recovery of Nicotiana benthamiana plants from a necrotic response induced by a nepovirus is associated with RNA silencing but not with reduced virus titer.

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Jovel, Juan; Walker, Melanie; Sanfaçon, Hélène

2007-11-01

Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed.

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

International Nuclear Information System (INIS)

Lim, Hyoun-Sub; Nam, Jiryun; Seo, Eun-Young; Nam, Moon; Vaira, Anna Maria; Bae, Hanhong; Jang, Chan-Yong; Lee, Cheol Ho; Kim, Hong Gi; Roh, Mark; Hammond, John

2014-01-01

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP SP ) with that from AltMV-Po (CP Po ) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP Po [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP SP but not CP Po interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP SP than CP Po in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein

Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana.

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Bi, Huiping; Zhang, Peng

2012-03-01

Sweepoviruses are important begomoviruses that infect Ipomoea plants worldwide and cause sweet potato yield losses and cultivar decline. Two sweepoviruses, sweet potato leaf curl virus-Jiangsu (SPLCV-JS) and sweet potato leaf curl China virus-Zhejiang (SPLCCNV-ZJ), were cloned from diseased sweet potato plants collected in the Jiangsu and Zhejiang provinces of China. Sequence characterization and phylogenetic analysis demonstrated that both are typical monopartite begomoviruses and have close relationships to several reported SPLCV and SPLCCNV isolates, respectively, from Asian countries. Analysis of the protein alignments and subcellular localizations of the six SPLCV-JS proteins was also conducted to verify their putative functions. In Nicotiana benthamiana, an infectivity assay of the infectious SPLCV-JS clone resulted in mild symptoms and weak viral DNA accumulation. Interestingly, SPLCV-JS, together with a heterologous betasatellite DNA (tomato yellow leaf curl China virus isolate Y10 [TYLCCNV-Y10] DNA-β), showed a synergistic effect on enhanced symptom severity and viral DNA accumulation. This is the first reported infectious SPLCV clone.

Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

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Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

2014-10-01

Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

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Lim, Hyoun-Sub, E-mail: hyounlim@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Jiryun, E-mail: jilyoon@naver.com [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Seo, Eun-Young, E-mail: sey22@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Moon, E-mail: moonlit51@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Vaira, Anna Maria, E-mail: a.vaira@ivv.cnr.it [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States); Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino 10135 (Italy); Bae, Hanhong, E-mail: hanhongbae@ynu.ac.kr [School of Biotechnology, Yeungnam University, Geongsan 712-749 (Korea, Republic of); Jang, Chan-Yong, E-mail: sunbispirit@gmail.com [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Cheol Ho, E-mail: chlee1219@hanmail.net [Department of Chemical and Biological Engineering, Seokyoung University, Seoul 136-704 (Korea, Republic of); Kim, Hong Gi, E-mail: hgkim@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Roh, Mark, E-mail: marksroh@gmail.com [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States); Laboratory of Floriculture and Plant Physiology, School of Bio-Resource Science, Dankook University, Cheonan, Chungnam 330-714 (Korea, Republic of); Hammond, John, E-mail: john.hammond@ars.usda.gov [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States)

2014-03-15

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP{sub SP}) with that from AltMV-Po (CP{sub Po}) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP{sub Po} [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP{sub SP} but not CP{sub Po} interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP{sub SP} than CP{sub Po} in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein.

Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana

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Mei, Yuzhen; Yang, Xiuling; Huang, Changjun

2018-01-01

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants. PMID:29293689

Jasmonate ZIM-domain (JAZ protein regulates host and nonhost pathogen-induced cell death in tomato and Nicotiana benthamiana.

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Yasuhiro Ishiga

Full Text Available The nonhost-specific phytotoxin coronatine (COR produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1 is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000, and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS, we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP, INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

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Sandra Fresquet-Corrales

Full Text Available Proanthocyanidins (PAs, or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (- catechin/g FW and 228.5 nmol (- epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass are discussed.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

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Kytidou, Kassiani; Beekwilder, Jules; Artola, Marta; van Meel, Eline; Wilbers, Ruud H P; Moolenaar, Geri F; Goosen, Nora; Ferraz, Maria J; Katzy, Rebecca; Voskamp, Patrick; Florea, Bogdan I; Hokke, Cornelis H; Overkleeft, Herman S; Schots, Arjen; Bosch, Dirk; Pannu, Navraj; Aerts, Johannes M F G

2018-04-19

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lysoGb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lysoGb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Production of Complex Multiantennary N-Glycans in Nicotiana benthamiana Plants1[W][OA

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Nagels, Bieke; Van Damme, Els J.M.; Pabst, Martin; Callewaert, Nico; Weterings, Koen

2011-01-01

In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions. PMID:21233332

Biologically active, magnICON®-expressed EPO-Fc from stably transformed Nicotiana benthamiana plants presenting tetra-antennary N-glycan structures.

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Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Zabeau, Lennart; Tavernier, Jan; Delanghe, Joris R; Boets, Annemie; Castilho, Alexandra; Weterings, Koen

2012-08-31

In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized. Here, we report the transient magnICON(®) expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (Nagels et al., 2011). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (Bitonti et al., 2004). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants. Copyright © 2012 Elsevier B.V. All rights reserved.

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

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Bianco Linda

2009-11-01

Full Text Available Abstract Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein. In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19 gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor

Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus.

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Reavy, B; Arif, M; Kashiwazaki, S; Webster, K D; Barker, H

1995-01-01

Nicotiana benthamiana stem tissue was transformed with Agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (PMTV) coat protein (CP) gene. PMTV CP was expressed in large amounts in some of the primary transformants. The five transgenic lines which produced the most CP were selected for resistance testing. Flowers on transformed plants were allowed to self-fertilize. Transgenic seedlings selected from the T1 seed were mechanically inoculated with two strains of PMTV. Virus multiplication, assayed by infectivity, was detected in only one transgenic plant of 98 inoculated. T1 plants were also highly resistant to graft inoculation; PMTV multiplied in only one plant of 45 inoculated. Transgenic T1 seedlings were challenged in a bait test in which they were grown in soil containing viruliferous spores of the vector fungus Spongospora subterranea. In these tests only two plants out of 99 became infected. Of the five transgenic lines tested, plants of three lines were immune to infection following manual, graft, or fungal inoculation.

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

Science.gov (United States)

Kamencayová, M; Košík, I; Hunková, J; Subr, Z W

2014-01-01

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Science.gov (United States)

Rattanapisit, Kaewta; Srijangwad, Anchalee; Chuanasa, Taksina; Sukrong, Suchada; Tantituvanont, Angkana; Mason, Hugh S; Nilubol, Dachrit; Phoolcharoen, Waranyoo

2017-12-01

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro . These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection. Georg Thieme Verlag KG Stuttgart · New York.

GhWRKY25, a group I WRKY gene from cotton, confers differential tolerance to abiotic and biotic stresses in transgenic Nicotiana benthamiana.

Science.gov (United States)

Liu, Xiufang; Song, Yunzhi; Xing, Fangyu; Wang, Ning; Wen, Fujiang; Zhu, Changxiang

2016-09-01

WRKY transcription factors are involved in various processes, ranging from plant growth to abiotic and biotic stress responses. Group I WRKY members have been rarely reported compared with group II or III members, particularly in cotton (Gossypium hirsutum). In this study, a group I WRKY gene, namely, GhWRKY25, was cloned from cotton and characterized. Expression analysis revealed that GhWRKY25 can be induced or deduced by the treatments of abiotic stresses and multiple defense-related signaling molecules. Overexpression of GhWRKY25 in Nicotiana benthamiana reduced plant tolerance to drought stress but enhanced tolerance to salt stress. Moreover, more MDA and ROS accumulated in transgenic plants after drought treatment with lower activities of SOD, POD, and CAT. Our study further demonstrated that GhWRKY25 overexpression in plants enhanced sensitivity to the fungal pathogen Botrytis cinerea by reducing the expression of SA or ET signaling related genes and inducing the expression of genes involved in the JA signaling pathway. These results indicated that GhWRKY25 plays negative or positive roles in response to abiotic stresses, and the reduced pathogen resistance may be related to the crosstalk of the SA and JA/ET signaling pathways.

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

Science.gov (United States)

Khakimov, Bekzod; Kuzina, Vera; Erthmann, Pernille Ø; Fukushima, Ery Odette; Augustin, Jörg M; Olsen, Carl Erik; Scholtalbers, Jelle; Volpin, Hanne; Andersen, Sven Bode; Hauser, Thure P; Muranaka, Toshiya; Bak, Søren

2015-11-01

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs). © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons

An arabinoxyloglucan isolated from the midrib of the leaves of Nicotiana tabacum

Energy Technology Data Exchange (ETDEWEB)

Eda, S; Kato, K

1978-01-01

The structure of an arabinoxyloglucan, separated from the hemicellulosic polysaccharides of the midrib of the leaves of Nicotiana tabacum, was investigated by methylation analyses before and after mild acid hydrolysis, acetolysis and cellulase-degradation. The arabinoxyloglucan consists of L-arabinose, D-xylose and D-glucose in a molar ratio of 13:33:54, and has a backbone of ..beta..-(1..-->..4)-linked D-glucopyranosyl residues. Some of the glucopyranosyl residues are attached at the 6 position by single ..cap alpha..-D-xylopyranosyl and ..cap alpha..-L-arabinofuranosyl-(1..-->..2)-..cap alpha..-D-xylopyranosyl side chains.

Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins.

Science.gov (United States)

Matsuo, Kouki; Matsumura, Takeshi

2017-08-01

The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (ΔD2, ΔD4, and ΔD2ΔD4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the ΔD2ΔD4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than ΔD2, ΔD4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in ΔD2ΔD4 plants than in ΔD2, ΔD4, and wild-type plants. These findings demonstrate that ΔD2ΔD4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

DEFF Research Database (Denmark)

Qian, W; Yu, C; Qin, H

2007-01-01

, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces...... was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral......-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM...

Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus.

Science.gov (United States)

Germundsson, Anna; Sandgren, Maria; Barker, Hugh; Savenkov, Eugene I; Valkonen, Jari P T

2002-05-01

Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.

Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.

Science.gov (United States)

Strasser, Richard; Stadlmann, Johannes; Schähs, Matthias; Stiegler, Gabriela; Quendler, Heribert; Mach, Lukas; Glössl, Josef; Weterings, Koen; Pabst, Martin; Steinkellner, Herta

2008-05-01

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

Highly Oxygenated Flavonoids from the Leaves of Nicotiana plumbaginifolia (Solanaceae

Directory of Open Access Journals (Sweden)

Md. Shafiullah Shajib

2017-11-01

Full Text Available Nicotiana plumbaginifolia Viv. is an annual herb of the family Solanaceae, which grows abundantly in the weedy lands of Bangladesh . This plant possesses analgesic, antibacterial, anti-anxiety and hepatoprotective properties, and produces various phenolic compounds including flavonoids. The present study afforded determination of total phenolic and flavonoid contents, and for the first time, the isolation and characterization of highly oxygenated flavonoids, e.g., 3,3' ,5,6,7,8-hexamethoxy- 4',5'-methylenedioxyflavone (1, 3,3' ,4' ,5',5,6,7,8-octamethoxyflavone (2, exoticin, 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone (3 and ( 3,3' ,4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone (4 from the leaves of N. plumbaginifolia . All these flavonoids are rather rare natural products, and only found in a few genera, e.g.,Polygonum and Murraya. The structures of the isolated flavonoids were elucidated by comprehensive spectroscopic analyses, e.g., UV, 1H, 13C NMR, DEPT, HSQC, HMBC and MS.

The potato virus x TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement

International Nuclear Information System (INIS)

Mitra, Ruchira; Krishnamurthy, Konduru; Blancaflor, Elison; Payton, Mark; Nelson, Richard S.; Verchot-Lubicz, Jeanmarie

2003-01-01

Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association

Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS

Science.gov (United States)

Virus-induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS v...

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).

Cross Kingdom Glyco Protein Engineering

DEFF Research Database (Denmark)

Möller, Svenning Rune

delivered CRISPR/Cas9’, describes the use of viral replicons to deliver the CRISPR/Cas9 components to leaves of Nicotiana benthamiana, which we have optimized by fusing a Gfp marker to the Cas9 protein combined with FACS mediated cell sorting of Cas9-Gfp expressing protoplast cells....

An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF.

Science.gov (United States)

Yang, Liu; Ye, Chaofei; Zhao, Yuting; Cheng, Xiaolin; Wang, Yiqiao; Jiang, Yuan-Qing; Yang, Bo

2018-06-01

Overexpression of BnaWGR1 causes ROS accumulation and promotes leaf senescence. BnaWGR1 binds to promoters of RbohD and RbohF and regulates their expression. Manipulation of leaf senescence process affects agricultural traits of crop plants, including biomass, seed yield and stress resistance. Since delayed leaf senescence usually enhances tolerance to multiple stresses, we analyzed the function of specific MAPK-WRKY cascades in abiotic and biotic stress tolerance as well as leaf senescence in oilseed rape (Brassica napus L.), one of the important oil crops. In the present study, we showed that expression of one WRKY gene from oilseed rape, BnaWGR1, induced an accumulation of reactive oxygen species (ROS), cell death and precocious leaf senescence both in Nicotiana benthamiana and transgenic Arabidopsis (Arabidopsis thaliana). BnaWGR1 regulates the transcription of two genes encoding key enzymes implicated in production of ROS, that is, respiratory burst oxidase homolog (Rboh) D and RbohF. A dual-luciferase reporter assay confirmed the transcriptional regulation of RbohD and RbohF by BnaWGR1. In vitro electrophoresis mobility shift assay (EMSA) showed that BnaWGR1 could bind to W-box cis-elements within promoters of RbohD and RbohF. Moreover, RbohD and RbohF were significantly upregulated in transgenic Arabidopsis overexpressing BnaWGR1. In summary, these results suggest that BnaWGR1 could positively regulate leaf senescence through regulating the expression of RbohD and RbohF genes.

Some physiological aspects of nitrate reductase-deficient Nicotiana plumbaginifolia plants

International Nuclear Information System (INIS)

Saux, C.; Morot-Gaudry, J.F.; Lemoine, Y.; Caboche, M.

1986-01-01

Chlorate-resistant Nicotiana plumbaginifolia (cv. Viviani) mutants were found to be defective in the nitrate reductase apoprotein (NR - nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild type Nicotiana tabacum. The grafts of NR - plants were found to contain less malate but more amino acids, sugars and starch than the wild type. Moreover they were chlorotic, with a slight increase of the proportion of LH Chl a/b protein complexes and they exhibited a lowering of the efficiency of energy transfer between the light-harvesting complexes and the active centers. After 14 CO 2 pulse and chase experiments. The total 14 C incorporation of the mutant leaves was approximately 20% of that of the control. The NR - leaves mainly accumulated 14 C in the whole intermediates of the Calvin-cycle and in sucrose. In the most deficient NR leaves, chloroplasts were stuffed with large starch inclusions disorganizing the lamellar system

Some physiological aspects of nitrate reductase-deficient Nicotiana plumbaginifolia plants

Energy Technology Data Exchange (ETDEWEB)

Saux, C.; Morot-Gaudry, J.F.; Lemoine, Y.; Caboche, M.

1986-04-01

Chlorate-resistant Nicotiana plumbaginifolia (cv. Viviani) mutants were found to be defective in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild type Nicotiana tabacum. The grafts of NR/sup -/ plants were found to contain less malate but more amino acids, sugars and starch than the wild type. Moreover they were chlorotic, with a slight increase of the proportion of LH Chl a/b protein complexes and they exhibited a lowering of the efficiency of energy transfer between the light-harvesting complexes and the active centers. After /sup 14/CO/sub 2/ pulse and chase experiments. The total /sup 14/C incorporation of the mutant leaves was approximately 20% of that of the control. The NR/sup -/ leaves mainly accumulated /sup 14/C in the whole intermediates of the Calvin-cycle and in sucrose. In the most deficient NR leaves, chloroplasts were stuffed with large starch inclusions disorganizing the lamellar system.

Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors

Directory of Open Access Journals (Sweden)

Maria R. Mendoza

2017-11-01

Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions

Czech Academy of Sciences Publication Activity Database

Dziurka, M.; Janeczko, A.; Juhasz, C.; Gullner, G.; Oklešťková, Jana; Novák, Ondřej; Saja, D.; Skoczowski, A.; Tobias, I.; Barna, B.

2016-01-01

Roč. 109, DEC (2016), s. 355-364 ISSN 0981-9428 R&D Projects: GA ČR GA14-34792S Institutional support: RVO:61389030 Keywords : tobacco-mosaic-virus * pathogenesis-related proteins * salicylic-acid * abscisic-acid * acquired- resistance * disease resistance * nicotiana-benthamiana * arabidopsis-thaliana * defense response * immune-responses * Brassinosteroids * Ethylene * Hormone * Pepper * Phenylalanine ammonia lyase * Progesterone * Salicylic acid * Tobamovirus Subject RIV: EF - Botanics Impact factor: 2.724, year: 2016

Highly Oxygenated Flavonoids from the Leaves of Nicotiana plumbaginifolia (Solanaceae)

OpenAIRE

Md. Shafiullah Shajib; Bidyut Kanti Datta; Md. Hossain Sohrab; Mohammad Abdur Rashid; Lutfun Nahar; Satyajit Dey Sarker

2017-01-01

Nicotiana plumbaginifolia Viv. is an annual herb of the family Solanaceae, which grows abundantly in the weedy lands of Bangladesh . This plant possesses analgesic, antibacterial, anti-anxiety and hepatoprotective properties, and produces various phenolic compounds including flavonoids. The present study afforded determination of total phenolic and flavonoid contents, and for the first time, the isolation and characterization of highly oxygenated flavonoids, e.g., 3,3' ,5,6,7,8-hexamethoxy- 4...

Acute toxicity of tobacco ( Nicotiana tobaccum ) leaf dust on ...

African Journals Online (AJOL)

Experiments were conducted using dry tobacco (Nicotiana tobaccum) leaves aqueous extract to determine the acute toxicity and sub lethal effects on some haematological indices of Oreochromis niloticus using static renewable bioassay method. The extract was found to be toxic with a 48-h LC50 value of 109.6 mg/l.

SolCyc: a database hub at the Sol Genomics Network (SGN) for the manual curation of metabolic networks in Solanum and Nicotiana specific databases

Science.gov (United States)

Foerster, Hartmut; Bombarely, Aureliano; Battey, James N D; Sierro, Nicolas; Ivanov, Nikolai V; Mueller, Lukas A

2018-01-01

Abstract SolCyc is the entry portal to pathway/genome databases (PGDBs) for major species of the Solanaceae family hosted at the Sol Genomics Network. Currently, SolCyc comprises six organism-specific PGDBs for tomato, potato, pepper, petunia, tobacco and one Rubiaceae, coffee. The metabolic networks of those PGDBs have been computationally predicted by the pathologic component of the pathway tools software using the manually curated multi-domain database MetaCyc (http://www.metacyc.org/) as reference. SolCyc has been recently extended by taxon-specific databases, i.e. the family-specific SolanaCyc database, containing only curated data pertinent to species of the nightshade family, and NicotianaCyc, a genus-specific database that stores all relevant metabolic data of the Nicotiana genus. Through manual curation of the published literature, new metabolic pathways have been created in those databases, which are complemented by the continuously updated, relevant species-specific pathways from MetaCyc. At present, SolanaCyc comprises 199 pathways and 29 superpathways and NicotianaCyc accounts for 72 pathways and 13 superpathways. Curator-maintained, taxon-specific databases such as SolanaCyc and NicotianaCyc are characterized by an enrichment of data specific to these taxa and free of falsely predicted pathways. Both databases have been used to update recently created Nicotiana-specific databases for Nicotiana tabacum, Nicotiana benthamiana, Nicotiana sylvestris and Nicotiana tomentosiformis by propagating verifiable data into those PGDBs. In addition, in-depth curation of the pathways in N.tabacum has been carried out which resulted in the elimination of 156 pathways from the 569 pathways predicted by pathway tools. Together, in-depth curation of the predicted pathway network and the supplementation with curated data from taxon-specific databases has substantially improved the curation status of the species–specific N.tabacum PGDB. The implementation of this

Omzettingen van koolhydraten in het blad van Nicotiana tabacum L.

NARCIS (Netherlands)

Tollenaar, D.

1925-01-01

Nicotiana tabacum L. was chosen as an experimental plant for several practical reasons. The plants were grown in large pots in a glasshouse at 22 °C and great humidity in February-March and September-October until 4 normal leaves were present. Each day at 16.00 h the plants were brought into

iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

Science.gov (United States)

Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

2016-01-15

Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress. Copyright © 2015 Yunnan Academy of Tobacco Agricultural Sciences. Published by Elsevier Inc. All rights reserved.

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa.

Science.gov (United States)

da Silva, Fernanda R; Erdtmann, Bernardo; Dalpiaz, Tiago; Nunes, Emilene; Ferraz, Alexandre; Martins, Tales L C; Dias, Johny F; da Rosa, Darlan P; Porawskie, Marilene; Bona, Silvia; da Silva, Juliana

2013-07-01

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

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Fernanda R. da Silva

2013-01-01

Full Text Available Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L leaves (control group. All of the snails received leaves (tobacco and lettuce leaves were the only food provided and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.

Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

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Ragusa, S; Cambria, M T; Scarpa, M; Di Paolo, M L; Falconi, M; Rigo, A; Cambria, A

2001-11-01

The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites. Copyright 2001 Academic Press.

De invloed van auxine, tryptofaan en enige anorganische zouten op de infectie van Nicotiana glutinosa met tabaksmozaiekvirus

NARCIS (Netherlands)

Liem, A.S.N.

1963-01-01

The number of necrotic spots arising on leaves of Nicotiana glutinosa after inoculation with tobacco mosaic virus was less than in controls without additives, if the water in which the leaves floated hadβ-indoleacetic acid (IAA),α-naphthylacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

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Fontaine, Jean-Xavier; Saladino, Francesca; Agrimonti, Caterina; Bedu, Magali; Tercé-Laforgue, Thérèse; Tétu, Thierry; Hirel, Bertrand; Restivo, Francesco M; Dubois, Frédéric

2006-03-01

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

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Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

2016-08-01

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

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Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

1987-01-01

Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR - nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second 14 CO 2 pulse, the total 14 C incorporation of the mutant leaves was approximately 20 5 of that of the control. The 14 C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second 14 CO 2 pulse followed by a 60 second chase with normal CO 2 , 14 C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus

Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

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Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

1987-05-01

Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second /sup 14/CO/sub 2/ pulse, the total /sup 14/C incorporation of the mutant leaves was approximately 20/sup 5/ of that of the control. The /sup 14/C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second /sup 14/CO/sub 2/ pulse followed by a 60 second chase with normal CO/sub 2/, /sup 14/C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.

Environmental and genetic factors associated with solanesol accumulation in potato leaves

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Raymond Campbell

2016-08-01

Full Text Available Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterised the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP and mevalonic acid (MVA pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production.

A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

Czech Academy of Sciences Publication Activity Database

Týcová, Anna; Piernikarczyk, R.J.J.; Kugler, M.; Lipovová, P.; Podzimek, T.; Steger, G.; Matoušek, Jaroslav

2016-01-01

Roč. 127, č. 2 (2016), s. 347-358 ISSN 0167-6857 Institutional support: RVO:60077344 Keywords : mRNA quantification * Nicotiana benthamiana * Nicotiana tabacum * Post-transcriptional gene silencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.002, year: 2016

Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 show increased susceptibility to a group of fungal and oomycete pathogens.

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Bultreys, Alain; Trombik, Tomasz; Drozak, Anna; Boutry, Marc

2009-09-01

SUMMARY The behaviour of Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 was investigated in response to fungal and oomycete infections. The importance of NpPDR1 in plant defence was demonstrated for two organs in which NpPDR1 is constitutively expressed: the roots and the petal epidermis. The roots of the plantlets of two lines silenced for NpPDR1 expression were clearly more sensitive than those of controls to the fungal pathogens Botrytis cinerea, Fusarium oxysporum sp., F. oxysporum f. sp. nicotianae, F. oxysporum f. sp. melonis and Rhizoctonia solani, as well as to the oomycete pathogen Phytophthora nicotianae race 0. The Ph gene-linked resistance of N. plumbaginifolia to P. nicotianae race 0 was totally ineffective in NpPDR1-silenced lines. In addition, the petals of the NpPDR1-silenced lines were spotted 15%-20% more rapidly by B. cinerea than were the controls. The rapid induction (after 2-4 days) of NpPDR1 expression in N. plumbaginifolia and N. tabacum mature leaves in response to pathogen presence was demonstrated for the first time with fungi and one oomycete: R. solani, F. oxysporum and P. nicotianae. With B. cinerea, such rapid expression was not observed in healthy mature leaves. NpPDR1 expression was not observed during latent infections of B. cinerea in N. plumbaginifolia and N. tabacum, but was induced when conditions facilitated B. cinerea development in leaves, such as leaf ageing or an initial root infection. This work demonstrates the increased sensitivity of NpPDR1-silenced N. plumbaginifolia plants to all of the fungal and oomycete pathogens investigated.

Effect of vermicompost on some physiological attributes involved in carbon and nitrogen metabolism as well as nutrient status in leaves of tobacco (nicotiana tabacum L.)

International Nuclear Information System (INIS)

Qin, C.; Zheng, P.; Akram, N.A.

2016-01-01

A pot experiment was carried out to examine the influence of vermicompost application on some key enzymes and metabolites involved in carbon (C) and nitrogen (N) metabolism as well as nutrient status in the leaves of tobacco (Nicotiana tabacum L.). Two types of vermicompost with two application rates were used in this study. Regardless of application rate, both types of vermicompost significantly increased total N, phosphorus (P) and potassium (K) contents in the leaves. They also caused enhancements in contents of total soluble carbohydrates, reducing sugars, starch and total organic C as well as amylase and invertase activities involved in C metabolism, contents of soluble protein and nicotine in N metabolism in the leaves. With an increase in application rate, each vermicompost type had an increasing effect on almost all measured parameters except nitrate reductase activity. Regardless of vermicompost type, the high rate (50%) of application showed the best effects compared with controls. The effects of V1 type vermicompost were superior to those of V2 at the same application rate. Therefore, the above effects might appear to be dependent on both type and dose. Vermicompost could be considered as an effective organic matter for attaining improved plant nutrition as well as C and N metabolism. (author)

Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

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Joseph Minu

2012-03-01

Full Text Available Abstract Background The N-terminal proline-rich domain (Zera of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER-derived protein bodies (PBs when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles. Results We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed. Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer. Conclusions This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of

Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv. tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis.

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Wei, Hai-Lei; Zhang, Wei; Collmer, Alan

2018-05-08

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a complex repertoire of type III secretion effector (T3E) proteins. Effectorless polymutant DC3000D36E was used with a modularized system for native delivery of the 29 DC3000 T3Es singly and in pairs. Assays of the performance of this T3E library in N. benthamiana leaves revealed a matrix of T3E interplay, with six T3Es eliciting death and eight others variously suppressing the death activity of the six. The T3E library was also interrogated for effects on DC3000D36E elicitation of a reactive oxygen species burst, for growth in planta, and for T3Es that reversed these effects. Pseudomonas fluorescens and Agrobacterium tumefaciens heterologous delivery systems yielded notably different sets of death-T3Es. The DC3000D36E T3E library system highlights the importance of 13 T3Es and their interplay in interactions with N. benthamiana. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity.

Science.gov (United States)

Iraklis, Boubourakas; Kanda, Hiroko; Nabeshima, Tomoyuki; Onda, Mayu; Ota, Nao; Koeda, Sota; Hosokawa, Munetaka

2016-08-01

CSVd could not infect Nicotiana benthamiana when the plants were pretreated with crude leaf extract of Capsicum chinense 'Sy-2'. C. chinense leaves were revealed to contain strong RNA-digesting activity. Several studies have identified active antiviral and antiviroid agents in plants. Capsicum plants are known to contain antiviral agents, but the mechanism of their activity has not been determined. We aimed to elucidate the mechanism of Capsicum extract's antiviroid activity. Chrysanthemum stunt viroid (CSVd) was inoculated into Nicotiana benthamiana plants before or after treating the plants with a leaf extract of Capsicum chinense 'Sy-2'. CSVd infection was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 3 weeks after inoculation. When Capsicum extract was sprayed or painted onto N. benthamiana before inoculation, it was effective in preventing infection by CSVd. To evaluate CSVd digestion activity in leaf extracts, CSVd was mixed with leaf extracts of Mirabilis, Phytolacca, Pelargonium and Capsicum. CSVd-digesting activities were examined by quantifying undigested CSVd using qRT-PCR, and RNA gel blotting permitted visualization of the digested CSVd. Only Capsicum leaf extract digested CSVd, and in the Capsicum treatment, small digested CSVd products were detected by RNA gel blot analysis. When the digesting experiment was performed for various cultivars and species of Capsicum, only cultivars of C. chinense showed strong CSVd-digesting activity. Our observations indicated that Capsicum extract contains strong RNA-digesting activity, leading to the conclusion that this activity is the main mechanism for protection from infection by CSVd through spraying or painting before inoculation. To our knowledge, this is the first report of a strong RNA-digesting activity by a plant extract.

Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

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Albertha R. van Zyl

2016-03-01

Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophyll a-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, in Nicotiana sp.*

Science.gov (United States)

Pinnola, Alberta; Ghin, Leonardo; Gecchele, Elisa; Merlin, Matilde; Alboresi, Alessandro; Avesani, Linda; Pezzotti, Mario; Capaldi, Stefano; Cazzaniga, Stefano; Bassi, Roberto

2015-01-01

Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies. PMID:26260788

Autophagy induction in tobacco leaves infected by potato virus Y{sup O} and its putative roles

Energy Technology Data Exchange (ETDEWEB)

Choi, Dabin; Park, Jaeyoung [Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of); Oh, Seonhee, E-mail: seonh@chosun.ac.kr [Department of Premedics, School of Medicine, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of); Cheong, Hyunsook, E-mail: hscheong@chosun.ac.kr [Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of)

2016-06-03

Autophagy plays a critical role in the innate immune response of plants to pathogen infection. In the present study, we examined autophagy induced by potato virus Y ordinary strain (PVY{sup O}) infection in tobacco (Nicotiana benthamiana). Enzyme-linked immunosorbent assays revealed that the number of virus particles in the plant peaked at 2 weeks post-inoculation and then gradually decreased. Additionally, the amount of virus increased significantly in the 3rd and 4th leaves distal to the inoculated leaf and decreased slightly in the 5th leaf. Within 2 weeks of PVY{sup O} inoculation, the tobacco leaves showed typical symptoms of Potyvirus inoculation, including mottling, yellowing, a mosaic pattern, and necrotic tissue changes at the inoculated site. Based on an ultrastructural analysis of the PVY{sup O}-infected tobacco leaves, virus aggregates appeared as longitudinal and transverse arrays and pinwheels, which are typical of Potyvirus inoculation. Moreover, PVY{sup O} infection caused changes in the number, size, and shape of chloroplasts, whereas the number of plastogranules increased markedly. Furthermore, double-membrane autophagosome-like vacuoles, including electron-dense materials, laminated structures, and cellular organelles, were found. The induction of autophagy after the PVY{sup O} infection of tobacco leaves was further confirmed by the expression of lipidated microtubule-associated protein 1 light chain 3 (LC3)-II, an autophagy marker and p62, an autophagy adaptor protein. The LC3-II levels increased daily over the 4-week period. Although virus inoculation was performed systemically on the basal leaves of the plants, LC3-II was expressed throughout the leaves and the expression was higher in leaves distal to the inoculated leaf. Moreover, PVY{sup O} infection caused the activation of stress-activated protein kinases/c-Jun N-terminal kinases. Therefore, PVY{sup O} infection-induced autophagy was positively correlated with the virus content

Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants

Science.gov (United States)

Lou, Yonggen; Baldwin, Ian T.

2003-01-01

Allopolyploid speciation occurs instantly when the genomes of different species combine to produce self-fertile offspring and has played a central role in the evolution of higher plants, but its consequences for adaptive responses are unknown. We compare herbivore-recognition and -resistance responses of the diploid species and putative ancestral parent Nicotiana attenuata with those of the two derived allopolyploid species Nicotiana clevelandii and Nicotiana bigelovii. Manduca sexta larvae attack all three species, and in N. attenuata attack is recognized when larval oral secretions are introduced to wounds during feeding, resulting in a jasmonate burst, a systemic amplification of trypsin inhibitor accumulation, and a release of volatile organic compounds, which function as a coordinated defense response that slows caterpillar growth and increases the probability of their being attacked. Most aspects of this recognition response are retained with modifications in one allotetraploid (N. bigelovii) but lost in the other (N. clevelandii). Differences between diploid and tetraploid species were apparent in delays (maximum 1 and 0.5 h, respectively) in the jasmonate burst, the elicitation of trypsin inhibitors and release of volatile organic compounds, and the constitutive levels of nicotine, trypsin inhibitors, diterpene glycosides, rutin, and caffeoylputrescine in the leaves. Resistance to M. sexta larvae attack was most strongly associated with diterpene glycosides, which were higher in the diploid than in the two allotetraploid species. Because M. sexta elicitors differentially regulate a large proportion of the N. attenuata transcriptome, we propose that these species are suited for the study of the evolution of adaptive responses requiring trans-activation mechanisms. PMID:14530394

Determination of gold and arsenic in Indian tobacco leaves

International Nuclear Information System (INIS)

Purkayastha, B.C.; Bhattacharyya, D.K.

1975-01-01

Two varieties of Indian Tobacco leaves have been analysed for gold and arsenic by neutron activation ( 76 As, 198 Au). Nicotiana rustica variety from North Bengal was found to contain 3.7x10 -1 ppm of gold and 4.0x10 -3 ppm of arsenic and the nicotiana tabaccum variety from Andhra Pradesh contains 1.26x10 -1 ppm of gold and 5.1x10 -3 ppm of arsenic, respectively. Unlike those in other countries Indian tobacco leaves seem to be enriched in the gold content and depleted in the arsenic content. The soil of North Bengal is richer in gold than the soil of Andhra Pradesh which requires further investigation, and the amount of arsenic in both soils is physiologically insignificant. Irradiation of leaf samples was done in a CIRUS reactor at a neutron flux of 10 13 n cm -2 s -1 for seven days. (F.G.)

Wild Nicotiana Species as a Source of Cytoplasmic Male Sterility in Nicotianatabacum

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Nikova V

2014-12-01

Full Text Available The results of our experiments executed to obtain tobacco male sterile lines through interspecific hybridization are summarized. Ten wild species from the genus Nicotiana: N. excelsior (exc, N. amplexicaulis (amp, N. rustica (rus, Nicotianaglauca (gla, N. velutina (vel, N. benthamiana (ben, N. maritima (mar, N. paniculata (pan, N. longiflora (lon and N. africana (afr were used as cytoplasmic donors and N. tabacum, cv. HarmanliiskaBasma (HB as a donor of the nucleus. Genetic effects of cytoplasmic-nuclear interaction of the studied species are discussed. Our results suggested that cytoplasmic male sterility (CMS was expressed when the cytoplasms of the above mentioned wild Nicotiana species were combined with the nucleus of N. tabacum. The 10 sources of CMS obtained in tobacco were characterized by altered flower phenotypes. Flowers are classified into types according the stamen, pistil and corolla modification. All these CMS sources were backcrossed to Oriental tobaccos, cvs. Tekne, Nevrokop B-12, Kroumovgrad 90 and Djebel 576, to develop corresponding CMS lines. The investigated cytoplasms produced compete male sterility in all those cultivars. The CMS lines preserved flower types, specific for every “sterile” cytoplasm. The extent of male organ modifications varied from apparently normal (but pollenless stamens in CMS (pan, (afr, some plants of (vel (mar through different degrees of malformations (shriveled anther on shortened filaments (lon, pinnate-like anthers on filaments of normal length (amp, petal - (ben, pistil- or stigma-like structures (rus, (gla to lack of male reproductive organs in (exc and in some plants of (vel, (mar, (rus and (gla. Most of the above mentioned cytoplasms had normal female gametophyte and good seed productivity. Alterations of the pistils were observed in CMS (rus, (exc and (ben causing reduction of the seed set. Electrophoresis of seed proteins of the tobacco cultivars and their CMS lines also suggested that

210Pb and 210Po concentrations determined in Nicotiana tabacum L., Burley variety, cultivated in Brazil

International Nuclear Information System (INIS)

Damatto, Sandra R.; Rocha, Rique J.; Da Silva, Carolina F.; Frujuele, Jonatan V.

2013-01-01

Tobacco products are extensively used throughout the world and the most consumed are cigarettes cigars and narghile. The damaging effects that these products cause to human health are discussed worldwide and many researches are performed with the aim of relating the use of these products with various illnesses. Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop year 2009/2010 production. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation (compression, filter and paper) and the temperature variation resulting from the incomplete combustion of tobacco. There is lack of information about the chemical and radiological characterization of the tobacco plant both in international and Brazilian literature. Thus a project was established with the objectives of characterizing chemically and radiologically the three varieties most cultivate in Brazil of Nicotiana tabacum L.; this paper presents the preliminary results of 210 Pb and 210 Po concentration for the Burley variety. Plants from this variety cultivated in open air, both in pots with special soil and fertilizer; and in small farms in natural conditions. The whole plant was analyzed; root, steam, leaves and flowers. The results obtained presented higher values for 210 Pb in leaves when compared with the other parts of the plant. (author)

Non-host Plant Resistance against Phytophthora capsici Is Mediated in Part by Members of the I2 R Gene Family in Nicotiana spp.

Science.gov (United States)

Vega-Arreguín, Julio C; Shimada-Beltrán, Harumi; Sevillano-Serrano, Jacobo; Moffett, Peter

2017-01-01

The identification of host genes associated with resistance to Phytophthora capsici is crucial to developing strategies of control against this oomycete pathogen. Since there are few sources of resistance to P. capsici in crop plants, non-host plants represent a promising source of resistance genes as well as excellent models to study P. capsici - plant interactions. We have previously shown that non-host resistance to P. capsici in Nicotiana spp. is mediated by the recognition of a specific P. capsici effector protein, PcAvr3a1 in a manner that suggests the involvement of a cognate disease resistance (R) genes. Here, we have used virus-induced gene silencing (VIGS) and transgenic tobacco plants expressing dsRNA in Nicotiana spp. to identify candidate R genes that mediate non-host resistance to P. capsici . Silencing of members of the I2 multigene family in the partially resistant plant N. edwardsonii and in the resistant N. tabacum resulted in compromised resistance to P. capsici . VIGS of two other components required for R gene-mediated resistance, EDS1 and SGT1 , also enhanced susceptibility to P. capsici in N. edwardsonii , as well as in the susceptible plants N. benthamiana and N. clevelandii . The silencing of I2 family members in N. tabacum also compromised the recognition of PcAvr3a1. These results indicate that in this case, non-host resistance is mediated by the same components normally associated with race-specific resistance.

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

KAUST Repository

Mahfouz, Magdy M.; Tashkandi, Manal; Ali, Zahir; Aljedaani, Fatimah R.; Shami, Ashwag

2017-01-01

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato

Expression studies of the zeaxanthin epoxidase gene in nicotiana plumbaginifolia

Science.gov (United States)

Audran; Borel; Frey; Sotta; Meyer; Simonneau; Marion-Poll

1998-11-01

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Directory of Open Access Journals (Sweden)

Yoomi Choi

Full Text Available Cucumber mosaic virus (CMV is a destructive pathogen affecting Capsicum annuum (pepper production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase. Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP. Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

Activity and specificity of TRV-mediated gene editing in plants

KAUST Repository

Ali, Zahir; Abulfaraj, Aala A.; Piatek, Marek J.; Mahfouz, Magdy M.

2015-01-01

editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.

Science.gov (United States)

Ruppert, Martin; Woll, Jörn; Giritch, Anatoli; Genady, Ezzat; Ma, Xueyan; Stöckigt, Joachim

2005-11-01

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

Science.gov (United States)

Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

2017-08-15

Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N

Expression Studies of the Zeaxanthin Epoxidase Gene in Nicotiana plumbaginifolia1

Science.gov (United States)

Audran, Corinne; Borel, Charlotte; Frey, Anne; Sotta, Bruno; Meyer, Christian; Simonneau, Thierry; Marion-Poll, Annie

1998-01-01

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors. PMID:9808747

Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants

OpenAIRE

Lou, Yonggen; Baldwin, Ian T.

2003-01-01

Allopolyploid speciation occurs instantly when the genomes of different species combine to produce self-fertile offspring and has played a central role in the evolution of higher plants, but its consequences for adaptive responses are unknown. We compare herbivore-recognition and -resistance responses of the diploid species and putative ancestral parent Nicotiana attenuata with those of the two derived allopolyploid species Nicotiana clevelandii and Nicotiana bigelovii. Manduca sexta larvae a...

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

Science.gov (United States)

Moureaux, T; Leydecker, M T; Meyer, C

1989-02-15

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

KAUST Repository

Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bé lair, Guy; Moffett, Peter

2015-01-01

in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

Science.gov (United States)

Liu, Hui; Ma, Xiao; Yu, Haiqin; Fang, Dunhuang; Li, Yongping; Wang, Xiao; Wang, Wen; Dong, Yang; Xiao, Bingguang

2016-01-01

Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive. We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome. These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.

Coat protein-mediated resistance against an Indian isolate of the ...

Indian Academy of Sciences (India)

Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were ...

Live cell CRISPR-imaging in plants reveals dynamic telomere movements

KAUST Repository

Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F.; Puchta, Holger; Houben, Andreas

2017-01-01

of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking

RNAi-mediated transgenic tospovirus resistance broken by intraspecies NSs complementation

NARCIS (Netherlands)

Hassani-Mehraban, A.; Brenkman, A.B.; Broek, N.F.J.; Goldbach, R.W.; Kormelink, R.J.M.

2009-01-01

Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants

Interactions with the actin cytoskeleton are required for cell wall localization of barley stripe mosaic virus TGB proteins

Science.gov (United States)

The host cytoskeleton and membrane system are the main routes by which plant viruses move within or between cells. Barley stripe mosaic virus (BSMV) -induced actin filament thickening was visualized in the cytoskeleton of agroinfiltrated Nicotiana benthamiana epidermal cells expressing DsRed:Talin. ...

Identification of a Xylogalacturonan Xylosyltransferase Involved in Pectin Biosynthesis in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Pauly, Markus; Sorensen, Susanne Oxenboll; Harholt, Jesper; Geshi, Naomi; Sakuragi, Yumiko; Moller, Isabel; Zandleven, Joris; Bernal, Adriana J.; Jensen, Niels Bjerg; Sorensen, Charlotte; Jensen, Jacob K.; Beldman, Gerrit; Willats, William G.T.; Scheller, Henrik

2009-08-19

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

Directory of Open Access Journals (Sweden)

Yanmei Shi

2015-12-01

Full Text Available Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

and C-terminus of

Indian Academy of Sciences (India)

amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2108-120-PVX CP was ...

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

DEFF Research Database (Denmark)

Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan

2016-01-01

rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole...

Growth of nicotiana in response to atmospheric CO sub 2 enrichment and various light regimes

Energy Technology Data Exchange (ETDEWEB)

Pope, S.; Thomas, J.F. (North Carolina State Univ., Raleigh (USA))

1989-04-01

Nicotiana tabacum NCTG-22, N. tabacum Petite Havana and N. plumbaginifolia were grown in chambers (24 C, 12-h light) under daytime atmospheric CO{sub 2} levels of 340 ppm (ambient) or 1000 ppm (enriched). All 3 types of tobacco grew faster and had open flowers sooner under CO2 enrichment, but patterns of dry weight distribution varied with type of tobacco. In N. plumbaginifolia significant proportions of dry weight were allocated to stems and branches, while in tabacum types, less was allocated to stems and more to leaves and roots. Increases in dry weight due to CO2 enrichment were accompanied by increases in leaf area and thickness. Plants given a far-red low intensity night break exhibited few differences from controls except having thinner leaves under ambient CO2; but under enriched CO2, had greater total dry weight and thicker leaves containing a higher proportion of spongy mesophyll than controls. A 50% reduction in light intensity led to a comparable reduction in dry weight and leaf area across treatments.

Assessment of 210Pb concentration in Nicotiana tabacum L., burley variety, cultivated in Brazil

International Nuclear Information System (INIS)

Rocha, Rique J.F.X.; Silva, Carolina F.; Frujuele, Jonatan V.; Bovolini, Raquel R.; Damatto, Sandra R.

2013-01-01

Tobacco products are extensively used throughout the world and the most consumed are cigarettes, cigars and narghile. The damaging effects that these products cause to human health are discussed worldwide and many researches are performed with the aim of relating the use of these products with various diseases. Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop year 2009/2010 production. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation and the temperature variations resulting from the tobacco incomplete combustion. There is lack of information about the chemical and radiological characterization of the tobacco plant both in international and Brazilian literature. Thus a project was established with the objectives of characterizing chemically and radiologically the three varieties most cultivated in Brazil of Nicotiana tobacum L., Virginia, Burley and Common; this paper presents the preliminary results of 210 Pb concentrations for the Burley variety. Plants from this variety were cultivated in pots with organic substrate and fertilizer and in a small farm in natural conditions. The entire plant was analyzed, the organic substrates, the fertilizers and the soil. The results obtained presented higher values for 210 Pb in leaves when compared with the other parts of the plant. Comparing the three study areas the highest results of 210 Pb concentration were obtained in the plants cultivated in the urban area probably due to its atmospheric deposition. (author)

Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes.

Science.gov (United States)

Raguso, Robert A; Schlumpberger, Boris O; Kaczorowski, Rainee L; Holtsford, Timothy P

2006-09-01

We analyzed floral volatiles from eight tobacco species (Nicotiana; Solanaceae) including newly discovered Brazilian taxa (Nicotiana mutabilis and "Rastroensis") in section Alatae. Eighty-four compounds were found, including mono- and sesquiterpenoids, nitrogenous compounds, benzenoid and aliphatic alcohols, aldehydes and esters. Floral scent from recent accessions of Nicotiana alata, Nicotiana bonariensis and Nicotiana langsdorffii differed from previously published data, suggesting intraspecific variation in scent composition at the level of biosynthetic class. Newly discovered taxa in Alatae, like their relatives, emit large amounts of 1,8-cineole and smaller amounts of monoterpenes on a nocturnal rhythm, constituting a chemical synapomorphy for this lineage. Fragrance data from three species of Nicotiana sect. Suaveolentes, the sister group of Alatae, (two Australian species: N. cavicola, N. ingulba; one African species: N. africana), were compared to previously reported data from their close relative, N. suaveolens. Like N. suaveolens, N. cavicola and N. ingulba emit fragrances dominated by benzenoids and phenylpropanoids, whereas the flowers of N. africana lacked a distinct floral scent and instead emitted only small amounts of an aliphatic methyl ester from foliage. Interestingly, this ester also is emitted from foliage of N. longiflora and N. plumbaginifolia (both in section Alatae s.l.), which share a common ancestor with N. africana. This result, combined with the synapomorphic pattern of 1,8 cineole emission in Alatae s.s., suggests that phylogenetic signal explains a major component of fragrance composition among tobacco species in sections Alatae and Suaveolentes. At the intraspecific level, interpopulational scent variation is widespread in sect. Alatae, and may reflect edaphic specialization, introgression, local pollinator shifts, genetic drift or artificial selection in cultivation. Further studies with genetically and geographically well

The movement protein and coat protein of alfalfa mosaic virus accumulate in structurally modified plasmodesmata

NARCIS (Netherlands)

van der Wel, N. N.; Goldbach, R. W.; van Lent, J. W.

1998-01-01

In systemically infected tissues of Nicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are

The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity.

Science.gov (United States)

Mural, Ravi V; Liu, Yao; Rosebrock, Tracy R; Brady, Jennifer J; Hamera, Sadia; Connor, Richard A; Martin, Gregory B; Zeng, Lirong

2013-09-01

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.

Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR.

Science.gov (United States)

Arif, Muhammad; Ali, Murad; Rehman, Anayatur; Fahim, Muhammad

2014-01-01

The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region. Copyright © 2013 Elsevier B.V. All rights reserved.

Assessment of {sup 210}Pb concentration in Nicotiana tabacum L., burley variety, cultivated in Brazil

Energy Technology Data Exchange (ETDEWEB)

Rocha, Rique J.F.X.; Silva, Carolina F.; Frujuele, Jonatan V.; Bovolini, Raquel R.; Damatto, Sandra R., E-mail: rjrocha@ipen.br, E-mail: cfsilva@ipen.br, E-mail: jonatanfrujuele@hotmail.com, E-mail: ra_bovolini@yahoo.com.br, E-mail: damatto@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Lab. de Radiometria Ambiental

2013-07-01

Tobacco products are extensively used throughout the world and the most consumed are cigarettes, cigars and narghile. The damaging effects that these products cause to human health are discussed worldwide and many researches are performed with the aim of relating the use of these products with various diseases. Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop year 2009/2010 production. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation and the temperature variations resulting from the tobacco incomplete combustion. There is lack of information about the chemical and radiological characterization of the tobacco plant both in international and Brazilian literature. Thus a project was established with the objectives of characterizing chemically and radiologically the three varieties most cultivated in Brazil of Nicotiana tobacum L., Virginia, Burley and Common; this paper presents the preliminary results of {sup 210}Pb concentrations for the Burley variety. Plants from this variety were cultivated in pots with organic substrate and fertilizer and in a small farm in natural conditions. The entire plant was analyzed, the organic substrates, the fertilizers and the soil. The results obtained presented higher values for {sup 210}Pb in leaves when compared with the other parts of the plant. Comparing the three study areas the highest results of {sup 210}Pb concentration were obtained in the plants cultivated in the urban area probably due to its atmospheric deposition. (author)

Identification and characterization of two RNA silencing suppressors encoded by ophioviruses

NARCIS (Netherlands)

Robles Luna, Gabriel; Reyes, Carina A.; Peña, Eduardo J.; Ocolotobiche, Eliana; Baeza, Cecilia; Borniego, Maria Belén; Kormelink, Richard; García, María Laura

2017-01-01

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the

Browse Title Index

African Journals Online (AJOL)

Items 301 - 333 of 333 ... Vol 29, No 1 (2006), The undifferentiated afromontane forest of Denkoro in the central highland of Ethiopia: a floristic and structural analysis, Abstract PDF. Abate Ayalew, Tamrat Bekele, Sebsebe Demissew. Vol 36, No 1 (2013), Tobacco (Nicotiana benthamiana Domin) Plant Transformation With and ...

Pathway of phloem unloading in tobacco sink leaves

International Nuclear Information System (INIS)

Turgeon, R.

1987-01-01

Phloem unloading in transition sink leaves of tobacco (Nicotiana tabacum L.) was analyzed by quantitative autoradiography. Source leaves were labeled with 14 CO 2 and experimental treatments were begun approximately 1 h later when label had entered the sink leaves. Autoradiographs were prepared from rapidly frozen, lyophilized sink tissue at the beginning and end of the treatments and the amount of label in veins and in surrounding cells was determined by microdensitometry. Photoassimilate unloaded from third order and larger, but not smaller, veins. Long-distance import and unloading did not respond the same way to all experimental treatments. Import was completely inhibited by cold, anaerobiosis or steam girdling the sink leaf petiole. Unloading was inhibited by cold but continued in an anaerobic atmosphere and after steam girdling. Uptake of exogenous [ 14 C]sucrose was inhibited by anaerobiosis. Since an apoplastic pathway of phloem unloading would involve solute uptake from the apoplast the results are most consistent with passive symplastic unloading of photoassimilates from phloem to surrounding cells

Sincronización de Células de Tabaco (Nicotiana tabacum) NT-1 Synchronization of tobacco cells (Nicotiana tabacum) NT-1

OpenAIRE

León F Ruiz; Ana E Higareda; Marco A Pardo

2010-01-01

Se ha evaluado la capacidad sincronizante de afidicolina e hidroxiurea en cultivos de células de tabaco (Nicotiana tabacum) NT-1. Los cultivos sincronizados son poderosas herramientas en estudios moleculares y bioquímicos relacionados al ciclo celular y comúnmente se utilizan químicos para bloquear el ciclo celular. La línea celular de tabaco (Nicotiana tabacum) NT-1 proviene de la línea celular TBY-2, caracterizándose NT-1 por su menor velocidad de crecimiento y tamaño celular heterogéneo. L...

Improved phytoaccumulation of cadmium by genetically modified tobacco plants (Nicotiana tabacum L.). Physiological and biochemical response of the transformants to cadmium toxicity

International Nuclear Information System (INIS)

Gorinova, N.; Nedkovska, M.; Todorovska, E.; Simova-Stoilova, L.; Stoyanova, Z.; Georgieva, K.; Demirevska-Kepova, K.; Atanassov, A.; Herzig, R.

2007-01-01

The response of tobacco plants (Nicotiana tabacum L.)-non-transformed and transformed with a metallothionein gene MThis from Silene vulgaris L. - to increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd both in the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with 300 μM CdCl 2 resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 μM CdCl 2 led to irreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptide pattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment both in transgenic and non-transformed plants. - Genetic transformation of Nicotiana tabacum L. by metallothionein gene improved phytoaccumulation of cadmium

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.

Optimum storage conditions for product of transiently expressed epitopes of Human papillomavirus using Potato virus X -based vector

Czech Academy of Sciences Publication Activity Database

Čeřovská, Noemi; Hoffmeisterová, Hana; Moravec, Tomáš; Plchová, Helena; Kmoníčková, Jitka; Hadámková, Renata

2008-01-01

Roč. 52, č. 1 (2008), s. 184-186 ISSN 0006-3134 R&D Projects: GA ČR GA521/06/0973 Institutional research plan: CEZ:AV0Z50380511 Keywords : edible vaccination * heterologous proteins * Nicotiana benthamiana * serological activity Subject RIV: EE - Microbiology, Virology Impact factor: 1.426, year: 2008

The influence of the N- and C- terminal modifications of Potato virus X coat protein on virus properties

Czech Academy of Sciences Publication Activity Database

Hoffmeisterová, Hana; Moravec, Tomáš; Plchová, Helena; Folwarczna, Jitka; Čeřovská, Noemi

2012-01-01

Roč. 56, č. 4 (2012), s. 775-779 ISSN 0006-3134 R&D Projects: GA ČR GA521/09/1525 Institutional research plan: CEZ:AV0Z50380511 Keywords : chimeric coat protein * expression of recombinant protein * Nicotiana benthamiana Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.692, year: 2012

Effects of the mutation of selected genes of cotton leaf curl Kokhran virus on infectivity, symptoms and the maintenance of cotton leaf curl Multan betasatellite

NARCIS (Netherlands)

Iqbal, Z.; Sattar, M.N.; Kvarnheden, A.; Mansoor, S.; Briddon, R.W.

2012-01-01

Cotton leaf curl Kokhran virus (CLCuKoV) is a cotton-infecting monopartite begomovirus (family Geminiviridae). The effects of mutation of the coat protein (CP), V2, C2 and C4 genes of CLCuKoV on infectivity and symptoms in Nicotiana benthamiana were investigated. Each mutation introduced a premature

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation.

Science.gov (United States)

Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

2015-12-01

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

The novel GrCEP12 peptide from the plant-parasitic nematode Globodera rostochiensis suppresses flg22-mediated PTI.

Science.gov (United States)

Chen, Shiyan; Chronis, Demosthenis; Wang, Xiaohong

2013-09-01

The potato cyst nematode Globodera rostochiensis is a biotrophic pathogen that secretes effector proteins into host root cells to promote successful plant parasitism. In addition to the role in generating within root tissue the feeding cells essential for nematode development, (1) nematode secreted effectors are becoming recognized as suppressors of plant immunity. (2)(-) (4) Recently we reported that the effector ubiquitin carboxyl extension protein (GrUBCEP12) from G. rostochiensis is processed into free ubiquitin and a 12-amino acid GrCEP12 peptide in planta. Transgenic potato lines overexpressing the derived GrCEP12 peptide showed increased susceptibility to G. rostochiensis and to an unrelated bacterial pathogen Streptomyces scabies, suggesting that GrCEP12 has a role in suppressing host basal defense or possibly pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) during the parasitic interaction. (3) To determine if GrCEP12 functions as a PTI suppressor we evaluated whether GrCEP12 suppresses flg22-induced PTI responses in Nicotiana benthamiana. Interestingly, we found that transient expression of GrCEP12 in N. benthamiana leaves suppressed reactive oxygen species (ROS) production and the induction of two PTI marker genes triggered by the bacterial PAMP flg22, providing direct evidence that GrCEP12 indeed has an activity in PTI suppression.

The Fusarium oxysporum effector Six6 contributes to virulence and suppresses I-2-mediated cell death.

Science.gov (United States)

Gawehns, F; Houterman, P M; Ichou, F Ait; Michielse, C B; Hijdra, M; Cornelissen, B J C; Rep, M; Takken, F L W

2014-04-01

Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death.

A jasmonate ZIM-domain protein NaJAZd regulates floral jasmonic acid levels and counteracts flower abscission in Nicotiana attenuata plants.

Directory of Open Access Journals (Sweden)

Youngjoo Oh

Full Text Available Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions

Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv. tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis

Directory of Open Access Journals (Sweden)

Hai-Lei Wei

2018-05-01

Full Text Available Summary: The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a complex repertoire of type III secretion effector (T3E proteins. Effectorless polymutant DC3000D36E was used with a modularized system for native delivery of the 29 DC3000 T3Es singly and in pairs. Assays of the performance of this T3E library in N. benthamiana leaves revealed a matrix of T3E interplay, with six T3Es eliciting death and eight others variously suppressing the death activity of the six. The T3E library was also interrogated for effects on DC3000D36E elicitation of a reactive oxygen species burst, for growth in planta, and for T3Es that reversed these effects. Pseudomonas fluorescens and Agrobacterium tumefaciens heterologous delivery systems yielded notably different sets of death-T3Es. The DC3000D36E T3E library system highlights the importance of 13 T3Es and their interplay in interactions with N. benthamiana. : Wei et al. used a Pseudomonas syringae strain lacking all known type III effectors with a modularized library expressing the 29 active effectors in the strain’s native repertoire, individually and in pairs, to comprehensively determine effector actions and interplay in inducing and suppressing responses associated with plant pathogenesis and immunity. Keywords: effector-triggered-immunity, pattern-triggered-immunity, Hop proteins, plant immunity, mini-Tn7

Genetic analysis of Phytophthora nicotianae populations from different hosts using microsatellite markers

Science.gov (United States)

Two hundred thirty-one isolates of P. nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa and Ruta) and other plant genera of lesser econo...

BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea

Directory of Open Access Journals (Sweden)

Wenjun Zhu

2017-09-01

Full Text Available We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.

Sequencing and phylogenetic analysis of tobacco virus 2, a polerovirus from Nicotiana tabacum.

Science.gov (United States)

Zhou, Benguo; Wang, Fang; Zhang, Xuesong; Zhang, Lina; Lin, Huafeng

2017-07-01

The complete genome sequence of a new virus, provisionally named tobacco virus 2 (TV2), was determined and identified from leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic, yellowing, and deformity, in Anhui Province, China. The genome sequence of TV2 comprises 5,979 nucleotides, with 87% nucleotide sequence identity to potato leafroll virus (PLRV). Its genome organization is similar to that of PLRV, containing six open reading frames (ORFs) that potentially encode proteins with putative functions in cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the nucleotide sequence placed TV2 alongside members of the genus Polerovirus in the family Luteoviridae. To the best our knowledge, this study is the first report of a complete genome sequence of a new polerovirus identified in tobacco.

Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume

DEFF Research Database (Denmark)

Heckmann, Anne Birgitte Lau; Lombardo, Fabien; Miwa, Hiroki

2006-01-01

and then increased following infection by M. loti. In hairy root transformations, LjNSP1 and MtNSP1 complemented both Mtnsp1-1 and Ljnsp1-1 mutants, demonstrating that these orthologous proteins have a conserved biochemical function. A Nicotiana benthamiana NSP1-like gene (NbNSP1) was shown to restore nodule...

Intracellular and extracellular phosphatidylinositol 3-phosphate produced by Phytophthora species is important for infection.

Science.gov (United States)

Lu, Shan; Chen, Linlin; Tao, Kai; Sun, Nannan; Wu, Yuren; Lu, Xiaoxue; Wang, Yuanchao; Dou, Daolong

2013-09-01

RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate (PtdIns(3)P) to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of PtdIns(3)P in plants are low, we examined whether Phytophthora species may produce PtdIns(3)P to promote infection. We observed that PtdIns(3)P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae during infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed PtdIns(3)P on their plasma membrane and/or secreted PtdIns(3)P. Silencing of the phosphatidylinositol 3-kinases (PI3K) genes, treatment with LY294002, or expression of PtdIns(3)P-binding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized PtdIns(3)P was required for full virulence. Secretion of PtdIns(3)P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resistance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external PtdIns(3)P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avr1b confirm that both the N-terminus and the C-terminus of this effector can bind PtdIns(3)P.

The Tomato Fni3 Lysine-63–Specific Ubiquitin-Conjugating Enzyme and Suv Ubiquitin E2 Variant Positively Regulate Plant Immunity[C][W

Science.gov (United States)

Mural, Ravi V.; Liu, Yao; Rosebrock, Tracy R.; Brady, Jennifer J.; Hamera, Sadia; Connor, Richard A.; Martin, Gregory B.; Zeng, Lirong

2013-01-01

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase–deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63–linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63–linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63–linked ubiquitination. PMID:24076975

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Science.gov (United States)

Dong, Lemeng; Miettinen, Karel; Goedbloed, Miriam; Verstappen, Francel W A; Voster, Alessandra; Jongsma, Maarten A; Memelink, Johan; van der Krol, Sander; Bouwmeester, Harro J

2013-11-01

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

Directory of Open Access Journals (Sweden)

Kittipong Rattanaporn

2011-08-01

Full Text Available Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin, CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA domain of human capillary morphogenesis 2 (CMG2, an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG. We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS: p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI, with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

Science.gov (United States)

Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M; McDonald, Karen A

2011-01-01

Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

Activity and specificity of TRV-mediated gene editing in plants

KAUST Repository

Ali, Zahir

2015-06-03

© 2015 Taylor and Francis Group, LLC. Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRVmediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

Biotechnological Reduction of Tobacco (Nicotiana Tabacum L. Toxicity

Directory of Open Access Journals (Sweden)

Samane Sattar

2012-11-01

Full Text Available Background: Nicotiana tobacco contains large amounts of alkaloid nicotine. Tobacco plant is used for smoking and causes many health problems since it is high in nicotine which is one of the widely-recognized toxic compounds with serious side effects for different body organs. Reducing nicotine content of this plant is a way to reduce its health hazards in cigarette smokers. Utilizing the new methods of genetic engineering can modify nicotine levels in the plant. In this study, through transferring the blocking gene, the pathway of nicotine biosynthesis was blocked to produce transgenic tobacco with low levels of nicotine. Methods: Transgenic plants carrying T DNA, and non-transgenic plants were grown on MS medium. Then their leaves were dried and powdered. The plants were extracted with alkali solution. Eventually, the nicotine content of the extract were analyzed using GC. Results: The analysis of extracts showed a reduction in the nicotine content of the transgenic plant (contain T-DNA in comparison with non-transgenic plants. Conclusion: Tobacco with lower nicotine reduction can reduce the toxic effects of smoking on smokers and can facilitate withdrawal from it.

Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

Science.gov (United States)

Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2015-02-02

Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of virus infection on symplastic transport of fluorescent tracers in Nicotiana clevelandii leaf epidermis.

Science.gov (United States)

Derrick, P M; Barker, H; Oparka, K J

1990-07-01

The molecular weight exclusion limit of plasmodesmata in subveinal epidermal cells of Nicotiana clevelandii (Gray) leaves was estimated by microinjection and fluorescence microscopy using fluorescein isothiocyanate-peptide conjugates, carboxyfluorescein and Lucifer Yellow CH. The largest fluorochrome which moved symplastically between cells had a molecular weight of 749, although movement did not appear to depend purely on molecular weight parameters. Systemic infection of plants by tobacco rattle tobravirus, tomato black ring nepovirus or potato Y potyvirus did not alter the limits of plasmodesmatal conductance of the fluorochromes. However, carrot mottle umbravirus and groundnut rosette umbravirus diminished the symplastic mobility of some fluorescent tracers. These results imply that intercellular movement of these viruses does not involve a long-lasting increase in the plasmodesmatal molecular size exclusion limit.

Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves

Directory of Open Access Journals (Sweden)

VanDoorn Arjen

2010-08-01

Full Text Available Abstract Background Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants. Results The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu, was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3 and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene in irlox2 plants. Conclusions The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential

Nicotiana plumbaginifolia: A Rich Antimicrobial and Antioxidant Source

International Nuclear Information System (INIS)

Ajaib, M.; Perveen, S.

2016-01-01

Antimicrobial and antioxidant activities of plant Nicotiana plumbaginifolia Viv. Were carried out using various techniques. The petroleum ether, chloroform, methanol and aqueous extracts of the N. plumbaginifolia were obtained by maceration technique. The maximum antibacterial potential was exhibited by chloroform leaves extract (76.3 ± 0.3 mm), methanolic root extract (69 ± 0.8 mm) and petroleum ether root extract (67 ± 1.7 mm) against P. aureginosa. Methanolic root extract possessed 64 ± 2.3 mm zone of inhibition against E. coli, whereas chloroform root extract displayed 49 ± 0.8 mm against B. subtilis. Chloroform root extract showed 48 ±1.2 against S. aureus. The maximum zone of inhibition of antifungal potential was displayed by methanolic extracts of leaves against A. niger (43 ± 0.8 mm) and F. solani (43 ± 1.6 mm). The MIC assay was determine for further analysis which showed the MIC value of methanolic root extract (0.04 ± 0.1 mg/mL) against E. coli and the MIC value was noticed (0.108 ± 0.04 mg/mL) against A. niger by methanolic root extract. Antioxidant potential was determined using four methods i.e. (1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, total antioxidant activity (TAA), total phenolic contents (TPC) and metal chelating activity. The highest value of percent DPPH was observed 90.56 at 1000 microL concentration in petroleum ether extract. The maximum values of TAA, TPC, FRAP and FTC were 1.352 ± 0.01, 1.683 ± 0.09 and 80.66 ± 0.08, respectively. (author)

The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen.

Science.gov (United States)

Roberts, Andy V

2007-12-01

"Bead beating" is commonly used to release DNA from cells for genomic studies but it was used here to prepare suspensions of plant nuclei for measurement of DNA amounts by flow cytometry. Plant material was placed in 2-ml screw-capped tubes containing beads of zirconia/silica (2.5 mm diameter) or glass (2.5 or 1.0 mm diameter) and 1 ml of lysis buffer. The tubes were mechanically shaken with an FP120 FastPrep Cell Disrupter to release intact nuclei from plant tissue by the impact of the beads. The nuclei were then stained with propidium iodide (PI) and analyzed by flow cytometry. The method was tested using fresh leaves, fresh petals and herbarium leaves of Rosa canina, leaves and pollen of R. rugosa, and fresh leaves of Petroselinum crispum, Nicotiana tabacum, and Allium cepa. Batches of 12 samples of fresh leaves were prepared, simultaneously, in 45 s by bead beating in the Cell Disrupter. In flow cytometry histograms, nuclei of fresh leaves gave G(1)/G(0) peaks with CVs of less than 3.0% and nuclei from fresh petals and herbarium leaves of R. canina, and pollen of the generative nuclei of R. rugosa gave peaks with coefficients of variation (CVs) of less than 4.0%. DNA amounts estimated from 24-month-old herbarium leaves, using P. crispum as an internal standard, were less than those of fresh leaves by a small but significant amount. Suspensions of nuclei can be prepared rapidly and conveniently from a diversity of tissues by bead beating. Exposure of laboratory workers to harmful substances in the lysis buffer is minimized. (c) 2007 International Society for Analytical Cytology

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles.

Science.gov (United States)

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

2008-03-28

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

Nicotiana glauca poisoning in ostriches (Struthio camelus)

CSIR Research Space (South Africa)

Botha, CJ

2011-01-01

Full Text Available Putative Nicotiana glauca (wild tobacco) poisoning was diagnosed in a flock of ostriches near Oudtshoorn, South Africa. Post mortem examinations (n = 7) were performed on ostriches (Struthio camelus) that had died. Suspicious leaf remnants (weighing...

Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

Czech Academy of Sciences Publication Activity Database

Vaculík, Petr; Plchová, Helena; Moravec, Tomáš; Hoffmeisterová, Hana; Čeřovská, Noemi; Šmahel, M.

2015-01-01

Roč. 120, č. 2 (2015), s. 671-680 ISSN 0167-6857 R&D Projects: GA ČR(CZ) GAP501/12/1761 Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Potato virus X * Human papillomavirus * Nicotiana benthamiana Subject RIV: EE - Microbiology, Virology Impact factor: 2.390, year: 2015

Pest and disease resistance enhanced by heterologous suppression of a Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2.

Science.gov (United States)

Smigocki, Ann C; Wilson, Dennis

2004-12-01

The functional role of the Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58 kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.

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.

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

Science.gov (United States)

Rossi, Marika; Vallino, Marta; Abbà, Simona; Ciuffo, Marina; Balestrini, Raffaella; Genre, Andrea; Turina, Massimo

2015-01-01

The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

Science.gov (United States)

Hérouart, D; Van Montagu, M; Inzé, D

1994-03-01

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.

Genome-Wide Identification of BAHD Acyltransferases and In vivo Characterization of HQT-like Enzymes Involved in Caffeoylquinic Acid Synthesis in Globe Artichoke

Science.gov (United States)

Moglia, Andrea; Acquadro, Alberto; Eljounaidi, Kaouthar; Milani, Anna M.; Cagliero, Cecilia; Rubiolo, Patrizia; Genre, Andrea; Cankar, Katarina; Beekwilder, Jules; Comino, Cinzia

2016-01-01

Globe artichoke (Cynara cardunculus L. var. scolymus) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong to the large family of BAHD acyltransferases. Following a survey of the globe artichoke genome, we identified 69 BAHD proteins carrying the catalytic site (HXXXD). Their phylogenetic analysis together with another 43 proteins, from 21 species, representative of the BAHD family, highlighted their grouping in seven major clades. Nine globe artichoke acyltransferases clustered in a sub-group of Clade V, with 3 belonging to hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) and 2 to hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) like proteins. We focused our attention on the former, HQT1, HQT2, and HQT3, as they are known to play a key role in CGA biosynthesis. The expression of genes coding for the three HQTs and correlation of expression with the CQA content is reported for different globe artichoke tissues. For the first time in the globe artichoke, we developed and applied the virus-induced gene silencing approach with the goal of assessing in vivo the effect of HQT1 silencing, which resulted in a marked reduction of both CGA and diCQAs. On the other hand, when the role of the three HQTs was assessed in leaves of Nicotiana benthamiana through their transient overexpression, significant increases in mono- and diCQAs content were observed. Using transient GFP fusion proteins expressed in N. benthamiana leaves we also established the sub-cellular localization of these three enzymes. PMID:27721818

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

KAUST Repository

Mahfouz, Magdy M.

2017-12-22

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

Mutagenesis for ACMV resistance in a Ghanian cassava cultivar `Bosom nsia`

Energy Technology Data Exchange (ETDEWEB)

Ahiabu, R K; Lokko, Y; Danso, K; Klu, G Y.P. [Biotechnology and Nuclear Agricultural Research Inst., Ghana Atomic Energy Commission, Legon (Ghana)

1997-07-01

Breeding for resistance to the African Cassava Mosaic Virus (ACMV) disease in the Ghanian cassava cultivar `Bosom nsia` has been on-going for the past four years at the Biotechnology and Nuclear Agricultural Research Institute. Protocols for in vitro culture from shoot meristems and acclimation of plantlets were established. Radiosensitivity tests on the regenerated plantlets indicated LD{sub 50} of 40 Gy, and doses of 25, 30 and 35 Gy were suitable for mutagenesis. These doses were applied to in vivo and in vitro grown plants, and selection was carried out in three propagations. Four variants, selected under field conditions with high viral incidence, were analyzed for virus particles with three virus indexing techniques. Polymerase chain reaction (PCR) product analysis of DNA extracts from Nicotiana benthamiana test plants, inoculated with sap from the leaves of variants, confirmed the presence of virus particles in all variants. Inoculation and ELISA tests suggested ACMV tolerance in selected variants. (author). 24 refs, 3 figs, 1 tab.

Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca).

Science.gov (United States)

Osorio, Sonia; Castillejo, Cristina; Quesada, Miguel A; Medina-Escobar, Nieves; Brownsey, Geoff J; Suau, Rafael; Heredia, Antonio; Botella, Miguel A; Valpuesta, Victoriano

2008-04-01

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

Mutagenesis for ACMV resistance in a Ghanian cassava cultivar 'Bosom nsia'

International Nuclear Information System (INIS)

Ahiabu, R.K.; Lokko, Y.; Danso, K.; Klu, G.Y.P.

1997-01-01

Breeding for resistance to the African Cassava Mosaic Virus (ACMV) disease in the Ghanian cassava cultivar 'Bosom nsia' has been on-going for the past four years at the Biotechnology and Nuclear Agricultural Research Institute. Protocols for in vitro culture from shoot meristems and acclimation of plantlets were established. Radiosensitivity tests on the regenerated plantlets indicated LD 50 of 40 Gy, and doses of 25, 30 and 35 Gy were suitable for mutagenesis. These doses were applied to in vivo and in vitro grown plants, and selection was carried out in three propagations. Four variants, selected under field conditions with high viral incidence, were analyzed for virus particles with three virus indexing techniques. Polymerase chain reaction (PCR) product analysis of DNA extracts from Nicotiana benthamiana test plants, inoculated with sap from the leaves of variants, confirmed the presence of virus particles in all variants. Inoculation and ELISA tests suggested ACMV tolerance in selected variants. (author). 24 refs, 3 figs, 1 tab

NaJAZh Regulates a Subset of Defense Responses against Herbivores and Spontaneous Leaf Necrosis in Nicotiana attenuata Plants[C][W][OA

Science.gov (United States)

Oh, Youngjoo; Baldwin, Ian T.; Gális, Ivan

2012-01-01

The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development. PMID:22496510

Uptake and translocation of 109Cd and stable Cd within tobacco plants (Nicotiana sylvestris)

International Nuclear Information System (INIS)

Rosén, K.; Eriksson, J.; Vinichuk, M.

2012-01-01

The availability, uptake, and translocation of recently added ( 109 Cd) and naturally occurring (stable) soil Cd within tobacco plants were compared. 109 Cd was added to soil in two treatments, A (0.25 MBq kg soil −1 DW) and B (eight-fold dose): stable Cd was measured in both treatments. Both the added and the stable Cd were higher in leaves and reproductive structures of the plant than in stalks and roots. The uptake of 109 Cd was 5.3 kBq plant −1 for treatment A and 36.7 kBq plant −1 for treatment B, and about 26 μg plant −1 for stable Cd. Leaves of the tobacco plants accumulated 40–45% of the total 109 Cd and about 50% of total stable Cd taken up by the plant. Cadmium concentration in the plant was three times higher than in roots and two times higher than the concentration in soil: the concentration in roots was lower than in the soil. - Capsule: The availability, uptake, and translocation of recently added ( 109 Cd) and naturally occurring (stable) soil Cd within tobacco plants (Nicotiana sylvestris) were investigated. - Highlights: ► We compared uptake recently added and naturally occurring soil Cd by tobacco plant. ► Both added and stable Cd display similar uptake and translocation within the plant. ► Leaves of tobacco plants accumulate half of the total Cd taken up by the plant. ► Recently added 109 Cd to soil is more available than naturally occurring cadmium.

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

Science.gov (United States)

Castresana, C; de Carvalho, F; Gheysen, G; Habets, M; Inzé, D; Van Montagu, M

1990-01-01

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection. PMID:2152158

Cloning and Molecular Analysis of HlbZip1 and HlbZip2 Transcription Factors Putatively Involved in the Regulation of the Lupulin Metabolome in Hop (Humulus lupulus L.)

Czech Academy of Sciences Publication Activity Database

Matoušek, Jaroslav; Kocábek, Tomáš; Patzak, J.; Stehlík, Jan; Füssy, Zoltán; Krofta, K.; Heyerick, A.; Roldán-Ruiz, I.; Maloukh, L.; De Keukeleire, D.

2010-01-01

Roč. 58, č. 2 (2010), s. 902-912 ISSN 0021-8561 R&D Projects: GA ČR GA521/08/0740; GA MZe QH81052; GA MŠk ME 940 Institutional research plan: CEZ:AV0Z50510513 Keywords : secondary metabolites transcriptional regulation * cDNA-AFLP analysis * hop cDNA library screening * Nicotiana benthamiana * Petunia hybrida Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.816, year: 2010

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

OpenAIRE

Bhoo, Seong Hee; Lai, Huafang; Ma, Julian; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

2011-01-01

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal hemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identify Ebola and Marburg viruses as “category A” pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their dev...

Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants.

Science.gov (United States)

Vincentz, M; Caboche, M

1991-01-01

A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of NR transcript and protein was restored for NR activity by transformation with a chimaeric NR gene. This gene was composed of a full-length tobacco NR cDNA fused to the CaMV 35S promoter and to termination signals from the tobacco NR gene. The transgenic plants we obtained were viable and fertile and expressed from one-fifth to three times the wild-type NR activity in their leaves. The analysis of chimeric NR gene expression in these plants showed, by comparison with wild-type plants, that the regulation of NR gene expression by light, nitrate and circadian rhythm takes place at the transcriptional level. However, unlike nitrate, light was required for the accumulation of NR protein in transgenic plants, suggesting that NR expression is also controlled at the translational and/or post-translational level. Images PMID:2022181

Sincronización de Células de Tabaco (Nicotiana tabacum) NT-1

OpenAIRE

Ruiz, León F; Higareda, Ana E; Pardo, Marco A

2010-01-01

Se ha evaluado la capacidad sincronizante de afidicolina e hidroxiurea en cultivos de células de tabaco (Nicotiana tabacum) NT-1. Los cultivos sincronizados son poderosas herramientas en estudios moleculares y bioquímicos relacionados al ciclo celular y comúnmente se utilizan químicos para bloquear el ciclo celular. La línea celular de tabaco (Nicotiana tabacum) NT-1 proviene de la línea celular TBY-2, caracterizándose NT-1 por su menor velocidad de crecimiento y tamaño celular heterogéneo. L...

Single amino acid changes in the 6K1-CI region can promote the alternative adaptation of Prunus- and Nicotiana-propagated Plum pox virus C isolates to either host.

Science.gov (United States)

Calvo, María; Malinowski, Tadeusz; García, Juan Antonio

2014-02-01

Plum pox virus (PPV) C is one of the less common PPV strains and specifically infects cherry trees in nature. Making use of two PPV-C isolates that display different pathogenicity features, i.e., SwCMp, which had been adapted to Nicotiana species, and BY101, which had been isolated from cherry rootstock L2 (Prunus lannesiana) and propagated only in cherry species, we have generated two infective full-length cDNA clones in order to determine which viral factors are involved in the adaptation to each host. According to our results, the C-P3(PIPO)/6K1/N-CI (cylindrical inclusion) region contains overlapping but not coincident viral determinants involved in symptoms development, local viral amplification, and systemic movement capacity. Amino acid changes in this region promoting the adaptation to N. benthamiana or P. avium have trade-off effects in the alternative host. In both cases, adaptation can be achieved through single amino acid changes in the NIapro protease recognition motif between 6K1 and CI or in nearby sequences. Thus, we hypothesize that the potyvirus polyprotein processing could depend on specific host factors and the adaptation of PPV-C isolates to particular hosts relies on a fine regulation of the proteolytic cleavage of the 6K1-CI junction.

Characterization of a Novel Polerovirus Infecting Maize in China.

Science.gov (United States)

Chen, Sha; Jiang, Guangzhuang; Wu, Jianxiang; Liu, Yong; Qian, Yajuan; Zhou, Xueping

2016-04-28

A novel virus, tentatively named Maize Yellow Mosaic Virus (MaYMV), was identified from the field-grown maize plants showing yellow mosaic symptoms on the leaves collected from the Yunnan Province of China by the deep sequencing of small RNAs. The complete 5642 nucleotide (nt)-long genome of the MaYMV shared the highest nucleotide sequence identity (73%) to Maize Yellow Dwarf Virus-RMV. Sequence comparisons and phylogenetic analyses suggested that MaYMV represents a new member of the genus Polerovirus in the family Luteoviridae. Furthermore, the P0 protein encoded by MaYMV was demonstrated to inhibit both local and systemic RNA silencing by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene, which further supported the identification of a new polerovirus. The biologically-active cDNA clone of MaYMV was generated by inserting the full-length cDNA of MaYMV into the binary vector pCB301. RT-PCR and Northern blot analyses showed that this clone was systemically infectious upon agro-inoculation into N. benthamiana. Subsequently, 13 different isolates of MaYMV from field-grown maize plants in different geographical locations of Yunnan and Guizhou provinces of China were sequenced. Analyses of their molecular variation indicate that the 3' half of P3-P5 read-through protein coding region was the most variable, whereas the coat protein- (CP-) and movement protein- (MP-)coding regions were the most conserved.

Characterization of a Novel Polerovirus Infecting Maize in China

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Sha Chen

2016-04-01

Full Text Available A novel virus, tentatively named Maize Yellow Mosaic Virus (MaYMV, was identified from the field-grown maize plants showing yellow mosaic symptoms on the leaves collected from the Yunnan Province of China by the deep sequencing of small RNAs. The complete 5642 nucleotide (nt-long genome of the MaYMV shared the highest nucleotide sequence identity (73% to Maize Yellow Dwarf Virus-RMV. Sequence comparisons and phylogenetic analyses suggested that MaYMV represents a new member of the genus Polerovirus in the family Luteoviridae. Furthermore, the P0 protein encoded by MaYMV was demonstrated to inhibit both local and systemic RNA silencing by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene, which further supported the identification of a new polerovirus. The biologically-active cDNA clone of MaYMV was generated by inserting the full-length cDNA of MaYMV into the binary vector pCB301. RT-PCR and Northern blot analyses showed that this clone was systemically infectious upon agro-inoculation into N. benthamiana. Subsequently, 13 different isolates of MaYMV from field-grown maize plants in different geographical locations of Yunnan and Guizhou provinces of China were sequenced. Analyses of their molecular variation indicate that the 3′ half of P3–P5 read-through protein coding region was the most variable, whereas the coat protein- (CP- and movement protein- (MP-coding regions were the most conserved.

Effect of Radiation Dosage on Efficiency of Chloroplast Transfer by Protoplast Fusion in Nicotiana

OpenAIRE

Menczel, László; Galiba, Gábor; Nagy, Ferenc; Maliga, Pál

1982-01-01

Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a 60Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protopla...

A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium

OpenAIRE

Hoshi, Ayaka; Oshima, Kenro; Kakizawa, Shigeyuki; Ishii, Yoshiko; Ozeki, Johji; Hashimoto, Masayoshi; Komatsu, Ken; Kagiwada, Satoshi; Yamaji, Yasuyuki; Namba, Shigetou

2009-01-01

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidop...

Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

Science.gov (United States)

Hettenhausen, Christian; Heinrich, Maria; Baldwin, Ian T; Wu, Jianqiang

2014-11-28

Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

Characterization of Phytophthora nicotianae isolates in southeast Spain and their detection and quantification through a real-time TaqMan PCR.

Science.gov (United States)

Blaya, Josefa; Lacasa, Carmen; Lacasa, Alfredo; Martínez, Victoriano; Santísima-Trinidad, Ana B; Pascual, Jose A; Ros, Margarita

2015-04-01

The soil-borne pathogens Phytophthora nicotianae and P. capsici are the causal agents of root and stem rot of many plant species. Although P. capsici was considered the causal agent in one of the main pepper production areas of Spain to date, evidence of the presence of P. nicotianae was found. We aimed to survey the presence of P. nicotianae and study the variability in its populations in this area in order to improve the management of Tristeza disease. A new specific primer and a TaqMan probe were designed based on the internal transcribed spacer regions of ribosomal DNA to detect and quantify P. nicotianae. Both morphological and molecular analysis showed its presence and confirmed it to be the causal agent of the Phytophthora disease symptoms in the studied area. The genetic characterization among P. nicotianae populations showed a low variability of genetic diversity among the isolates. Only isolates of the A2 mating type were detected. Not only is a specific and early detection of P. nicotianae essential but also the study of genetic variability among isolates for the appropriate management of the disease, above all, in producing areas with favorable conditions for the advance of the disease. © 2014 Society of Chemical Industry.

Ecological Roles and Biological Activities of Specialized Metabolites from the Genus Nicotiana.

Science.gov (United States)

Jassbi, Amir Reza; Zare, Somayeh; Asadollahi, Mojtaba; Schuman, Meredith C

2017-10-11

Species of Nicotiana grow naturally in different parts of the world and have long been used both medicinally and recreationally by human societies. More recently in our history, Nicotiana tabacum has attracted interest as one of the most economically important industrial crops. Nicotiana species are frequently investigated for their bioactive natural products, and the ecological role of their specialized metabolites in responses to abiotic stress or biotic stress factors like pathogens and herbivores. The interest of tobacco companies in genetic information as well as the success of a few wild tobacco species as experimental model organisms have resulted in growing knowledge about the molecular biology and ecology of these plants and functional studies of the plant's natural products. Although a large number of reviews and books on biologically active natural products already exists, mostly from N. tabacum, we focus our attention on the ecological roles and biological activity of natural products, versus products from cured and processed material, in this Review. The studied compounds include alkaloids, aromatic compounds, flavonoids, volatiles, sesquiterpenoids, diterpenes alcohols, and sugar esters from trichomes of the plants, and recently characterized acyclic hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). In this Review (1800s-2017), we describe the above-mentioned classes of natural products, emphasizing their biological activities and functions as they have been determined either in bioassay-guided purification approaches or in bioassays with plants in which the expression of specific biosynthetic genes has been genetically manipulated. Additionally, a review on the history, taxonomy, ecology, and medicinal application of different Nicotiana species growing around the globe presented in this Review may be of interest for pharmacognosists, natural products, and ecological chemists.

Transient expression of human papillomavirus type 16 virus-like particles in tobacco and tomato using a tobacco rattle virus expression vector

Czech Academy of Sciences Publication Activity Database

Šmídková, Markéta; Müller, M.; Thönes, N.; Piuko, K.; Angelisová, Pavla; Velemínský, Jiří; Angelis, Karel

2010-01-01

Roč. 54, č. 3 (2010), s. 451-460 ISSN 0006-3134 R&D Projects: GA ČR GA521/01/1418; GA ČR GA521/04/0971; GA AV ČR IAA6038201; GA AV ČR IBS5038304; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514 Keywords : ELISA * Lycopersicon esculentum * Nicotiana benthamiana Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.582, year: 2010

Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR.

Science.gov (United States)

Li, Mingzhu; Inada, Minoru; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

2013-01-01

Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

Science.gov (United States)

Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

2016-01-01

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

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.

Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

Directory of Open Access Journals (Sweden)

Mi Sa Vo Phan

2014-06-01

Full Text Available Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV from Glycine soja (wild soybean, named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean and Pisum sativum (pea as well as N. benthamiana, but not the other legume species.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

Energy Technology Data Exchange (ETDEWEB)

Hipp, Katharina; Rau, Peter; Schäfer, Benjamin [Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart (Germany); Gronenborn, Bruno [Institut des Sciences du Végétal, CNRS, 91198 Gif-sur-Yvette (France); Jeske, Holger, E-mail: holger.jeske@bio.uni-stuttgart.de [Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart (Germany)

2014-08-15

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

International Nuclear Information System (INIS)

Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Gronenborn, Bruno; Jeske, Holger

2014-01-01

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis

Differences in the Detoxification Metabolism between Two clonal Lineages of the Aphid Myzus persicae (Sulzer (Hemiptera: Aphididae Reared on Tobacco (Nicotiana tabacum L. Diferencias en el Metabolismo de Detoxificación entre dos Linajes Clonales del Áfido Myzus persicae (Sulzer (Hemiptera: Aphididae creados sobre tabaco (Nicotiana tabacum L.

Directory of Open Access Journals (Sweden)

Marco A Cabrera-Brandt

2010-12-01

Full Text Available Myzus persicae (Sulzer is a highly polyphagous aphid species, with a subspecies (M. persicae nicotianae well adapted to tobacco (Nicotiana tabacum L.. We evaluated the effect of this host plant on the aphid performance and detoxification enzymes, in order to test the participation of xenobiotic metabolism on the ability of this aphid to overcome the tobacco chemical defences. Two genotypes, one corresponding to the only M. persicae nicotianae genotype reported in Chile on tobacco, and one genotype belonging to M. persicae sensu stricto were reared on tobacco and pepper (Capsicum annuum L., respectively. M. persicae nicotianae showed a significantly higher intrinsic rate of increase (r m on pepper than on tobacco, and M. persicae s.s. performed similarly, but with no reproduction at all on tobacco. In order to evaluate the effect of tobacco on detoxification enzymes, esterases, glutathione S-transferases (GST and cytochrome P-450 monooxygenases (MO were determined in both selected aphid genotypes after 12, 24, 36, 48 and 72 h of rearing on tobacco and pepper. M. persicae nicotianae exhibited the higher total esterase activities when reared on tobacco than on pepper after 48 h of rearing, while the activities of GST and MO did not show any significant difference between host-plants and duration of treatment. For M. persicae s.s., no significant differences were observed among host-plants for the studied enzymes. These results suggest a participation of the esterases, on the ability of this M. persicae nicotianae to overcome the tobacco defences.Myzus persicae (Sulzer es un áfido polífago que incluye a Myzus persicae nicotianae, una subespecie altamente adaptada sobre tabaco (Nicotiana tabacum L.. Evaluamos el efecto del tabaco sobre el desempeño biológico y sobre determinadas enzimas de detoxificación en áfidos, para estudiar su participación en la capacidad de M. persicae nicotianae de superar las defensas químicas del tabaco. Dos

Evaluation of 5P12-RANTES analogue expression in Nicotiana benthamiana

CSIR Research Space (South Africa)

Mawela, KG

2012-10-10

Full Text Available 100 200 300 400 500 600 700 Apoplast Cytosol pICH11599 (-ve control) 5P 12 (? g /k g F W ) 3 dpi 6 dpi 9 dpi (B) Bacterial vector system 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Cytosol ER Chloroplast pICH11599 (-ve... MTS tetrazolium compound, {3-(4,5-dimethyl-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium} Efficacy testing Table 1 Summary of IC50 values of the HIV-1 pseudovirions screened. Sample identification ZM53 (targets CCR5) IC50 (?g...

Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum

OpenAIRE

Xin, Shan; Tao, Chengcheng; Li, Hongbin

2016-01-01

Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibite...

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

Energy Technology Data Exchange (ETDEWEB)

Rodriguez-Medina, Caren; Boissinot, Sylvaine [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Chapuis, Sophie [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Revers, Frédéric [INRA, Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, 33882 Villenave d’Ornon (France); Ziegler-Graff, Véronique [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Brault, Véronique, E-mail: veronique.brault@colmar.inra.fr [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France)

2015-12-15

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT{sub Cter}) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT{sub Cter}. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT{sub Cter} in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

Directory of Open Access Journals (Sweden)

Zainul A Khan

Full Text Available Cotton leaf curl Burewala virus (CLCuBuV, belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV were fused with β-glucuronidase (GUS and green fluorescent protein (GFP reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

Molecular characterization of natural hybrids of Phytophthora nicotianae and P. cactorum

NARCIS (Netherlands)

Bonants, P.J.M.; Hagenaar-de Weerdt, M.; Man in 't Veld, W.A.; Baayen, R.P.

2000-01-01

Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by

Changes in Air CO2 Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves

Directory of Open Access Journals (Sweden)

Francesca Secchi

2016-04-01

Full Text Available The aquaporin specific control on water versus carbon pathways in leaves is pivotal in controlling gas exchange and leaf hydraulics. We investigated whether Nicotiana tabacum aquaporin 1 (NtAQP1 and Nicotiana tabacum plasma membrane intrinsic protein 2;1 (NtPIP2;1 gene expression varies in tobacco leaves subjected to treatments with different CO2 concentrations (ranging from 0 to 800 ppm, inducing changes in photosynthesis, stomatal regulation and water evaporation from the leaf. Changes in air CO2 concentration ([CO2] affected net photosynthesis (Pn and leaf substomatal [CO2] (Ci. Pn was slightly negative at 0 ppm air CO2; it was one-third that of ambient controls at 200 ppm, and not different from controls at 800 ppm. Leaves fed with 800 ppm [CO2] showed one-third reduced stomatal conductance (gs and transpiration (E, and their gs was in turn slightly lower than in 200 ppm– and in 0 ppm–treated leaves. The 800 ppm air [CO2] strongly impaired both NtAQP1 and NtPIP2;1 gene expression, whereas 0 ppm air [CO2], a concentration below any in vivo possible conditions and specifically chosen to maximize the gene expression alteration, increased only the NtAQP1 transcript level. We propose that NtAQP1 expression, an aquaporin devoted to CO2 transport, positively responds to CO2 scarcity in the air in the whole range 0–800 ppm. On the contrary, expression of NtPIP2;1, an aquaporin not devoted to CO2 transport, is related to water balance in the leaf, and changes in parallel with gs. These observations fit in a model where upregulation of leaf aquaporins is activated at low Ci, while downregulation occurs when high Ci saturates photosynthesis and causes stomatal closure.

An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants

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Cannon, Gordon C.; Van, K. Tran Thanh; Heinhorst, Sabine; Trinh, T. H.; Weissbach, Arthur

1989-01-01

DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome. Images Figure 1 Figure 2 PMID:16666781

Susceptibility of peach GF 305 seedlings and selected herbaceous plants to plum pox virus isolates from western Slovakia.

Science.gov (United States)

Glasa, M; Matisová, J; Hricovský, I; Kúdela, O

1997-12-01

The susceptibility of peach GF 305 seedlings and herbaceous plants to five plum pox virus (PPV) isolates from orchards of western Slovakia was investigated. PPV was isolated from diseased plum, apricot and peach trees, and transmitted by chip-budding to peach GF 305. The herbaceous plants were infected by mechanical inoculation. The transmission was analysed by symptomatology and double sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Infected peaches developed leaf distortion, tissue clearing along the veins and small chlorotic spots (isolate BOR-3). With exception of BOR-3, the PPV isolates transmitted from peach caused local chlorotic spots on Chenopodium foetidum. The character of symptoms changed when a sap from PPV-infected Nicotiana benthamiana was used as virus inoculum. From N. benthamiana, the PPV isolates could be transmitted to Pisum sativum, cv. Colmo (light green mosaic), N. clevelandii and N. clevelandii x N. glutinosa hybrid (latent infection or chlorotic spots).

Imaging of elements in leaves of tobacco by solid sampling–electrothermal vaporization–inductively coupled plasma-optical emission spectrometry

Energy Technology Data Exchange (ETDEWEB)

Masson, Pierre, E-mail: masson@bordeaux.inra.fr

2014-12-01

Plants take up and store elements according to the environment in which they are growing. Because plants are at the base of the food chain, the determination of essential elements or toxic elements in plant materials is of importance. However, it is assumed that the element content determined on selected tissues may provide more specific information than that derived from the whole plant analysis. In this work, we assessed the feasibility of solid sampling–electrothermal vaporization–inductively coupled plasma-optical emission spectrometry analyses for quantitative imaging of Cd and Mg in plant leaves. Leaves of tobacco (Nicotiana tabacum) were selected to be used as samples. To produce a two dimensional image, sections cut from leaf samples were analyzed. Cellulose doped with multi-element solution standards was used as calibration samples. Two certified reference materials (NIST SRM 1547 Peach Leaves and NIST SRM 1573a Tomato leaves) were used to verify the accuracy of measurements with good agreement between the measured concentrations and the certified values. Quantitative imaging revealed the inhomogeneous distribution of the selected elements. Excess of Cd and Mg tended to be focused on peripheral regions and the tip of the leaf.

Characterization of a new potyvirus causing mosaic and flower variegation in Catharanthus roseus in Brazil

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Sheila Conceição Maciel

2011-12-01

Full Text Available Catharanthus roseus is a perennial, evergreen herb in the family Apocynaceae, which is used as ornamental and for popular medicine to treat a wide assortment of human diseases. This paper describes a new potyvirus found causing mosaic symptom, foliar malformation and flower variegation in C. roseus. Of 28 test-plants inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae. When the nucleotide sequence of the CP gene (768nt was compared with other members of the Potyviridae family, the highest identities varied from 67 to 76 %. For the 3' UTR (286nt, identities varied from 16.8 to 28.6 %. The name Catharanthus mosaic virus (CatMV is proposed for this new potyvirus.

Effect of lethal and sub-lethal concentrations of tobacco (Nicotiana ...

African Journals Online (AJOL)

Lethal and sub-lethal bioassays on Clarias gariepinus were conducted to evaluate the toxicity of tobacco (Nicotiana tobaccum) leaf dust on weight gain and haematological indices of Clarias gariepinus (mean weight 10.5±0.70g) in glass aquaria with aeration system. The concentrations used during the lethal exposure are: ...

Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport.

Science.gov (United States)

Godon, C; Krapp, A; Leydecker, M T; Daniel-Vedele, F; Caboche, M

1996-02-25

This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.

The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana.

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Hancock, C Nathan; Kent, Lia; McClure, Bruce A

2005-09-01

S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia x SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.

In Vitro Assay of Ethanolic Heat Reflux Extract of Nicotiana tabacum L. var Virginia Against Nosocomial Bacteria Pathogen

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Pramono, Andri; Fauzantoro, Ahmad; Rizki Hidayati, Irma; Hygea, Arina; Puspita, Oktaviani Sandra; Muktamiroh, Hikmah; Simanjuntak, Kristina; Gozan, Misri

2018-03-01

Tobacco plays an important role in international trade as one of the export commodities. Indonesia is one of the good quality export contributors of tobacco leaves in the world. Nevertheless, tobacco is used only as a raw material for the cigarette industries, and the rise on anti-cigarette regulations prompted the exploration of alternative product from tobacco plants. The content of alkaloids, flavonoids, terpenoids and steroids in tobacco leaves were reported in literatures as antibacterial. Therefore, this study proposed in vitro assay of the ethanolic heat reflux extract (EHRE) of Nicotiana tabacum var. Virginia against nosocomial bacteria pathogen ((Pseudomonas aeruginosa (ATCC 27853), Eschericia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212)). Kirby-bauer diffusion method was used for this assay. The concentration of the EHRE for Kirby-bauer assay were 20; 40; 60; 80; and 100%. The presence of clear zones on Kirby-bauer test, against the growth of each nosocomial bacteria pathogen show that tobacco extract has antibacterial effect. Statistical analysis result showed that each extract concentration had significant difference value (p steroids) of tobacco leaf extracts (N. tabacum) has potential as antibacterial against nosocomial bacteria pathogen. Nevertheless, optimization of tobacco leaf extract to obtain maximum active ingredient still needs to be done. This study is important for further development of the tobacco leaf extract as antibacterial

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif.

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Ala-Poikela, Marjo; Goytia, Elisa; Haikonen, Tuuli; Rajamäki, Minna-Liisa; Valkonen, Jari P T

2011-07-01

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.

A Rice Gene Homologous to Arabidopsis AGD2-LIKE DEFENSE1 Participates in Disease Resistance Response against Infection with Magnaporthe oryzae

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Ga Young Jung

2016-08-01

Full Text Available ALD1 (ABERRANT GROWTH AND DEATH2 [AGD2]-LIKE DEFENSE1 is one of the key defense regulators in Arabidopsis thaliana and Nicotiana benthamiana. In these model plants, ALD1 is responsible for triggering basal defense response and systemic resistance against bacterial infection. As well ALD1 is involved in the production of pipecolic acid and an unidentified compound(s for systemic resistance and priming syndrome, respectively. These previous studies proposed that ALD1 is a potential candidate for developing genetically modified (GM plants that may be resistant to pathogen infection. Here we introduce a role of ALD1-LIKE gene of Oryza sativa, named as OsALD1, during plant immunity. OsALD1 mRNA was strongly transcribed in the infected leaves of rice plants by Magnaporthe oryzae, the rice blast fungus. OsALD1 proteins predominantly localized at the chloroplast in the plant cells. GM rice plants over-expressing OsALD1 were resistant to the fungal infection. The stable expression of OsALD1 also triggered strong mRNA expression of PATHOGENESIS-RELATED PROTEIN1 genes in the leaves of rice plants during infection. Taken together, we conclude that OsALD1 plays a role in disease resistance response of rice against the infection with rice blast fungus.

Antinuclear human autoantibodies as markers in Nicotiana tabacum pollen tubes

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C. Poggialini

2014-01-01

Full Text Available In the present paper we report on the use of antinuclear human autoantibodies as specific markers in Nicotiana tabacum pollen tubes. The antibodies have been tested by fluorescence techniques using a confocal laser scanning microscope. All the antibodies showed specifc labelling pattern and the results, although preliminary in nature, could open new perspectives of research.

Modulation of sulfur metabolism enables efficient glucosinolate engineering

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Geu-Flores Fernando

2011-01-01

Full Text Available Abstract Background Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1, resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization. Results To optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2 alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold. Conclusion Adjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in

Nectar Sugar Modulation and Cell Wall Invertases in the Nectaries of Day- and Night- Flowering Nicotiana.

Science.gov (United States)

Tiedge, Kira; Lohaus, Gertrud

2018-01-01

Nectar composition varies between species, depending on flowering time and pollinator type, among others. Various models of the biochemical and molecular mechanisms underlying nectar production and secretion have been proposed. To gain insights into these mechanisms, day- and night-flowering tobacco ( Nicotiana ) species with high or low proportions of hexoses in the nectar were analyzed. Nectar and nectaries were simultaneously collected, throughout the day and night. Soluble sugars and starch were determined and the activity and expression level of cell wall invertase (CW-INVs) were measured in nectaries. Nectaries and nectar of the five Nicotiana species contained different amounts of sucrose, glucose, and fructose. CW-INV activity was detected in the nectaries of all Nicotiana species and is probably involved in the hydrolysis of sucrose in the nectary tissue and during nectar secretion. The larger differences in the sucrose-to-hexose-ratio between nectaries and nectar in diurnal species compared to nocturnal species can be explained by higher sucrose cleavage within the nectaries in night-flowering species, and during secretion in day-flowering species. However, cell wall invertase alone cannot be responsible for the differences in sugar concentrations. Within the nectaries of the Nicotiana species, a portion of the sugars is transiently stored as starch. In general, night-flowering species showed higher starch contents in the nectaries compared to day-flowering species. Moreover, in night flowering species, the starch content decreased during the first half of the dark period, when nectar production peaks. The sucrose concentrations in the cytoplasm of nectarial cells were extrapolated from nectary sucrose contents. In day-flowering species, the sucrose concentration in the nectary cytoplasm was about twice as high as in nectar, whereas in night-flowering species the situation was the opposite, which implies different secretion mechanisms. The secreted nectar

Co-expression of peppermint geranyl diphosphate synthase small subunit enhances monoterpene production in transgenic tobacco plants.

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Yin, Jun-Lin; Wong, Woon-Seng; Jang, In-Cheol; Chua, Nam-Hai

2017-02-01

Monoterpenes are important for plant survival and useful to humans. In addition to their function in plant defense, monoterpenes are also used as flavors, fragrances and medicines. Several metabolic engineering strategies have been explored to produce monoterpene in tobacco but only trace amounts of monoterpenes have been detected. We investigated the effects of Solanum lycopersicum 1-deoxy-d-xylulose-5-phosphate synthase (SlDXS), Arabidopsis thaliana geranyl diphosphate synthase 1 (AtGPS) and Mentha × piperita geranyl diphosphate synthase small subunit (MpGPS.SSU) on production of monoterpene and geranylgeranyl diphosphate (GGPP) diversities, and plant morphology by transient expression in Nicotiana benthamiana and overexpression in transgenic Nicotiana tabacum. We showed that MpGPS.SSU could enhance the production of various monoterpenes such as (-)-limonene, (-)-linalool, (-)-α-pinene/β-pinene or myrcene, in transgenic tobacco by elevating geranyl diphosphate synthase (GPS) activity. In addition, overexpression of MpGPS.SSU in tobacco caused early flowering phenotype and increased shoot branching by elevating contents of GA 3 and cytokinins due to upregulated transcript levels of several plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway genes, geranylgeranyl diphosphate synthases 3 (GGPPS3) and GGPPS4. Our method would allow the identification of new monoterpene synthase genes using transient expression in N. benthamiana and the improvement of monoterpene production in transgenic tobacco plants. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells

DEFF Research Database (Denmark)

Reuter, Lauri J.; Shahbazi, Mohammad-Ali; Makila, Ermei M.

2017-01-01

can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration...... to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of concept for the functionalization...

Molecular and biological characterization of Potato mop-top virus (PMTV, Pomovirus) isolates from potato-growing regions in Colombia

DEFF Research Database (Denmark)

Gil, José; Adams, Ian; Boonham, Neil

2016-01-01

Potato mop-top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil...... samples were taken from the main potato-producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons...

Localization and in-vivo characterization of thapsia garganica CYP76AE2 indicates a role in thapsigargin biosynthesis

DEFF Research Database (Denmark)

Andersen, Trine Bundgaard; Martinez-Swatson, Karen Agatha; Rasmussen, Silas Anselm

2017-01-01

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+ -ATPase calcium pump in mammals and is of industrial importance as the active...... in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts...

Cowpea mosaic virus RNA-1 acts as an amplicon whose effects can be counteracted by a RNA-2-encoded suppressor of silencing

International Nuclear Information System (INIS)

Liu Li; Grainger, Jef; Canizares, M. Carmen; Angell, Susan M.; Lomonossoff, George P.

2004-01-01

Lines of Nicotiana benthamiana transgenic for full-length copies of both Cowpea mosaic virus (CPMV) genomic RNAs, either singly or together, have been produced. Plants transgenic for both RNAs developed symptoms characteristic of a CPMV infection. When plants transgenic for RNA-1 were agro-inoculated with RNA-2, no infection developed and the plants were also resistant to challenge with CPMV. By contrast, plants transgenic for RNA-2 became infected when agro-inoculated with RNA-1 and were fully susceptible to CPMV infection. The resistance of RNA-1 transgenic plants was shown to be related to the ability of RNA-1 to self-replicate and act as an amplicon. The ability of transgenically expressed RNA-2 to counteract the amplicon effect suggested that it encodes a suppressor of posttranscriptional gene silencing (PTGS). By examining the ability of portions of RNA-2 to reverse PTGS in N. benthamiana, we have identified the small (S) coat protein as the CPMV RNA-2-encoded suppressor of PTGS

An antiviral RISC isolated from Tobacco rattle virus-infected plants.

Science.gov (United States)

Ciomperlik, Jessica J; Omarov, Rustem T; Scholthof, Herman B

2011-03-30

The RNAi model predicts that during antiviral defense a RNA-induced silencing complex (RISC) is programmed with viral short-interfering RNAs (siRNAs) to target the cognate viral RNA for degradation. We show that infection of Nicotiana benthamiana with Tobacco rattle virus (TRV) activates an antiviral nuclease that specifically cleaves TRV RNA in vitro. In agreement with known RISC properties, the nuclease activity was inhibited by NaCl and EDTA and stimulated by divalent metal cations; a novel property was its preferential targeting of elongated RNA molecules. Intriguingly, the specificity of the TRV RISC could be reprogrammed by exogenous addition of RNA (containing siRNAs) from plants infected with an unrelated virus, resulting in a newly acquired ability of RISC to target this heterologous genome in vitro. Evidently the virus-specific nuclease complex from N. benthamiana represents a genuine RISC that functions as a readily employable and reprogrammable antiviral defense unit. Copyright © 2011 Elsevier Inc. All rights reserved.

Population structure and genetic diversity of Phytophthora nicotianae from tobacco in Georgia

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Black shank caused by Phytophthora nicotianae occurs worldwide and is responsible for significant yield loss in tobacco production in Georgia. Management of the disease has primarily relied on utilization of tobacco cultivars with resistance to race 0 of the pathogen and application of the fungicide...

Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

Science.gov (United States)

Lowery, D Thomas; Vickers, Patricia M; Bittner, Lori A; Stobbs, Lorne W; Foottit, Robert G

2015-10-01

Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs. © Her Majesty in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. Published by Oxford University Press on behalf of Entomological Society of America.

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

International Nuclear Information System (INIS)

Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

2015-01-01

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT_C_t_e_r) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT_C_t_e_r. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT_C_t_e_r in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

Map-based cloning and expression analysis of BMR-6 in sorghum.

Science.gov (United States)

Li, Jieqin; Wang, Lihua; Zhang, Qiuwen; Liu, Yanlong

2015-09-01

Brown midrib mutants in sorghum are associated with reduced lignin content and increased cell wall digestibility. In this study, we characterized a bmr-6 sorghum mutant, which shows reddish pigment in the midrib and stem after the fifth-leaf stage. Compared to wild type, Kalson lignin content of bmr-6 is decreased significantly. We used histological analysis to determine that the mutant exhibited a modified pattern of lignin staining and found an increased polysaccharide content. We cloned BMR-6 gene, a gene encoded a cinnamyl alcohol dehydrogenase (CAD), using a map-based cloning approach. Genetic complementation confirmed that CAD is responsible for the BMR-6 phenotype. BMR-6 gene was expressed in all tested sorghum tissues, with the highest being in midrib and stem. Transient expression assays in Nicotiana benthamiana leaves demonstrated cytomplasmic localization of BMR-6. We found that the expression level of bmr-6 was significantly decreased in the mutant but expression of SbCAD3 and SbCAD5 were significantly increased. Our results indicate that BMR-6 not only affects the distribution of lignin but also the biosynthesis of lignin in sorghum.

Identification and Expression Profiling of the Auxin Response Factors in Dendrobium officinale under Abiotic Stresses.

Science.gov (United States)

Chen, Zhehao; Yuan, Ye; Fu, Di; Shen, Chenjia; Yang, Yanjun

2017-05-04

Auxin response factor (ARF) proteins play roles in plant responses to diverse environmental stresses by binding specifically to the auxin response element in the promoters of target genes. Using our latest public Dendrobium transcriptomes, a comprehensive characterization and analysis of 14 DnARF genes were performed. Three selected DnARFs , including DnARF1 , DnARF4 , and DnARF6 , were confirmed to be nuclear proteins according to their transient expression in epidermal cells of Nicotiana benthamiana leaves. Furthermore, the transcription activation abilities of DnARF1 , DnARF4 , and DnARF6 were tested in a yeast system. Our data showed that DnARF6 is a transcriptional activator in Dendrobium officinale . To uncover the basic information of DnARF gene responses to abiotic stresses, we analyzed their expression patterns under various hormones and abiotic treatments. Based on our data, several hormones and significant stress responsive DnARF genes have been identified. Since auxin and ARF genes have been identified in many plant species, our data is imperative to reveal the function of ARF mediated auxin signaling in the adaptation to the challenging Dendrobium environment.

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

Science.gov (United States)

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

Ontwikkeling en demonstratie van een geintegreerd bestrijdingssysteem voor de rode luis Myzus nicotianae

NARCIS (Netherlands)

Dijken, van M.J.

1998-01-01

De geïntegreerde plaagbestrijding van de paprikateelt onder glas, wordt sinds 1993 verstoord door de opkomst van de rode luis, Myzus nicotianae. Deze luis is namelijk resistent tegen het selectieve chemische correctiemiddel pirimicarb en een effectieve biologische bestrijding was onvoldoende

Inter Simple Sequence Repeat DNA (ISSR) Polymorphism Utility in Haploid Nicotiana Alata Irradiated Plants for Finding Markers Associated with Gamma Irradiation and Salinity

International Nuclear Information System (INIS)

El-Fiki, A.; Adly, M.; El-Metabteb, G.

2017-01-01

Nicotiana alata is an ornamental plant. It is a member of family Solanasea. Tobacco (Nicotiana spp.) is one of the most important commercial crops in the world. Wild Nicotiana species, as a store house of genes for several diseases and pests, in addition to genes for several important phytochemicals and quality traits which are not present in cultivated varieties. Inter simple sequence repeat DNA (ISSR) analysis was used to determine the degree of genetic variation in treated haploid Nicotiana alata plants. Total genomic DNAs from different treated haploid plant lets were amplified using five specific primers. All primers were polymorphic. A total of 209 bands were amplified of which 135 (59.47%) polymorphic across the radiation treatments. Whilst, the level of polymorphism among the salinity treatments were 181 (85.6 %). Whereas, the polymorphism among the combined effects between gamma radiation doses and salinity concentrations were 283 ( 73.95% ). Treatments relationships were estimated through cluster analysis (UPGMA) based on ISSR data

The influence of light quality on the accumulation of flavonoids in tobacco (Nicotiana tabacum L.) leaves.

Science.gov (United States)

Fu, Bo; Ji, Xiaoming; Zhao, Mingqin; He, Fan; Wang, Xiaoli; Wang, Yiding; Liu, Pengfei; Niu, Lu

2016-09-01

Flavonoids are important secondary metabolites in plants regulated by the environment. To analyze the effect of light quality on the accumulation of flavonoids, we performed a rapid analysis of flavonoids in extracts of tobacco leaves using UHPLC-QTOF. A total of 12 flavonoids were detected and identified in tobacco leaves, which were classified into flavonoid methyl derivatives and flavonoid glycoside derivatives according to the groups linked to the flavonoid core. Correlation analysis was further conducted to investigate the effect of different wavelengths of light on their accumulation. The content of flavonoid methyl derivatives was positively correlated with the proportions of far-red light (FR; 716-810nm) and near-infrared light (NIR; 810-2200nm) in the sunlight spectrum and negatively correlated with the proportion of ultraviolet (UV-A; 350-400nm) and the red/far-red ratio (R/FR). By contrast, the content of flavonoid glycoside derivatives was positively correlated with the proportion of UV-A and the R/FR, and negatively correlated with FR and NIR. The results indicated that light quality with higher proportions of FR and NIR increases the activity of flavonoid methyltransferases but suppresses the activity of flavonoid glycoside transferases. While a high proportion of UV-A and a high R/FR can increase flavonoid glycoside transferase activity but suppress flavonoid methyltransferase activity. Copyright © 2016 Elsevier B.V. All rights reserved.

The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

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VERONIKA DOUBNEROVÁ

2009-08-01

Full Text Available The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME, which catalyzes the oxidative decarboxylation of L-malate and NADP+ to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (a-ketoglutarate, succinate, fumarate, metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate, compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA and some amino acids (glutamate, glutamine, aspartate did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn2+ or Mg2+, required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP+ and mixed type to L-malate were found.

An ANNEXIN-like protein from the cereal cyst nematode Heterodera avenae suppresses plant defense.

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Changlong Chen

Full Text Available Parasitism genes encoding secreted effector proteins of plant-parasitic nematodes play important roles in facilitating parasitism. An annexin-like gene was isolated from the cereal cyst nematode Heterodera avenae (termed Ha-annexin and had high similarity to annexin 2, which encodes a secreted protein of Globodera pallida. Ha-annexin encodes a predicted 326 amino acid protein containing four conserved annexin domains. Southern blotting revealed that there are at least two homologies in the H. avenae genome. Ha-annexin transcripts were expressed within the subventral gland cells of the pre-parasitic second-stage juveniles by in situ hybridization. Additionally, expression of these transcripts were relatively higher in the parasitic second-stage juveniles by quantitative real-time RT-PCR analysis, coinciding with the time when feeding cell formation is initiated. Knockdown of Ha-annexin by method of barley stripe mosaic virus-based host-induced gene silencing (BSMV-HIGS caused impaired nematode infections at 7 dpi and reduced females at 40 dpi, indicating important roles of the gene in parasitism at least in early stage in vivo. Transiently expression of Ha-ANNEXIN in onion epidermal cells and Nicotiana benthamiana leaf cells showed the whole cell-localization. Using transient expression assays in N. benthamiana, we found that Ha-ANNEXIN could suppress programmed cell death triggered by the pro-apoptotic mouse protein BAX and the induction of marker genes of PAMP-triggered immunity (PTI in N. benthamiana. In addition, Ha-ANNEXIN targeted a point in the mitogen-activated protein kinase (MAPK signaling pathway downstream of two kinases MKK1 and NPK1 in N. benthamiana.

Zoospore exudates from Phytophthora nicotianae affect immune responses in Arabidopsis.

Science.gov (United States)

Kong, Ping; McDowell, John M; Hong, Chuanxue

2017-01-01

Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by Phytophthora. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF) and zoospores of P. nicotianae were investigated comparatively for effects on resistance of Arabidopsis thaliana Col-0 and mutants that affect signaling mediated by salicylic acid (SA) and jasmonic acid (JA): eds16 (enhanced disease susceptibility16), pad4 (phytoalexin deficient4), and npr1 (nonexpressor of pathogenesis-related genes1). Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the PR1 and PDF1.2 marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating PR1 when SA signaling or synthesis is deficient, which may be responsible for Arabidopsis mutant plants more susceptible to infection by high concentration of P. nicotianae zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by Phytophthora.

Anaerobic soil disinfestation reduces survival and infectivity of Phytophthora nicotianae chlamydospores in pepper

Science.gov (United States)

Phytophthora nicotianae is the principal causal agent of root and crown rot disease of pepper plants in Extremadura (western Spain), a spring-summer crop in this region. Preplant soil treatment by anaerobic soil disinfestation (ASD) may effectively control plant pathogens in many crop production sys...

Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata

NARCIS (Netherlands)

Glawe, G.A.; Zavala, J.A.; Kessler, A.; Van Dam, N.M.; Baldwin, I.T.

2003-01-01

Genotypes of the wild tobacco Nicotiana attenuata from different geographic regions in North America vary considerably in the level of constitutive and inducible trypsin protease inhibitors (TrypPIs), a potent direct defense, as well as in the production of herbivore-induced volatiles that function

Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension.

Science.gov (United States)

Gillet; Mesnard; Fliniaux; Monti; Fliniaux

1999-11-01

A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension. This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography. Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA). The influence of culture conditions on the accumulation of this metabolite by N. plumbaginifolia cell suspensions has been studied. Darkness strongly inhibits the CA accumulation. Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content. This one was not influenced by a supplementation with quinic acid.

Overexpression of Nictaba-Like Lectin Genes from Glycine max Confers Tolerance towards Pseudomonas syringae Infection, Aphid Infestation and Salt Stress in Transgenic Arabidopsis Plants

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Sofie Van Holle

2016-10-01

Full Text Available Plants have evolved a sophisticated immune system that allows them to recognize invading pathogens by specialized receptors. Carbohydrate-binding proteins or lectins are part of this immune system and especially the lectins that reside in the nucleocytoplasmic compartment are known to be implicated in biotic and abiotic stress responses. The class of Nictaba-like lectins (NLL groups all proteins with homology to the tobacco (Nicotiana tabacum lectin, known as a stress-inducible lectin. Here we focus on two Nictaba homologs from soybean (Glycine max, referred to as GmNLL1 and GmNLL2. Confocal laser scanning microscopy of fusion constructs with the green fluorescent protein either transiently expressed in Nicotiana benthamiana leaves or stably transformed in tobacco BY-2 suspension cells revealed a nucleocytoplasmic localization for the GmNLLs under study. RT-qPCR analysis of the transcript levels for the Nictaba-like lectins in soybean demonstrated that the genes are expressed in several tissues throughout the development of the plant. Furthermore, it was shown that salt treatment, Phytophthora sojae infection and Aphis glycines infestation trigger the expression of particular NLL genes. Stress experiments with Arabidopsis lines overexpressing the NLLs from soybean yielded an enhanced tolerance of the plant towards bacterial infection (Pseudomonas syringae, insect infestation (Myzus persicae and salinity. Our data showed a better performance of the transgenic lines compared to wild type plants, indicating that the NLLs from soybean are implicated in the stress response. These data can help to further elucidate the physiological importance of the Nictaba-like lectins from soybean, which can ultimately lead to the design of crop plants with a better tolerance to changing environmental conditions.

Transient glyco-engineering to produce recombinant IgA1 with defined N- and O-glycans in plants

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Martina eDicker

2016-01-01

Full Text Available The production of therapeutic antibodies to combat pathogens and treat diseases such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG, less effort has been undertaken to express immunoglobulin A (IgA, which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumour activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered deltaXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that deltaXT/FT Nicotiana benthamiana plants can be engineered towards the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

Evaluation of the minimal replication time of Cauliflower mosaic virus in different hosts

International Nuclear Information System (INIS)

Khelifa, Mounia; Masse, Delphine; Blanc, Stephane; Drucker, Martin

2010-01-01

Though the duration of a single round of replication is an important biological parameter, it has been determined for only few viruses. Here, this parameter was determined for Cauliflower mosaic virus (CaMV) in transfected protoplasts from different hosts: the highly susceptible Arabidopsis and turnip, and Nicotiana benthamiana, where CaMV accumulates only slowly. Four methods of differing sensitivity were employed: labelling of (1) progeny DNA and (2) capsid protein, (3) immunocapture PCR,, and (4) progeny-specific PCR. The first progeny virus was detected about 21 h after transfection. This value was confirmed by all methods, indicating that our estimate was not biased by the sensitivity of the detection method, and approximated the actual time required for one round of CaMV replication. Unexpectedly, the replication kinetics were similar in the three hosts; suggesting that slow accumulation of CaMV in Nicotiana plants is determined by non-optimal interactions in other steps of the infection cycle.

Molecular Characterization of Tomato Yellow Leaf Curl Virus in Korea and the Construction of an Infectious Clone

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Bong Choon Lee

2015-06-01

Full Text Available Several tomato production regions in Korea were surveyed for tomato yellow leaf curl disease (TYLCD. Tomato leaf samples showing TYLCD-like symptoms were collected from Tongyeong (To, Geoje (Gi, and Gimhae (Gh cities of the southern part of Korea. Tomato yellow leaf curl virus (TYLCV was detected and the full-length genomes of the isolates were sequenced. The TYLCV isolates found in Korea shared high sequence identity (> 99% with TYLCV-IL [JR:Omu:Ng] (AB110217. Phylogenetic relationship analysis revealed that they formed two groups (with little genetic variability, and the To, Gj, and Gh isolates belonged to the TYLCV-IL group. An infectious clone of TYLCV-To (JQ013089 was constructed and agroinoculated into Nicotiana benthamiana, Nicotiana tabacum var. Xanthi, Petunia hybrida, Capsicum annuum, and Lycopersicon esculentum cv. Hausumomotaro. Agroinfection with a dimeric infectious clone of TYLCV-To induced severe leaf curling and stunting symptoms in these plants, excluding C. annuum. Tomato plants then developed typical yellow leaf curl symptoms.

Hazardous impact of toxic metals on tobacco leaves grown in contaminated soil by ultrasonic assisted pseudo-digestion: Multivariate study

International Nuclear Information System (INIS)

Arain, Mohammad Balal; Kazi, Tasneem Gul; Jamali, Mohammad Khan; Jalbani, Nusrat; Afridi, Hassan Imran; Kandhro, Ghulam Abbas; Ansari, Rehana; Sarfraz, Raja Adil

2008-01-01

Tobacco leaves (Nicotiana tabacum L.), agricultural soil and pollute irrigated lake water samples were collected during 2005-2006 and analyzed for Cd and Ni by electrothermal atomic absorption spectrometry (ETAAS). A simple and efficient procedure was investigated for the complete decomposition of tobacco leaves using ultrasonic assisted acid pseudo-digestion method (UPDM). A Plackett-Burman experimental design was used as a multivariate strategy for the evaluation of seven factors/variables at once, while central composite were used to found optimum values of significant variables. The accuracy of the proposed methods was assessed by analyzing certified reference (CRM); Virginia tobacco leaves (CTA-VTL-2). The results being compared with those obtained by conventional wet acid digestion method. The result obtained by optimized method showed good agreement with the certified values and sufficiently high recovery 97.8 and 98.7% for Cd and Ni, respectively. Under optimal conditions, the detection limits (3σ) were evaluated to be 0.019 μg g -1 for Cd and 0.37 μg g -1 for Ni. The proposed method was successfully applied to the determination of Cd and Ni in raw, processed tobacco and different branded cigarettes samples

Hazardous impact of toxic metals on tobacco leaves grown in contaminated soil by ultrasonic assisted pseudo-digestion: Multivariate study

Energy Technology Data Exchange (ETDEWEB)

Arain, Mohammad Balal [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: bilal_ku2004@yahoo.com; Kazi, Tasneem Gul [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: tgkazi@yahoo.com; Jamali, Mohammad Khan [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: mkhanjamali@yahoo.com; Jalbani, Nusrat [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: nussaratjalbani_21@yahoo.com; Afridi, Hassan Imran [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: hassanimranafridi@yahoo.com; Kandhro, Ghulam Abbas [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: gakandhro@yahoo.com; Ansari, Rehana [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: rehana_ansari_57@yahoo.com; Sarfraz, Raja Adil [Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)], E-mail: rajaadilsarfraz@gmail.com

2008-06-30

Tobacco leaves (Nicotiana tabacum L.), agricultural soil and pollute irrigated lake water samples were collected during 2005-2006 and analyzed for Cd and Ni by electrothermal atomic absorption spectrometry (ETAAS). A simple and efficient procedure was investigated for the complete decomposition of tobacco leaves using ultrasonic assisted acid pseudo-digestion method (UPDM). A Plackett-Burman experimental design was used as a multivariate strategy for the evaluation of seven factors/variables at once, while central composite were used to found optimum values of significant variables. The accuracy of the proposed methods was assessed by analyzing certified reference (CRM); Virginia tobacco leaves (CTA-VTL-2). The results being compared with those obtained by conventional wet acid digestion method. The result obtained by optimized method showed good agreement with the certified values and sufficiently high recovery 97.8 and 98.7% for Cd and Ni, respectively. Under optimal conditions, the detection limits (3{sigma}) were evaluated to be 0.019 {mu}g g{sup -1} for Cd and 0.37 {mu}g g{sup -1} for Ni. The proposed method was successfully applied to the determination of Cd and Ni in raw, processed tobacco and different branded cigarettes samples.

Management of chili pepper root rot and wilt (caused by Phytophthora nicotianae by grafting onto resistant rootstock

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Mourad SAADOUN

2013-05-01

Full Text Available Root rot and plant wilting caused by Phytophthora nicotianae is a severe disease of chili pepper (Capsicum annuum L. in open fields and under greenhouse production in Tunisia. Chili pepper grafting for disease manage- ment is attracting increased interest in recent years. Using the tube grafting technique, different compatible scion/rootstock combinations were obtained with the wild-type pepper SCM334 and the local chili pepper cultivars ‘Beldi’ and ‘Baker’. SCM334 was resistant to P. nicotianae, while the cultivars Beldi and Baker were susceptible. Plant inoculations were performed with P. nicotianae zoospores, and severity of root rot was rated 30 days post- inoculation using a 0 (healthy plant to 5 (dead plant severity score. On SCM334 rootstock and with ‘Beldi’ or ‘Baker’ scions, the intensity of root rot was very low (mean score 0.1–0.2 and plants were healthy. However, with Baker or Beldi rootstocks and SCM334 scions, root rot was severe (mean score 3.1–4.6, leading to high numbers of wilting and dead plants. This severe root rot was similar to that observed on non-grafted plants of ‘Baker’ and ‘Beldi’ inoculated with the pathogen. Under greenhouse conditions, measurements of agronomic characters indicated non-consistent improvement of these features on the scion cultivar when SCM334 was the rootstock. Since plant foliage is not attacked by this pathogen, these results show that susceptible chili pepper scions grafted onto SCM334 rootstocks could be used for root rot management and improvement of pepper yields in P. nicotianae infested soils.

Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization

International Nuclear Information System (INIS)

Parokonny, A.S.; Kenton, A.Y.; Gleba, Y.Y.; Bennett, M.D.

1992-01-01

In situ hybridization was used to examine genome reorganization in asymmetric somatic hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by fusion of gamma-irradiated protoplasts from one of the parents (donor) with non-irradiated protoplasts from the other (recipient). Probing with biotinylated total genomic DNA from either the donor or the recipient species unequivocally identified genetic material from both parents in 31 regenerant plants, each originating from a different nuclear hybrid colony. This method, termed genomic in situ hybridization (GISH), allowed intergenomic translocations containing chromosome segments from both species to be recognized in four regenerants. A probe homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat (5'-TTTAGGG-3')n, identified telomeres on all chromosomes, including 'mini-chromosomes' originating from the irradiated donor genome. Genomic in situ hybridization to plant chromosomes provides a rapid and reliable means of screening for recombinant genotypes in asymmetric somatic hybrids. Used in combination with other DNA probes, it also contributes to a greater understanding of the events responsible for genomic recovery and restabilization following genetic manipulation in vitro

Molecular cloning of a catalase cDNA from Nicotiana glutinosa L. and its repression by tobacco mosaic virus infection.

Science.gov (United States)

Yi, S Y; Yu, S H; Choi, D

1999-06-30

Recent reports revealed that catalase has a role in the plant defense mechanism against a broad range of pathogens through being inhibited by salicylic acid (SA). During an effort to clone disease resistance-responsive genes, a cDNA encoding catalase (Ngcat1; Nicotiana glutinosa cat1) was isolated from a tobacco cDNA library. In N. glutinosa, catalase is encoded by a small gene family. The deduced amino acid sequence of the Ngcat1 cDNA has 98% homology with the cat1 gene of N. plumbaginifolia. The Ngcat1 expression is controlled by the circadian clock, and its mRNA level is the most abundant in leaves. Both the expression of Ngcat1 mRNA and its enzyme activity in the tobacco plant undergoing a hypersensitive response (HR) to TMV infection were repressed. The repression of the mRNA level was also observed following treatment with SA. These results imply that SA may act as an inhibitor of catalase transcription during the HR of tobacco. Cloning and expression of the Ngcat1 in tobacco following pathogen infection and SA treatment are presented.

The role of Nicotiana gluca Graham (paraguayan herbs) as an adjuvant in immunomodulation of Newcastle disease vaccine for broilers Estudo da ação de Nicotiana glauca Graham (erva paraguaia) como coadjuvante em vacina contra a doença de Newcastle em frangos de corte

OpenAIRE

Fabiane Pereira Gentilini; Telmo Vidor; Rogério Freitag; Marcos Antônio Anciuti; Caren Gularte Quincozes; Marlete Brum Cleff; Geferson Fischer; Carlos Eduardo Nogueira

2008-01-01

The Nicotiana glauca is a native plant species from Argentina, but found all over South América, being used against headaches, rheumatism, injuries, ulcers, and so on. Researchers have considered it as having immunomodulation effect. This study was conducted to investigate the use of a aqueous extract of Nicotiana glauca Graham as an immunomodulator (adjuvant) of a Newcastle disease vaccine.. A total of 56 broilers were distributed into 4 ...

High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

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Natalia V. Petukhova

2014-04-01

Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

Functional analysis of the cloverleaf-like structure in the 3' untranslated region of bamboo mosaic potexvirus RNA revealed dual roles in viral RNA replication and long distance movement

International Nuclear Information System (INIS)

Chen, I-H.; Meng Hsiao; Hsu, Y.-H.; Tsai, C.-H.

2003-01-01

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/ΔABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40B and C each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40B and C were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMVA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain

Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

Science.gov (United States)

Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

2015-12-01

Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

Plant viruses as scaffolds for the presentation of vaccine epitopes

Czech Academy of Sciences Publication Activity Database

Plchová, Helena; Čeřovská, Noemi; Vaculík, Petr; Moravec, Tomáš

2017-01-01

Roč. 61, č. 1 (2017), s. 1-12 ISSN 0006-3134 R&D Projects: GA ČR(CZ) GA15-10768S; GA ČR(CZ) GAP501/12/1761 Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : tobacco-mosaic-virus * x coat protein * human-papillomavirus type-16 * green fluorescent protein * n-terminal segment * triple gene block * cell-to-cell * transient expression * nicotiana-benthamiana * viral vector * transient expression * plant viral expression vectors Subject RIV: GE - Plant Breeding OBOR OECD: Virology Impact factor: 1.551, year: 2016

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5

DEFF Research Database (Denmark)

Madsen, Esben B; Antolín-Llovera, Meritxell; Grossmann, Christina

2011-01-01

and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression...... of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through...

The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity

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Zhenwei Zhang

2017-12-01

Full Text Available Cucumber green mottle mosaic virus (CGMMV is one of the most devastating viruses infecting members of the family Cucurbitaceae. The assembly initiation site of CGMMV is located in the coding region of the coat protein, which is not only involved in virion assembly but is also a key factor determining the long-distance movement of the virus. To understand the effect of assembly initiation site and the adjacent region on CGMMV infectivity, we created a GTT deletion mutation in the GAGGTTG assembly initiation site of the infectious clone of CGMMV, which we termed V97 (deletion mutation at residue 97 of coat protein, followed by the construction of the V94A and T104A mutants. We observed that these three mutations caused mosaic after Agrobacterium-mediated transformation in Nicotiana benthamiana, albeit with a significant delay compared to the wild type clone. The mutants also had a common spontaneous E96K mutation in the coat protein. These results indicated that the initial assembly site and the sequence of the adjacent region affected the infectivity of the virus and that E96 might play an essential role in this process. We constructed two single point mutants—E96A and E96K—and three double mutants—V94A-E96K, V97-E96K and T104A-E96K—to further understand the role of E96 in CGMMV pathogenesis. After inoculation in N. benthamiana, E96A showed delayed systemic symptoms, but the E96K and three double mutants exhibited typical symptoms of mosaic at seven days post-infection. Then, sap from CGMMV-infected N. benthamiana leaves was mechanically inoculated on watermelon plants. We confirmed that E96 affected CGMMV infection using double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA, reverse transcription-polymerase chain reaction (RT-PCR, and sequencing, which further confirmed the successful infection of the related mutants, and that E96K can compensate the effect of the V94, V97, and T104 mutations on virus infectivity. In

Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection.

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Pandolfini, Tiziana; Molesini, Barbara; Avesani, Linda; Spena, Angelo; Polverari, Annalisa

2003-06-25

Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in

The Valine Anticodon and Valylatability of Peanut Clump Virus RNAs Are Not Essential but Provide a Modest Competitive Advantage in Plants†

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Matsuda, Daiki; Dunoyer, Patrice; Hemmer, Odile; Fritsch, Christiane; Dreher, Theo W.

2000-01-01

The role of valine aminoacylation of the two genomic RNAs of Peanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GΔC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3′ end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GΔC anticodon that does not support valylation. The presence in RNA1 of GΔC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GΔC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV. PMID:10954573

Characterization of secretory phospholipase A₂ with phospholipase A₁ activity in tobacco, Nicotiana tabacum (L.).

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Fujikawa, Yukichi; Fujikawa, Ritsuko; Iijima, Noriaki; Esaka, Muneharu

2012-03-01

A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

Microspores irradiation in anther culture: testing a new technique to obtain mutations immediatly detected and fixed (Application to Nicotiana tabacum)

International Nuclear Information System (INIS)

Mondeil, Fanja

1974-01-01

In order to consider the effects of microspores irradiation on embryo development, and in order to observe the morphological responses of haploid plantlets derived from androgenetic anthers to ionizing irradiation, 1000, 1500 and 2000r of gamma rays were delivered on anthers of Nicotiana tabacum (DL 50 range calculated: 1500r). The cytological studies of embryo development revealed an apparent increase in irradiated microspores: cell division is stimulated but followed by an early mortality. A sharp rise in lethality effects was observed when gamma rays were applied beyond the seventh day of culture, when the proembryo contains an average of 4 cells. Morphological aberrations and colour changes in the Mo progeny derived from irradiated microspores are diverse. But after chromosome doubling and mutation checking out, all the plants were not recorded to have transmitted their aberrant characters. Thus, heritable character 'mutations) and not heritable character (variations) were obtained. The variations characters include dwarfing, excessive branching, fasciation and dichotomy of the stems, altered flower form, especially of petals. As to the leaves, they usually show induced changes in their colour (chlorotic areas, mosaic-colour changes, or an over-all colour changes), in their form (irregularity in outline) and in their texture (thickening, hairless leaf). Among the mutants, a monster tobacco, with excrescences on the leaves and the flowers is certainly the most conspicuous. But mutants also include altered leaf colour (over-all pale green) and altered flower colour, (dark red, clear pink, white) [fr

Zoospore exudates from Phytophthora nicotianae affect immune responses in Arabidopsis.

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Ping Kong

Full Text Available Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by Phytophthora. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF and zoospores of P. nicotianae were investigated comparatively for effects on resistance of Arabidopsis thaliana Col-0 and mutants that affect signaling mediated by salicylic acid (SA and jasmonic acid (JA: eds16 (enhanced disease susceptibility16, pad4 (phytoalexin deficient4, and npr1 (nonexpressor of pathogenesis-related genes1. Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the PR1 and PDF1.2 marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating PR1 when SA signaling or synthesis is deficient, which may be responsible for Arabidopsis mutant plants more susceptible to infection by high concentration of P. nicotianae zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by Phytophthora.

Synthesis of 'cineole cassette' monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis.

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Fähnrich, Anke; Brosemann, Anne; Teske, Laura; Neumann, Madeleine; Piechulla, Birgit

2012-08-01

The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, β-myrcene, α- and β-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.

Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

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Ann C Smigocki

Full Text Available Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance.

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Fragoso, Variluska; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

2014-06-01

While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied. To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature. Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In N. attenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus. We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses. © 2014 Max Planck Society. New Phytologist © 2014 New Phytologist Trust.

[Molecular and structural-biological analysis of Nicotiana plumbaginifolia mutants for identification of the site of beta-tubulins interaction with dinitroanilines and phosphorotioamidates].

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Emets, A I; Baiard, U V; Nyporko, A Iu; Swire-Clark, G A; Blium, Ia B

2009-01-01

The identification of point mutation locations on beta-tubulin molecules of amiprophosmethyl- and trifluralin-resistant Nicotiana plumbaginifolia lines have described in the work. It was shown that in the first case this mutation is connected with the substitution ofserine residue on proline in position 248; in the second case--with the substitution of phenilalanine on serine in position 317 of beta-tubulin amino acid sequence. Three-dimensional models of beta-tubulin molecule from Chlamydomonas with well-known location of mutations conferring dinitroaniline- and phosphorotioamidate resistance (substitution of lysine residue to methionine on position 350), and beta-tubulin from Nicotiana plumbaginifolia have been reconstructed. On the basis of analysis of site of interaction with dinitroanilines and phosphorotioamides on Chlamydomonas beta-tubulin molecule it was concluded that the revealed mutations on Nicotiana plumbaginifolia beta-tubulin affect amino acid residues participating in formation of this site.

The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

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Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

2017-11-24

To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus , family Closteroviridae ) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana . Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

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Kawamura, Ryusuke; Shimura, Hanako; Mochizuki, Tomofumi; Ohki, Satoshi T; Masuta, Chikara

2014-09-01

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Suitability of Nicotiana tabacum 'Bel W3' for biomonitoring ozone in Sao Paulo, Brazil

International Nuclear Information System (INIS)

Sant'Anna, Silvia M.R.; Esposito, Marisia P.; Domingos, Marisa; Souza, Silvia R.

2008-01-01

Nicotiana tabacum 'Bel W3' is a widely used sensitive bioindicator for ambient ozone, but it is rarely used in tropical countries. Our goal was to determine the suitability of this plant for biomonitoring ozone in the city of Sao Paulo by evaluating the relationships between leaf necroses and ozone under field conditions and measurements of chlorophyll a fluorescence and antioxidants in plants exposed to different concentrations of ozone in closed chambers. While a weak linear relationship between leaf injury and ozone concentrations (R 2 = 0.10) was determined in the field, a strong linear relationship was observed in the chamber experiments. Maximum leaf injury was observed in plants submitted to 40 ppb, which coincided with a significant decrease in fluorescence and total ascorbic acid. The relationship between leaf damage observed in the field and ozone was improved when the concentrations were limited to 40 ppb (R 2 = 0.28). - Nicotiana tabacum 'Bel W3' is suitable for indicating low ozone levels in Brazil

Tissue-specific expression of telomerase reverse transcriptase gene variants in Nicotiana tabacum

Czech Academy of Sciences Publication Activity Database

Jurečková, J.; Sýkorová, Eva; Hafidh, Said; Honys, David; Fajkus, Jiří; Fojtová, M.

2017-01-01

Roč. 245, č. 3 (2017), s. 549-561 ISSN 0032-0935 R&D Projects: GA ČR GA13-06943S; GA MŠk(CZ) LQ1601 Institutional support: RVO:68081707 ; RVO:61389030 Keywords : male gametophyte development * tobacco male gametophyte * allotetraploid nicotiana Subject RIV: EF - Botanics; EF - Botanics (UEB-Q) OBOR OECD: Plant sciences, botany; Plant sciences, botany (UEB-Q) Impact factor: 3.361, year: 2016

Molecular identification of catalases from Nicotiana plumbaginifolia (L.).

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Willekens, H; Villarroel, R; Van Montagu, M; Inzé, D; Van Camp, W

1994-09-19

We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3-aminotriazole, and salicylic acid. 3-Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

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Mazars, C; Thion, L; Thuleau, P; Graziana, A; Knight, M R; Moreau, M; Ranjeva, R

1997-11-01

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

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Kiran Zahid

2015-01-01

Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies.

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Prins, Anneke; van Heerden, Philippus D R; Olmos, Enrique; Kunert, Karl J; Foyer, Christine H

2008-01-01

The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

Science.gov (United States)

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

An Easy Method for Plant Polysome Profiling.

Science.gov (United States)

Lecampion, Cécile; Floris, Maïna; Fantino, Jean Raphaël; Robaglia, Christophe; Laloi, Christophe

2016-08-28

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

Science.gov (United States)

Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg

2015-01-01

Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

Isolation and characterization of the betalain biosynthesis gene involved in hypocotyl pigmentation of the allotetraploid Chenopodium quinoa.

Science.gov (United States)

Imamura, Tomohiro; Takagi, Hiroki; Miyazato, Akio; Ohki, Shinya; Mizukoshi, Hiroharu; Mori, Masashi

2018-02-05

In quinoa seedlings, the pigment betalain accumulates in the hypocotyl. To isolate the genes involved in betalain biosynthesis in the hypocotyl, we performed ethyl methanesulfonate (EMS) mutagenesis on the CQ127 variety of quinoa seedlings. While putative amaranthin and celosianin II primarily accumulate in the hypocotyls, this process produced a green hypocotyl mutant (ghy). This MutMap+ method using the quinoa draft genome revealed that the causative gene of the mutant is CqCYP76AD1-1. Our results indicated that the expression of CqCYP76AD1-1 was light-dependent. In addition, the transient expression of CqCYP76AD1-1 in Nicotiana benthamiana leaves resulted in the accumulation of betanin but not isobetanin, and the presence of a polymorphism in CqCYP76A1-2 in the CQ127 variety was shown to have resulted in its loss of function. These findings suggested that CqCYP76AD1-1 is involved in betalain biosynthesis during the hypocotyl pigmentation process in quinoa. To our knowledge, CqCYP76AD1-1 is the first quinoa gene identified by EMS mutagenesis using a draft gene sequence. Copyright © 2018. Published by Elsevier Inc.

The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae)

Czech Academy of Sciences Publication Activity Database

Leitch, I.J.; Hanson, L.; Lim, K.Y.; Kovařík, Aleš; Chase, M.W.; Clarkson, J.J.; Leitch, A.R.

2008-01-01

Roč. 101, č. 6 (2008), s. 805-814 ISSN 0305-7364 R&D Projects: GA ČR(CZ) GA521/07/0116 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : genome size * allopolyploidy * evolution-Nicotiana Subject RIV: BO - Biophysics Impact factor: 2.755, year: 2008

Differential modulation of plant immune responses by diverse members of the Pseudomonas savastanoi pv. savastanoi HopAF type III effector family.

Science.gov (United States)

Castañeda-Ojeda, M Pilar; López-Solanilla, Emilia; Ramos, Cayo

2017-06-01

The Pseudomonas savastanoi pv. savastanoi NCPPB 3335 type III secretion system (T3SS) effector repertoire includes 33 candidates, seven of which translocate into host cells and interfere with plant defences. The present study was performed to investigate the co-existence of both plasmid- and chromosomal-encoded members of the HopAF effector family, HopAF1-1 and HopAF1-2, respectively, in the genome of NCPPB 3335. Here, we show that the HopAF1 paralogues are widely distributed in the Pseudomonas syringae complex, where HopAF1-1 is most similar to the homologues encoded by other P. syringae pathovars infecting woody hosts that belong to phylogroups 1 and 3. We show that the expression of both HopAF1-1 and HopAF-2 is transcriptionally dependent on HrpL and demonstrate their delivery into Nicotiana tabacum leaves. Although the heterologous delivery of either HopAF1-1 or HopAF1-2 significantly suppressed the production of defence-associated reactive oxygen species levels, only HopAF1-2 reduced the levels of callose deposition. Moreover, the expression of HopAF1-2 by functionally effectorless P. syringae pv. tomato DC3000D28E completely inhibited the hypersensitive response in tobacco and significantly increased the competitiveness of the strain in Nicotiana benthamiana. Despite their functional differences, subcellular localization studies reveal that green fluorescent protein (GFP) fusions to either HopAF1-1 or HopAF1-2 are targeted to the plasma membrane when they are expressed in plant cells, a process that is completely dependent on the integrity of their N-myristoylation motif. Our results further support the notion that highly similar T3SS effectors might differentially interact with diverse plant targets, even when they co-localize in the same cell compartment. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Transient Expression of Lumbrokinase (PI239 in Tobacco (Nicotiana tabacum Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots

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Alexia Dickey

2017-01-01

Full Text Available Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239 was produced from a plant system. Both wild-type (WT and plant codon-optimized (OP PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production

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Zhou Xue-Rong

2010-03-01

Full Text Available Abstract Background Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. Results In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2 gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. Conclusions The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.

O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection.

Science.gov (United States)

Pérez, José de Jesús; Udeshi, Namrata D; Shabanowitz, Jeffrey; Ciordia, Sergio; Juárez, Silvia; Scott, Cheryl L; Olszewski, Neil E; Hunt, Donald F; García, Juan Antonio

2013-08-01

O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus. Copyright © 2013 Elsevier Inc. All rights reserved.

Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals.

Science.gov (United States)

Lyubenova, Lyudmila; Nehnevajova, Erika; Herzig, Rolf; Schröder, Peter

2009-07-01

Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the

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.

REMINDER Saved Leave Scheme (SLS) : Transfer of leave to saved leave accounts

CERN Multimedia

HR Division

2002-01-01

Under the provisions of the voluntary saved leave scheme (SLS), a maximum total of 10 days'*) annual and compensatory leave (excluding saved leave accumulated in accordance with the provisions of Administrative Circular No. 22B) can be transferred to the saved leave account at the end of the leave year (30 September). We remind you that, since last year, unused leave of all those taking part in the saved leave scheme at the closure of the leave-year accounts is transferred automatically to the saved leave account on that date. Therefore, staff members have no administrative steps to take. In addition, the transfer, which eliminates the risk of omitting to request leave transfers and rules out calculation errors in transfer requests, will be clearly shown in the list of leave transactions that can be consulted in EDH from October 2002 onwards. Furthermore, this automatic leave transfer optimizes staff members' chances of benefiting from a saved leave bonus provided that they are still participants in the schem...

The receptor-like kinase SOBIR1 interacts with Brassica napus LepR3 and is required for Leptosphaeria maculans AvrLm1-triggered immunity

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Lisong eMa

2015-10-01

Full Text Available AbstractThe fungus Leptosphaeria maculans (L. maculans is the causal agent of blackleg disease of canola/oilseed rape (Brassica napus worldwide. We previously reported cloning of the B. napus blackleg resistance gene, LepR3, which encodes a receptor-like protein. LepR3 triggers localised cell death upon recognition of its cognate Avr protein, AvrLm1. Here, we exploited the Nicotiana benthamiana model plant to investigate the recognition mechanism of AvrLm1 by LepR3. Co-expression of the LepR3/AvrLm1 gene pair in N. benthamiana resulted in development of a hypersensitive response (HR. However, a truncated AvrLm1 lacking its indigenous signal peptide was compromised in its ability to induce LepR3-mediated HR, indicating that AvrLm1 is perceived by LepR3 extracellularly. Structure-function analysis of the AvrLm1 protein revealed that the C-terminal region of AvrLm1 was required for LepR3-mediated HR in N. benthamiana and for resistance to L. maculans in B. napus. LepR3 was shown to be physically interacting with the B. napus receptor like kinase, SOBIR1 (BnSOBIR1. Silencing of NbSOBIR1 or NbSERK3 (BAK1 compromised LepR3-AvrLm1-dependent HR in N. benthamiana, suggesting that LepR3-mediated resistance to L. maculans in B. napus requires SOBIR1 and BAK1/SERK3. Using this model system, we determined that BnSOBIR1 and SERK3/BAK1 are essential partners in the LepR3 signalling complex and were able to define the AvrLm1 effector domain.

Fall and rise of satellite repeats in allopolyploids of Nicotiana over c. 5 million years

Czech Academy of Sciences Publication Activity Database

Koukalová, Blažena; Moraes, A.P.; Renny-Byfield, S.; Matyášek, Roman; Leitch, A.R.; Kovařík, Aleš

2010-01-01

Roč. 186, č. 1 (2010), s. 148-160 ISSN 0028-646X R&D Projects: GA ČR(CZ) GA521/07/0116 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : concerted evolution * interlocus homogenization * Nicotiana Subject RIV: BO - Biophysics Impact factor: 6.516, year: 2010

Expression of ACC oxidase promoter-GUS fusions in tomato and Nicotiana plumbaginifolia regulated by developmental and environmental stimuli.

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Blume, B; Grierson, D

1997-10-01

The enzyme ACC oxidase, catalysing the last step in the biosynthesis of the plant hormone ethylene, is encoded by a small multigene family in tomato, comprising three members, LEACO1, LEACO2 and LEACO3. LEACO1 is the major gene expressed during ripening, leaf senescence, and wounding (Barry et al., 1996). To investigate the transcriptional regulation of ACC oxidase gene expression, chimeric fusions between the beta-glucuronidase reporter gene and 97 bp of 5' UTR plus 124, 396 and 1825 bp, respectively, of 5' untranscribed LEACO1 sequence were constructed and introduced into Lycopersicon esculentum (Mill cv. Ailsa Craig) and Nicotiana plumbaginifolia. Analysis of transgenic tomatoes indicated that the region containing nucleotides -124 to +97 of the LEACO1 gene is sufficient to confer a marked increase in GUS activity during fruit ripening, albeit at very low levels. Fusion of 396 and 1825 bp of LEACO1 upstream sequence resulted in strong and specific induction of GUS expression in situations known to be accompanied by enhanced ethylene production. Reporter gene expression was similar to that of the endogenous LEACO1 gene, with major increases especially during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers. Analysis of transgenic N. plumbaginifolia plants confirmed the pattern of LEACO1 promoter activity detected in tomato leaves and flowers. Reporter gene expression was also induced following wounding, treatment with ethylene, and pathogen infection. Histochemical analysis illustrated localized GUS activity in the pericarp of ripening fruit, abscission zones of senescent petioles and unfertilized flowers, and at wound sites. These results demonstrate that ACC oxidase is regulated at the transcriptional level in a wide range of cell types at different developmental stages and in response to several external stimuli.

Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units.

Science.gov (United States)

Lim, K Y; Kovarik, A; Matýăsek, R; Bezdĕk, M; Lichtenstein, C P; Leitch, A R

2000-06-01

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n = 2x = 24) and N. tomentosiformis (2n = 2x = 24) and compared these with patterns in N. tabacum (tobacco, 2n = 4x = 48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N

UV irradiation as a tool for obtaining asymmetric somatic hybrids between Nicotiana plumbaginifolia and Lycopersicon esculentum

International Nuclear Information System (INIS)

Vlahova, M.; Hinnisdaels, S.; Frulleux, F.; Claeys, M.; Atanassov, A.; Jacobs, M.

1997-01-01

UV-irradiated kanamycin-resistant Lycopersicon esculentum leaf protoplasts were fused with wild-type Nicotiana plumbaginifolia leaf protoplasts. Hybrid calli were recovered after selection in kanamycin-containing medium and subsequently regenerated. Cytological analysis of these regenerants showed that several (2–4) tomato chromosomes, or chromosome fragments, were present in addition to a polyploid Nicotiana genome complement. All lines tested had neomycin phosphotransferase (NPTII) activity and the presence of the kanamycin gene was shown by Southern blotting. In two cases a different hybridization profile for the kanamycin gene, compared to the tomato donor partner, was observed, suggesting the occurence of intergenomic recombination events. The hybrid nature of the regenerants was further confirmed by Southernblotting experiments using either a ribosomal DNA sequence or a tomato-specific repeat as probes. The hybrids were partially fertile and some progeny could be obtained. Our results demonstrate that UV irradiation is a valuable alternative for asymmetric cell-hybridization experiments. (author)

NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

Science.gov (United States)

Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

2013-01-01

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

Identification of a Nicotiana plumbaginifolia plasma membrane H(+)-ATPase gene expressed in the pollen tube.

Science.gov (United States)

Lefebvre, Benoit; Arango, Miguel; Oufattole, Mohammed; Crouzet, Jérôme; Purnelle, Bénédicte; Boutry, Marc

2005-08-01

In Nicotiana plumbaginifolia, plasma membrane H(+)-ATPases (PMAs) are encoded by a gene family of nine members. Here, we report on the characterization of a new isogene, NpPMA5 (belonging to subfamily IV), and the determination of its expression pattern using the beta-glucuronidase (gusA) reporter gene. pNpPMA5-gusA was expressed in cotyledons, in vascular tissues of the stem (mainly in nodal zones), and in the flower and fruit. In the flower, high expression was found in the pollen tube after in vitro or in vivo germination. Northern blotting analysis confirmed that NpPMA5 was expressed in the pollen tube contrary to NpPMA2 (subfamily I) or NpPMA4 (subfamily II), two genes highly expressed in other tissues. The subcellular localization of PM H(+)-ATPase in the pollen tube was analyzed by immunocytodecoration. As expected, this enzyme was localized to the plasma membrane. However, neither the tip nor the base of the pollen tube was labeled, showing an asymmetrical distribution of this enzyme. This observation supports the hypothesis that the PM H(+)-ATPase is involved in creating the pH gradient that is observed along the pollen tube and is implicated in cell elongation. Compared to other plant PM H(+)-ATPases, the C-terminal region of NpPMA5 is shorter by 26 amino acid residues and is modified in the last 6 residues, due to a sequence rearrangement, which was also found in the orthologous gene of Nicotiana glutinosa, a Nicotiana species distant from N. plumbaginifolia and Petunia hybrida and Lycopersicon esculentum, other Solanacae species. This modification alters part of the PM H(+)-ATPase regulatory domain and raises the question whether this isoform is still regulated.

Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

Science.gov (United States)

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications.

Science.gov (United States)

Rice, J Hollis; Mundell, Richard E; Millwood, Reginald J; Chambers, Orlando D; Stewart, C Neal; Davies, H Maelor

2013-08-06

The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes. Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field. N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

Science.gov (United States)

Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

2014-02-13

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance.

Science.gov (United States)

Qin, Xiaoqiong; Zeevaart, Jan A D

2002-02-01

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.

Resistencia transgénica para el control del virus motoso del enanismo de la frambuesa (raspberry bushy dwarf virus-rbdv Transgenic resistance for the control of raspberry bushy dwarf virus-rbdv

Directory of Open Access Journals (Sweden)

Angel J.

1998-06-01

by manually inoculating expanded leaves and assaying for virus titre by ELISA 5 and 10 days after inoculation. More than 20 lines were obtained from transformations with each type of construct and the results showed that transformation with each construct had some effect on virus multiplication. Evidence of resistance induced by coat protein (CP sense, CP anti-sense and polymerase gene sequences was obtained and several degrees of effectiveness of protection were identified among these constructtransgenes. Construct pJAD-10 (RBDV R-15 CP gene sequence induced the highest level of protection and some N. benthamiana lines transformed with this construct also showed inhibition or delay in the development of the symptoms of infection by RBDV. Also, the results of challenging transgenic Nicotiana lines transformed with untranslatable CP and CP antisense sequences suggest that resistance can be generated by the expression of RBDV CP and also by mRNA CP transcripts. These constructs are now available to be inserted into raspberry germplasm to provide resistance against R-15 strains of RBDV.

High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

DEFF Research Database (Denmark)

Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan

2014-01-01

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized...

Optimization of engineered production of the glucoraphanin precursor dihomomethionine in Nicotiana benthamiana

DEFF Research Database (Denmark)

Crocoll, Christoph; Mirza, Nadia Muhammad Akram; Reichelt, Michael

2016-01-01

Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our...

Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

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Spena Angelo

2003-06-01

Full Text Available Abstract Background Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS, is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana. Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. Results In the ihprolC-PP197 gene (intron hair pin rolC PPV 197, a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330 was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80% transgenic plants are virus free and symptomless. Some plants (20% contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation

Evolution of rDNA in Nicotiana allopolyploids: A potential link between rDNa homogenization and epigenetics

Czech Academy of Sciences Publication Activity Database

Kovařík, Aleš; Nešpor Dadejová, Martina; Lim, Y.K.; Chase, M.W.; Clarkson, J.J.; Knapp, S.; Leitch, A.R.

2008-01-01

Roč. 101, č. 6 (2008), s. 815-823 ISSN 0305-7364 R&D Projects: GA ČR(CZ) GA521/07/0116 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : rDNA * allopolyploidy * evolution-Nicotiana Subject RIV: BO - Biophysics Impact factor: 2.755, year: 2008

Overexpression of a 9-cis-Epoxycarotenoid Dioxygenase Gene in Nicotiana plumbaginifolia Increases Abscisic Acid and Phaseic Acid Levels and Enhances Drought Tolerance1

Science.gov (United States)

Qin, Xiaoqiong; Zeevaart, Jan A.D.

2002-01-01

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels. PMID:11842158

In vitro and in vivo activities of eugenol against tobacco black shank caused by Phytophthora nicotianae.

Science.gov (United States)

Jing, Changliang; Gou, Jianyu; Han, Xiaobin; Wu, Qian; Zhang, Chengsheng

2017-10-01

Phytophthora nicotianae causes serious black shank disease in tobacco. Syringa oblata essential oil and its main components were evaluated to develop an effective and environmentally friendly biocontrol agent. Eugenol, which exhibited the strongest activity, was intensively investigated in vitro and in vivo. The mycelial growth of P. nicotianae was inhibited by eugenol at a minimum inhibitory concentration of 200μgmL -1 , and inhibition occurred in a dose-dependent manner. Extracellular pH and extracellular conductivity results indicated that eugenol increased membrane permeability. Flow cytometry and fluorescent staining results further showed that eugenol disrupted mycelial membranes but did not affect spore membrane integrity. The in vivo results confirmed that treatment of tobacco with various concentrations of eugenol formulations reduced disease incidence and better controlled against the disease. Our results suggested that the ability of eugenol to control tobacco black shank depended on its ability to damage mycelial membranes and that eugenol formulations have potential as an eco-friendly antifungal agent for controlling tobacco blank shank. Copyright © 2017 Elsevier B.V. All rights reserved.

Suitability of Nicotiana tabacum 'Bel W3' for biomonitoring ozone in Sao Paulo, Brazil

Energy Technology Data Exchange (ETDEWEB)

Sant' Anna, Silvia M.R.; Esposito, Marisia P.; Domingos, Marisa [Instituto de Botanica, Secao de Ecologia, Caixa Postal 3005, 01061-970 Sao Paulo, SP (Brazil); Souza, Silvia R. [Instituto de Botanica, Secao de Ecologia, Caixa Postal 3005, 01061-970 Sao Paulo, SP (Brazil)], E-mail: souzasrd@terra.com.br

2008-01-15

Nicotiana tabacum 'Bel W3' is a widely used sensitive bioindicator for ambient ozone, but it is rarely used in tropical countries. Our goal was to determine the suitability of this plant for biomonitoring ozone in the city of Sao Paulo by evaluating the relationships between leaf necroses and ozone under field conditions and measurements of chlorophyll a fluorescence and antioxidants in plants exposed to different concentrations of ozone in closed chambers. While a weak linear relationship between leaf injury and ozone concentrations (R{sup 2} = 0.10) was determined in the field, a strong linear relationship was observed in the chamber experiments. Maximum leaf injury was observed in plants submitted to 40 ppb, which coincided with a significant decrease in fluorescence and total ascorbic acid. The relationship between leaf damage observed in the field and ozone was improved when the concentrations were limited to 40 ppb (R{sup 2} = 0.28). - Nicotiana tabacum 'Bel W3' is suitable for indicating low ozone levels in Brazil.

Involvement of C4 protein of beet severe curly top virus (family Geminiviridae in virus movement.

Directory of Open Access Journals (Sweden)

Kunling Teng

Full Text Available BACKGROUND: Beet severe curly top virus (BSCTV is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction. METHODS AND FINDINGS: To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties. CONCLUSIONS: Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.

Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

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Kasaras Alexis

2012-04-01

Full Text Available Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER and the vacuole. Induction of spherical structures (“bulbs”, changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1

Nitrite reductase expression is regulated at the post-transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana.

Science.gov (United States)

Crété, P; Caboche, M; Meyer, C

1997-04-01

Higher plant nitrite reductase (NiR) is a monomeric chloroplastic protein catalysing the reduction of nitrite, the product of nitrate reduction, to ammonium. The expression of this enzyme is controlled at the transcriptional level by light and by the nitrogen source. In order to study the post-transcriptional regulation of NiR, Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed with a chimaeric NiR construct containing the tobacco leaf NiR1 coding sequence driven by the CaMV 35S RNA promoter. Transformed plants did not show any phenotypic difference when compared with the wild-type, although they overexpressed NiR activity in the leaves. When these plants were grown in vitro on media containing either nitrate or ammonium as sole nitrogen source, NiR mRNA derived from transgene expression was constitutively expressed, whereas NiR activity and protein level were strongly reduced on ammonium-containing medium. These results suggest that, together with transcriptional control, post-transcriptional regulation by the nitrogen source is operating on NiR expression. This post-transcriptional regulation of tobacco leaf NiR1 expression was observed not only in the closely related species N. plumbaginifolia but also in the more distant species A. thaliana.

Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis.

Science.gov (United States)

Eljounaidi, K; Comino, C; Moglia, A; Cankar, K; Genre, A; Hehn, A; Bourgaud, F; Beekwilder, J; Lanteri, S

2015-10-01

Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Tomato SlRbohB, a member of the NADPH oxidase family, is required for disease resistance against Botrytis cinerea and tolerance to drought stress

Directory of Open Access Journals (Sweden)

Xiaohui eLi

2015-06-01

Full Text Available NADPH oxidases (also known as respiratory burst oxidase homologues, Rbohs are the enzymes that catalyze the generation of reactive oxygen species (ROS in plants. In the present study, eight SlRboh genes were identified in tomato and their possible involvement in resistance to Botrytis cinerea and drought tolerance was examined. Expression of SlRbohs was induced by B. cinerea and Pseudomonas syringae pv. tomato but displayed distinct patterns. Virus-induced gene silencing (VIGS-based silencing of SlRbohB resulted in reduced resistance to B. cinerea but silencing of each of other SlRbohs did not affect the resistance. The SlRbohB-silenced plants accumulated more ROS and attenuated expression of defense genes after infection of B. cinerea than the nonsilenced plants. Silencing of SlRbohB also suppressed flg22-induced ROS burst and the expression of SlLrr22, a marker gene related to PAMP-triggered immunity (PTI. Transient expression of SlRbohB in Nicotiana benthamiana led to enhanced resistance to B. cinerea. Furthermore, silencing of SlRbohB resulted in decreased drought tolerance, accelerated water loss in leaves and altered expression of drought-responsive genes. Our data demonstrate that SlRbohB positively regulates the resistance to B. cinerea, flg22-induced PTI and drought tolerance in tomato.

A Rice Immunophilin Gene, OsFKBP16-3, Confers Tolerance to Environmental Stress in Arabidopsis and Rice

Directory of Open Access Journals (Sweden)

Jun Cheul Ahn

2013-03-01

Full Text Available The putative thylakoid lumen immunophilin, FKBP16-3, has not yet been characterized, although this protein is known to be regulated by thioredoxin and possesses a well-conserved CxxxC motif in photosynthetic organisms. Here, we characterized rice OsFKBP16-3 and examined the role of this gene in the regulation of abiotic stress in plants. FKBP16-3s are well conserved in eukaryotic photosynthetic organisms, including the presence of a unique disulfide-forming CxxxC motif in their N-terminal regions. OsFKBP16-3 was mainly expressed in rice leaf tissues and was upregulated by various abiotic stresses, including salt, drought, high light, hydrogen peroxide, heat and methyl viologen. The chloroplast localization of OsFKBP16-3-GFP was confirmed through the transient expression of OsFKBP16-3 in Nicotiana benthamiana leaves. Transgenic Arabidopsis and transgenic rice plants that constitutively expressed OsFKBP16-3 exhibited increased tolerance to salinity, drought and oxidative stresses, but showed no change in growth or phenotype, compared with vector control plants, when grown under non-stressed conditions. This is the first report to demonstrate the potential role of FKBP16-3 in the environmental stress response, which may be regulated by a redox relay process in the thylakoid lumen, suggesting that artificial regulation of FKBP16-3 expression is a candidate for stress-tolerant crop breeding.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

Science.gov (United States)

Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

2016-10-01

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Energy Technology Data Exchange (ETDEWEB)

Bejerman, Nicolás, E-mail: n.bejerman@uq.edu.au [Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria INTA, Camino a 60 Cuadras k 5,5, Córdoba X5020ICA (Argentina); Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072 (Australia); Giolitti, Fabián; Breuil, Soledad de; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio [Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria INTA, Camino a 60 Cuadras k 5,5, Córdoba X5020ICA (Argentina); Dietzgen, Ralf G. [Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072 (Australia)

2015-09-15

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

International Nuclear Information System (INIS)

Bejerman, Nicolás; Giolitti, Fabián; Breuil, Soledad de; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio; Dietzgen, Ralf G.

2015-01-01

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses

The NB-LRR gene Pm60 confers powdery mildew resistance in wheat.

Science.gov (United States)

Zou, Shenghao; Wang, Huan; Li, Yiwen; Kong, Zhaosheng; Tang, Dingzhong

2018-04-01

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T. urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucine-rich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiled-coil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

KAUST Repository

Wibowo, Anjar

2011-06-06

Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.

Identification, Characterization and Full-Length Sequence Analysis of a Novel Polerovirus Associated with Wheat Leaf Yellowing Disease.

Science.gov (United States)

Zhang, Peipei; Liu, Yan; Liu, Wenwen; Cao, Mengji; Massart, Sebastien; Wang, Xifeng

2017-01-01

To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV) (most likely pathogens) using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV). The full genome of WLYaV corresponds to 5,772 nucleotides (nt), with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame, and three untranslated regions, showing typical features of the family Luteoviridae . Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV), but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP) were 64.9 and 86.2%, respectively, below the species demarcation thresholds (90%) in the family Luteoviridae . Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat.

Identification, Characterization and Full-Length Sequence Analysis of a Novel Polerovirus Associated with Wheat Leaf Yellowing Disease

Directory of Open Access Journals (Sweden)

Peipei Zhang

2017-09-01

Full Text Available To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV (most likely pathogens using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV. The full genome of WLYaV corresponds to 5,772 nucleotides (nt, with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame, and three untranslated regions, showing typical features of the family Luteoviridae. Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV, but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP were 64.9 and 86.2%, respectively, below the species demarcation thresholds (90% in the family Luteoviridae. Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat.

Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta

NARCIS (Netherlands)

Voelckel, C.; Krugel, T.; Gase, K.; Heidrich, N.; Van Dam, N.M.; Winz, R.; Baldwin, I.T.

2001-01-01

Several lines of evidence support the defensive function of nicotine production in the Nicotiana genus against a range of herbivores, but the evidence is largely correlative. To suppress nicotine production in planta and to test its defensive function, we expressed DNA of putrescine N-methyl

Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants

NARCIS (Netherlands)

Moravcikova, J.; Matusikova, I.; Libantova, J.; Bauer, M.; Mlynarova, L.

2004-01-01

The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) breeding line 116/86 using Agrobacterium tumefaciens. For both transgenes the number of integrated copies and level of RNA expression

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

NARCIS (Netherlands)

Tempelaar, MJ; Drenth - Diephuis, L.J.; SAAT, TAWM; Jacobsen, E.

Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used

Lupines, poison-hemlock and Nicotiana spp: toxicity and teratogenicity in livestock.

Science.gov (United States)

Panter, K E; James, L F; Gardner, D R

1999-02-01

Many species of lupines contain quinolizidine or piperidine alkaloids known to be toxic or teratogenic to livestock. Poison-hemlock (Conium maculatum) and Nicotiana spp. including N. tabacum and N. glauca contain toxic and teratogenic piperidine alkaloids. The toxic and teratogenic effects from these plant species have distinct similarities including maternal muscular weakness and ataxia and fetal contracture-type skeletal defects and cleft palate. It is believed that the mechanism of action of the piperidine and quinolizidine alkaloid-induced teratogenesis is the same; however, there are some differences in incidence, susceptible gestational periods, and severity between livestock species. Wildlife species have also been poisoned after eating poison-hemlock but no terata have been reported. The most widespread problem for livestock producers in recent times has been lupine-induced "crooked calf disease." Crooked calf disease is characterized as skeletal contracture-type malformations and occasional cleft palate in calves after maternal ingestion of lupines containing the quinolizidine alkaloid anagyrine during gestation days 40-100. Similar malformations have been induced in cattle and goats with lupines containing the piperidine alkaloids ammodendrine, N-methyl ammodendrine, and N-acetyl hystrine and in cattle, sheep, goats, and pigs with poison-hemlock containing predominantly coniine or gamma-coniceine and N. glauca containing anabasine. Toxic and teratogenic effects have been linked to structural aspects of these alkaloids, and the mechanism of action is believed to be associated with an alkaloid-induced inhibition of fetal movement during specific gestational periods. This review presents a historical perspective, description and distribution of lupines, poison-hemlock and Nicotiana spp., toxic and teratogenic effects and management information to reduce losses.

Saved Leave Scheme (SLS) : Simplified procedure for the transfer of leave to saved leave accounts

CERN Multimedia

HR Division

2001-01-01

As part of the process of streamlining procedures, the HR and AS Divisions have jointly developed a system whereby annual and compensatory leave will henceforth be automatically transferred1) to saved leave accounts. Under the provisions of the voluntary saved leave scheme (SLS), a maximum total of 10 days'2) annual and compensatory leave (excluding saved leave accumulated in accordance with the provisions of Administrative Circular No. 22 B) can be transferred to the saved leave account at the end of the leave year (30 September). Previously, every person taking part in the scheme has been individually issued with a form for the purposes of requesting the transfer of leave to the leave account and the transfer has then had to be done manually by HR Division. To streamline the procedure, unused leave of all those taking part in the saved leave scheme at the closure of of the leave-year accounts will henceforth be transferred automatically to the saved leave account on that date. This simplification is in the ...

Effects of different potting growing media for Petunia grandiflora and Nicotiana alata Link & Otto on photosynthetic capacity, leaf area, and flowering potential

Directory of Open Access Journals (Sweden)

Gheorghe Cristian Popescu

2015-03-01

Full Text Available Petunia grandiflora Juss. and Nicotiana alata Link & Otto are two of the most widely spread plants on the market for annual potted ornamental plants. In order to identify the most adequate substrate formula we analyzed the effects of different potting growing media used for P. hybrida grandiflora 'Bravo' and N. alata 'Dinamo' on their photosynthetic capacity, leaf area, and flowering potential. Optimization of growing media formula for petunia and ornamental tobacco was performed by preparing four growing media mixing fallow soil (FS, Biolan peat (BP, acid peat (AP, leaf compost (C, and perlite (P in different proportions. The physiological potential of petunia and ornamental tobacco was investigated by photosynthesis and respiration rate and chlorophyll pigments in leaves, while the vegetative and flowering phenological stages were evaluated by number of leaves per plant, leaf area, number of flowers per plant and leaf area/flowers ratio. These measurements were significantly influenced by the different potting growing media used in this study. In the flowering stage, the highest photosynthesis rates (8.612 μmol CO2 m-2 s-1 as well as leaf area (1.766 dm² of petunias were obtained on growing media with 60% biolan peat, 30% acid peat and 10% perlite (BP60-AP30-P10. Flowering responses to growing conditions vary greatly among plants and the biggest number of ornamental tobacco flowers (22 flowers plant-1 was registered as an effect of BP60-AP30-P10 media. Growing media with the BP60-AP30-P10 formula seem to be the most adequate growth substrate to develop profitable crops for petunias and ornamental tobacco with high decorative value.

Paid Family Leave, Fathers' Leave-Taking, and Leave-Sharing in Dual-Earner Households.

Science.gov (United States)

Bartel, Anne P; Rossin-Slater, Maya; Ruhm, Christopher J; Stearns, Jenna; Waldfogel, Jane

Using difference-in-difference and difference-in-difference-in-difference designs, we study California's Paid Family Leave (CA-PFL) program, the first source of government-provided paid parental leave available to fathers in the Unites States. Relative to the pre-treatment mean, fathers of infants in California are 46 percent more likely to be on leave when CA-PFL is available. In households where both parents work, we find suggestive evidence that CA-PFL increases both father-only leave-taking (i.e., father on leave while mother is at work) and joint leave-taking (i.e., both parents on leave at the same time). Effects are larger for fathers of first-born children than for fathers of later-born children.

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

Science.gov (United States)

Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

A Functional Link between RNA Replication and Virion Assembly in the Potyvirus Plum Pox Virus.

Science.gov (United States)

Gallo, Araiz; Valli, Adrian; Calvo, María; García, Juan Antonio

2018-05-01

Accurate assembly of viral particles in the potyvirus Plum pox virus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in Nicotiana benthamiana leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied in trans , was assembled into virus-like particles and remained stable after in vitro incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in N. benthamiana leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection. IMPORTANCE Viruses of the family Potyviridae are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of Plum pox virus (genus

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes1[W][OA

Science.gov (United States)

Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

2013-01-01

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown. PMID:23150644

Mutability of the self-incompatibility locus and identification of the S-bearing chromosome in Nicotiana alata

International Nuclear Information System (INIS)

Gastel, A.J.G. van.

1976-01-01

γ rays, X rays, fast neutrons and ethyl methanesulfonate (EMS) were used for inducing mutations at the self-incompatibility locus of Nicotiana alata. Chronic gamma irradiation and EMS treatment neither induced self-compatability mutations nor led to changes from one S allele to another. X rays and fast neutrons induced many self-compatibility mutations, but did not generate new self-incompatibility alleles. (Auth.)

Paid Family Leave, Fathers' Leave-Taking, and Leave-Sharing in Dual-Earner Households

OpenAIRE

Bartel, Ann P.; Rossin-Slater, Maya; Ruhm, Christopher J.; Stearns, Jenna; Waldfogel, Jane

2015-01-01

This paper provides quasi-experimental evidence on the impact of paid leave legislation on fathers' leave-taking, as well as on the division of leave between mothers and fathers in dual-earner households. Using difference-in-difference and difference-in-difference-in-difference designs, we study California's Paid Family Leave (CA-PFL) program, which is the first source of government-provided paid parental leave available to fathers in the United States. Our results show that fathers in Califo...

The role of Nicotiana gluca Graham (paraguayan herbs as an adjuvant in immunomodulation of Newcastle disease vaccine for broilers Estudo da ação de Nicotiana glauca Graham (erva paraguaia como coadjuvante em vacina contra a doença de Newcastle em frangos de corte

Directory of Open Access Journals (Sweden)

Fabiane Pereira Gentilini

2008-07-01

Full Text Available The Nicotiana glauca is a native plant species from Argentina, but found all over South América, being used against headaches, rheumatism, injuries, ulcers, and so on. Researchers have considered it as having immunomodulation effect. This study was conducted to investigate the use of a aqueous extract of Nicotiana glauca Graham as an immunomodulator (adjuvant of a Newcastle disease vaccine.. A total of 56 broilers were distributed into 4 experimental groups. Each one of them received 3 dosages of this vaccine with or without the addition of different concentrations of the extract Using hemmoaglutination inhibition techniques , the results have shown differences (P<0.05 in the third sera collection. An increase in the antibody titer with the inclusion of 5 mg/dosage of the extract (Treatment 3 as compared to 1 mg/dosage (Treatment 2 and 10 mg/dosage of the extract (Treatment 4 was observed, However, birds from Treatment 3 did not differ (P> 0.05 from Treatment 1. These results indicated that further investigations are required, including the use of cytotoxicity tests in vitro, to evaluate the immunomodulation effect of this extract.

KEY WORDS: Immunomodulation effect, Nicotiana glauca Graham, vaccine.

Effect of gamma-radiation on callus initiation and oraganogenesis in the tissue culture of Nicotiana tabaccum L

International Nuclear Information System (INIS)

Shin, S. H.; Kim, J. G.; Song, H. S.

2004-01-01

It is generally agreed that ionizing radiations stimulate cell division, growth and development in various organisms including animals and plants. Differentiating tissues are the most sensitive to radiation. The present experiment was carried out to investigate the effects of ionizing radiation on callus initiation and organogenesis from the stem in the culture of Nicotiana tabaccum L. cv. When the stem segments were cultured on a Murashige and Skoog (MS) medium with 2 mg/L kinetin, with 1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), with 2 mg/L kinetin and 1 mg/L 2,4-D, the shoots and callus were differentiated 14 days after cultivation. Callus was especially formed on the MS medium with 2,4-D and/or kinetin and the formation was promoted by 1 Gy and 5 Gy of gamma radiation. The formation of the shoot clusters on the MS medium with 2 mg/L kinetin were prominent in the 5 Gy-irradiated groups. It is concluded that that gamma radiation enhanced the callus initiation and organogenesis in the tissue culture of Nicotiana tabaccum L

Ozone, sulfur dioxide, and ultraviolet B have similar effects on mRNA accumulation of antioxidant genes in Nicotiana plumbaginifolia L.

Energy Technology Data Exchange (ETDEWEB)

Willekens, H.; Van Camp, W.; Van Montagu, M.; Inze, D. [Laboratoire Associe de l`Institut National de la Recherche Agronomique (France); Langebartels, C.; Sandermann, H. Jr. [Universiteit Gent (Belgium)]|[Institut fuer Biochemische Pflanzenpathologie, Oberschleissheim (Germany)

1994-11-01

We have studied the expression of antioxidant genes in response to near ambient conditions of O{sub 3}, SO{sub 2}, and ultraviolet B (UV-B) in Nicotiana plumbaginifolia L. The genes analyzed encode four different superoxide dismutases (SODs), three catalases (Cat1, Cat2, and Cat3), the cytosolic ascorbate peroxidase (cyt APx), and glutathione peroxidase (GPx). The experimental setup for each treatment was essentially the same and caused no visible damage, thus allowing direct comparison of the different stress responses. Our data showed that the effects of O{sub 3}, SO{sub 2}, and UV-B on the antioxidant genes are very similar, although the response to SO{sub 2} is generally less pronounced and delayed. The effects of the different stresses are characterized by a decline in Cat1, a moderate increase in Cat3, and a strong increase in Cat2 and GPx. Remarkably, SODs and cyt APx were not affected. Analysis of SOD and APx expression in the ozone-sensitive Nicotiana tabacum L. cv PBD6 revealed that induction of the cytosolic copper/zinc SOD and cyt APx occurs only with the onset of visible damage. It is proposed that alterations in mRNA levels of catalases and GPx, but not of SODs and cyt APx, form part of the initial antioxidant response to O{sub 3}, SO{sub 2}, and UV-B in Nicotiana. 57 refs., 4 figs.

Evolutionary time-scale of the begomoviruses: evidence from integrated sequences in the Nicotiana genome.

Directory of Open Access Journals (Sweden)

Pierre Lefeuvre

Full Text Available Despite having single stranded DNA genomes that are replicated by host DNA polymerases, viruses in the family Geminiviridae are apparently evolving as rapidly as some RNA viruses. The observed substitution rates of geminiviruses in the genera Begomovirus and Mastrevirus are so high that the entire family could conceivably have originated less than a million years ago (MYA. However, the existence of geminivirus related DNA (GRD integrated within the genomes of various Nicotiana species suggests that the geminiviruses probably originated >10 MYA. Some have even suggested that a distinct New-World (NW lineage of begomoviruses may have arisen following the separation by continental drift of African and American proto-begomoviruses ∼110 MYA. We evaluate these various geminivirus origin hypotheses using Bayesian coalescent-based approaches to date firstly the Nicotiana GRD integration events, and then the divergence of the NW and Old-World (OW begomoviruses. Besides rejecting the possibility of a<2 MYA OW-NW begomovirus split, we could also discount that it may have occurred concomitantly with the breakup of Gondwanaland 110 MYA. Although we could only confidently narrow the date of the split down to between 2 and 80 MYA, the most plausible (and best supported date for the split is between 20 and 30 MYA--a time when global cooling ended the dispersal of temperate species between Asia and North America via the Beringian land bridge.

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls

Czech Academy of Sciences Publication Activity Database

Nováková, Martina; Macková, M.; Antošová, Z.; Viktorová, J.; Szekeres, M.; Demnerová, K.; Macek, Tomáš

2010-01-01

Roč. 1, č. 6 (2010), s. 419-423 ISSN 1949-1018 R&D Projects: GA MŠk 1M06030 Grant - others:GA MŠk(CZ) ME09024 Institutional research plan: CEZ:AV0Z40550506 Keywords : phytoremediation * transgenic plant * Nicotiana tabacum * bphC Subject RIV: EI - Biotechnology ; Bionics

Silencing of a Germin-Like Gene in Nicotiana attenuata Improves Performance of Native Herbivores1[W

Science.gov (United States)

Lou, Yonggen; Baldwin, Ian T.

2006-01-01

Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval oral secretions (OS) to wounds up-regulates transcripts of a GLP. To understand the function of this gene, which occurs as a single copy, we cloned the full-length NaGLP and silenced its expression in N. attenuata by expressing a 250-bp fragment in an antisense orientation with an Agrobacterium-based transformation system and by virus-induced gene silencing (VIGS). Homozygous lines harboring a single insert and VIGS plants had significantly reduced constitutive (measured in roots) and elicited NaGLP transcript levels (in leaves). Silencing NaGLP improved M. sexta larval performance and Tupiocoris notatus preference, two native herbivores of N. attenuata. Silencing NaGLP also attenuated the OS-induced hydrogen peroxide (H2O2), diterpene glycosides, and trypsin proteinase inhibitor responses, which may explain the observed susceptibility of antisense or VIGS plants to herbivore attack and increased nicotine contents, but did not influence the OS-elicited jasmonate and salicylate bursts, or the release of the volatile organic compounds (limonene, cis-α-bergamotene, and germacrene-A) that function as an indirect defense. This suggests that NaGLP is involved in H2O2 production and might also be related to ethylene production and/or perception, which in turn influences the defense responses of N. attenuata via H2O2 and ethylene-signaling pathways. PMID:16461381

Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris.

Science.gov (United States)

Pellny, Till K; Van Aken, Olivier; Dutilleul, Christelle; Wolff, Tonja; Groten, Karin; Bor, Melike; De Paepe, Rosine; Reyss, Agnès; Van Breusegem, Frank; Noctor, Graham; Foyer, Christine H

2008-06-01

Mitochondrial electron transport pathways exert effects on carbon-nitrogen (C/N) relationships. To examine whether mitochondria-N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered 'nitrate-sensing' phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.

Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

Science.gov (United States)

Vatsa, Parul; Chiltz, Annick; Luini, Estelle; Vandelle, Elodie; Pugin, Alain; Roblin, Gabriel

2011-07-01

The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²⁺](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 μM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²⁺](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl₃, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²⁺](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Taking Leave?

CERN Multimedia

2000-01-01

Planning a holiday? Then if you're a member of the personnel, you'll need to use the Laboratory's new leave system that will be put in place on 1 October. Leave allocations don't change - you are entitled to just as much holiday as before - but instead of being credited annually, your leave will be credited on a monthly basis, and this information will be communicated on your salary slip. The reason for the change is that with the various new leave schemes such as Recruitment by Saved Leave (RSL) and the Progressive Retirement Programme (PRP), a streamlined procedure was required for dealing with all kinds of leave. In the new system, each member of the personnel will have leave accounts to which leave will be credited monthly from the payroll and debited each time an absence is registered in the CERN Electronic Document Handling system (EDH). Leave balances will appear on monthly pay slips, and full details of leave transactions and balances will be available through EDH at all times. As the leave will be c...

Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

Science.gov (United States)

Mitter, Neena; Koundal, Vikas; Williams, Sarah; Pappu, Hanu

2013-01-01

Background Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. Principal Findings Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. Significance Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral

Differential expression of tomato spotted wilt virus-derived viral small RNAs in infected commercial and experimental host plants.

Directory of Open Access Journals (Sweden)

Neena Mitter

Full Text Available BACKGROUND: Viral small RNAs (vsiRNAs in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV, a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. PRINCIPAL FINDINGS: Tomato spotted wilt virus (TSWV-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1 higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. SIGNIFICANCE: Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsi

Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata

Czech Academy of Sciences Publication Activity Database

Bruetting, C.; Schaefer, N.; Vaňková, Radomíra; Gase, K.; Baldwin, I.T.; Meldau, S.

2017-01-01

Roč. 89, č. 1 (2017), s. 15-30 ISSN 0960-7412 R&D Projects: GA MŠk LD14120 Institutional support: RVO:61389030 Keywords : proteinase-inhibitor production * plant defense * arabidopsis-thaliana * leaf senescence * insect interactions * tobacco plants * jasmonic acid * manduca-sexta * cis-zeatin * responses * cytokinins * optimal defense * herbivores * inducible defense * Nicotiana attenuata * Manduca sexta * plant development * immunosenescence * phytohormones Subject RIV: ED - Physiology OBOR OECD: Plant sciences, botany Impact factor: 5.901, year: 2016

Supplementary Material for: CRISPR/Cas9-mediated viral interference in plants

KAUST Repository

Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shakila; Tashkandi, Manal; Mahfouz, Magdy

2015-01-01

Abstract Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

CRISPR/Cas9-mediated viral interference in plants

KAUST Repository

Ali, Zahir

2015-11-11

Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

Leaf curl diseases of two solanaceous species in Southwest Arabia are caused by a monopartite begomovirus evolutionarily most closely related to a species from the Nile Basin and unique suite of betasatellites

KAUST Repository

Idris, Ali

2012-10-01

The complete genome of 2780 bases was amplified using rolling circle amplification, and cloned, and sequenced for two distinct strains of the monopartite begomovirus Tomato leaf curl Sudan virus (ToLCSDV). The two strains shared 86-91% identity with the previously described ToLCSDV from the Nile Basin, and 90-91% identity with one another. One strain was cloned from symptomatic tomato plants from Tihamah (ToLCSDV-YE[YE:Tih:05]) while the other was cloned from symptomatic tobacco plants collected from Wadi Hadramaut (ToLCSDV-YE[YE:Had:89]). A distinct full-length betasatellite molecule (1352 bases) was cloned from the respective field-infected tomato and tobacco plants. Agro-inoculation of tomato and Nicotiana benthamiana plants with cloned partial tandem repeats of ToLCSDV-YE[YE:Tih11:05]) and the associated betasatellite, Tomato leaf curl Yemen betasatellite (ToLCYEB-[Tih:tom:137:05]), resulted in the reproduction of leaf curl disease symptoms in test plants like those observed in the field-infected plants. The betasatellite contributed to symptom severity in N. benthamiana test plants when it was co-inoculated with ToLCSDV-YE, compared to the milder symptoms that were observed in tobacco plants infected with the helper virus alone. © 2012 Elsevier B.V.

Leaf curl diseases of two solanaceous species in Southwest Arabia are caused by a monopartite begomovirus evolutionarily most closely related to a species from the Nile Basin and unique suite of betasatellites

KAUST Repository

Idris, Ali; Abdullah, N.; Brown, Judith K.

2012-01-01

The complete genome of 2780 bases was amplified using rolling circle amplification, and cloned, and sequenced for two distinct strains of the monopartite begomovirus Tomato leaf curl Sudan virus (ToLCSDV). The two strains shared 86-91% identity with the previously described ToLCSDV from the Nile Basin, and 90-91% identity with one another. One strain was cloned from symptomatic tomato plants from Tihamah (ToLCSDV-YE[YE:Tih:05]) while the other was cloned from symptomatic tobacco plants collected from Wadi Hadramaut (ToLCSDV-YE[YE:Had:89]). A distinct full-length betasatellite molecule (1352 bases) was cloned from the respective field-infected tomato and tobacco plants. Agro-inoculation of tomato and Nicotiana benthamiana plants with cloned partial tandem repeats of ToLCSDV-YE[YE:Tih11:05]) and the associated betasatellite, Tomato leaf curl Yemen betasatellite (ToLCYEB-[Tih:tom:137:05]), resulted in the reproduction of leaf curl disease symptoms in test plants like those observed in the field-infected plants. The betasatellite contributed to symptom severity in N. benthamiana test plants when it was co-inoculated with ToLCSDV-YE, compared to the milder symptoms that were observed in tobacco plants infected with the helper virus alone. © 2012 Elsevier B.V.

5 CFR 630.1204 - Intermittent leave or reduced leave schedule.

Science.gov (United States)

2010-01-01

... insurance, health benefits, retirement coverage, and leave accrual). (e) The agency shall determine the... REGULATIONS ABSENCE AND LEAVE Family and Medical Leave § 630.1204 Intermittent leave or reduced leave schedule... reduced leave schedule unless the employee and the agency agree to do so. (b) Leave under § 630.1203(a) (3...

Differential expression of catalase genes in Nicotiana plumbaginifolia (L.).

Science.gov (United States)

Willekens, H; Langebartels, C; Tiré, C; Van Montagu, M; Inzé, D; Van Camp, W

1994-10-25

We have analyzed the expression of three catalase (Cat; EC 1.11.1.6) genes from Nicotiana plumbaginifolia by means of RNA blot and in situ hybridizations. Our data demonstrate that the expression of each catalase is associated with a particular H2O2-producing process. Cat1 appears to be specifically involved in the scavenging of photorespiratory H2O2 and is under control of a circadian rhythm, Cat2 is uniformly expressed in different organs with a cellular preference for vascular tissues, and the expression profile of Cat3 points to a role in glyoxysomal processes. Differential expression of these catalases is also manifested in response to temperature changes. DNA sequence comparison with other dicotyledonous catalases led to the identification of at least three distinct classes, which indicates that the functional organization of catalases is generally conserved in dicotyledonous plants.

Indoor and outdoor genotoxic load detected by the Comet assay in leaves of Nicotiana tabacum cultivars Bel B and Bel W3.

Science.gov (United States)

Restivo, Francesco Maria; Laccone, Maria Concetta; Buschini, Annamaria; Rossi, Carlo; Poli, Paola

2002-03-01

Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. The use of plant material for a more complete picture of environmental health appears to be particularly appealing. Here we validate a previous plant-adapted Comet assay on leaf tissues of Nicotiana tabacum cultivars Bel B and Bel W3. The effects of H(2)O(2) on DNA damage in Bel B and Bel W3 agree with the hypothesis that some component of the machinery that protects DNA integrity from oxidative stress may be impaired in cv. Bel W3. Exposure in the field on sunny summer days (peak ozone concentration >80 p.p.b.) showed significantly higher DNA damage in cv. Bel W3 if plants were collected and subjected to the Comet assay when the air ozone concentration was reaching its peak value, but not when plants were sampled early in the morning and hence after a period of low ozone concentration. The different results suggest that Bel W3 possesses a less efficient recovery apparatus that requires a longer period of activity to be effective and/or is less protected against reactive oxygen species production during exposure to ozone. However, it cannot be excluded that the increase in mean DNA damage is the result of the presence of a genotoxic agent(s) other than ozone. Interestingly, Bel W3 also appears to be more responsive, compared with Bel B, when exposed to ambient indoor pollutants. The use of cv. Bel W3 increases the sensitivity of the assay under both indoor and field conditions. However, different classes of mutagens should be tested to define the range of profitable utilization of this tobacco cultivar for environmental genotoxicity detection.

Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

Science.gov (United States)

Wang, Yan; Cordewener, Jan H G; America, Antoine H P; Shan, Weixing; Bouwmeester, Klaas; Govers, Francine

2015-09-01

L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors.

Science.gov (United States)

Wei, Hai-Lei; Collmer, Alan

2017-12-25

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Ageratum enation virus—A Begomovirus of Weeds with the Potential to Infect Crops

Directory of Open Access Journals (Sweden)

Muhammad Tahir

2015-02-01

Full Text Available Samples of two Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India, and Pakistan—including the isolates identified here and India (isolates occurring only in India strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch’s postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.

Ageratum enation virus—A Begomovirus of Weeds with the Potential to Infect Crops

Science.gov (United States)

Tahir, Muhammad; Amin, Imran; Haider, Muhammad Saleem; Mansoor, Shahid; Briddon, Rob W.

2015-01-01

Samples of two Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India, and Pakistan—including the isolates identified here) and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch’s postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite. PMID:25674770

Ageratum enation virus-a begomovirus of weeds with the potential to infect crops.

Science.gov (United States)

Tahir, Muhammad; Amin, Imran; Haider, Muhammad Saleem; Mansoor, Shahid; Briddon, Rob W

2015-02-10

Samples of two Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India, and Pakistan-including the isolates identified here) and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch's postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.

Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus.

Science.gov (United States)

Yazhisai, Uthaman; Rajagopalan, Prem Anand; Raja, Joseph A J; Chen, Tsung-Chi; Yeh, Shyi-Dong

2015-08-01

Tospoviruses cause severe damages to important crops worldwide. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5' half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15-17 transgenic N. benthamiana lines carrying individual transgenes were evaluated against WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops.

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

Science.gov (United States)

McClure, B; Mou, B; Canevascini, S; Bernatzky, R

1999-11-09

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

REMINDER Saved Leave Scheme (SLS) : Simplified procedure for the transfer of leave to saved leave accounts

CERN Multimedia

HR Division

2001-01-01

As part of the process of streamlining procedures, the HR and AS Divisions have jointly developed a system whereby annual and compensatory leave will henceforth be automatically transferred1) to saved leave accounts. Under the provisions of the voluntary saved leave scheme (SLS), a maximum total of 10 days'2)Previously, every person taking part in the scheme has been individually issued with a form for the purposes of requesting the transfer of leave to the leave account and the transfer has then had to be done manually by HR Division. To streamline the procedure, unused leave of all those taking part in the saved leave scheme at the closure of the leave-year accounts will henceforth be transferred automatically to the saved leave account on that date. This simplification is in the interest of all parties concerned. This automatic transfer procedure has a number of advantages for participants in the SLS scheme. First, staff members will no longer have to take any administrative steps. Secondly, the new proced...

Parental leave: the impact of recent legislation on parents' leave taking.

Science.gov (United States)

Han, Wen-Jui; Waldfogel, Jane

2003-02-01

We use data from the Survey of Income and Program Participation to examine the impact of leave entitlements on unpaid leave usage by men and women after the birth of a child from 1991 to 1999. The results indicate that legislation providing the right to unpaid leave has not affected men's leave usage. The results for women are mixed: in some specifications, leave entitlements are associated with increased leave taking or longer leaves, but the results depend on how we define leave coverage. Our results point to the limited impact of unpaid leave policies and the potential importance of paid-leave policies.

Cauliflower mosaic virus protein P6 inhibits signaling responses to salicylic acid and regulates innate immunity.

Directory of Open Access Journals (Sweden)

Andrew J Love

Full Text Available Cauliflower mosaic virus (CaMV encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA- and jasmonic acid (JA-dependent signaling and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst. Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants

Phosphoenolpyruvate carboxylase, NADP-malic enzyme, and pyruvate, phosphate dikinase are involved in the acclimation of Nicotiana tabacum L. to drought stress

Czech Academy of Sciences Publication Activity Database

Doubnerová-Hýsková, V.; Miedzińska, L.; Dobrá, Jana; Vaňková, Radomíra; Ryšlavá, H.

2014-01-01

Roč. 171, č. 5 (2014), s. 19-25 ISSN 0176-1617 R&D Projects: GA MŠk 1M0505 Institutional support: RVO:61389030 Keywords : Drought * NADP-malic enzyme * Nicotiana tabacum L. Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.557, year: 2014

Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum.

Directory of Open Access Journals (Sweden)

Shan Xin

Full Text Available Apoplastic ascorbate oxidase (AO plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1 gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana showed that the GhAO1 promoter exhibited high activity, driving strong reporter gene expression in tobacco trichomes, leaves and roots. Promoter 5'-deletion analysis demonstrated that truncated GhAO1 promoters with serial 5'-end deletions had different GUS activities. A 360-bp fragment was sufficient to activate GUS expression. The P-1040 region had less GUS activity than the P-720 region, suggesting that the 320-bp region from nucleotide -720 to -1040 might include a cis-element acting as a silencer. Interestingly, an auxin-responsive cis-acting element (TGA-element was uncovered in the promoter. To analyze the function of the TGA-element, tobacco leaves transformed with promoters with different 5' truncations were treated with indole-3-acetic acid (IAA. Tobacco leaves transformed with the promoter regions containing the TGA-element showed significantly increased GUS activity after IAA treatment, implying that the fragment spanning nucleotides -1760 to -1600 (which includes the TGA-element might be a key component for IAA responsiveness. Analyses of the AO promoter region and AO expression pattern in Gossypium arboreum (Ga, diploid cotton with an AA genome, Gossypium raimondii (Gr, diploid cotton with a DD genome and Gossypium hirsutum (Gh, tetraploid cotton with an AADD genome indicated that AO promoter activation and AO transcription were detected together only in D genome/sub-genome (Gr and Gh cotton. Taken together, these results suggest that the 1,920-bp GhAO1 promoter is a functional sequence

Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum.

Science.gov (United States)

Xin, Shan; Tao, Chengcheng; Li, Hongbin

2016-01-01

Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibited high activity, driving strong reporter gene expression in tobacco trichomes, leaves and roots. Promoter 5'-deletion analysis demonstrated that truncated GhAO1 promoters with serial 5'-end deletions had different GUS activities. A 360-bp fragment was sufficient to activate GUS expression. The P-1040 region had less GUS activity than the P-720 region, suggesting that the 320-bp region from nucleotide -720 to -1040 might include a cis-element acting as a silencer. Interestingly, an auxin-responsive cis-acting element (TGA-element) was uncovered in the promoter. To analyze the function of the TGA-element, tobacco leaves transformed with promoters with different 5' truncations were treated with indole-3-acetic acid (IAA). Tobacco leaves transformed with the promoter regions containing the TGA-element showed significantly increased GUS activity after IAA treatment, implying that the fragment spanning nucleotides -1760 to -1600 (which includes the TGA-element) might be a key component for IAA responsiveness. Analyses of the AO promoter region and AO expression pattern in Gossypium arboreum (Ga, diploid cotton with an AA genome), Gossypium raimondii (Gr, diploid cotton with a DD genome) and Gossypium hirsutum (Gh, tetraploid cotton with an AADD genome) indicated that AO promoter activation and AO transcription were detected together only in D genome/sub-genome (Gr and Gh) cotton. Taken together, these results suggest that the 1,920-bp GhAO1 promoter is a functional sequence with a

Back-transmission of a virus associated with apple stem pitting and pear vein yellows from Nicotiana occidentalis to apple and pear indicators

NARCIS (Netherlands)

Leone, G.; Lindner, J.L.; Jongedijk, G.; Meer, van der F.

1995-01-01

The successful back-transmission of the mechanically transmissible virus associated with apple stem pitting and pear vein yellows, from Nicotiana occidentalis to apple seedlings "Golden Delicious" under greenhouse conditions is reported. This result enabled a field experiment where isolates of apple

29 CFR 825.202 - Intermittent leave or reduced leave schedule.

Science.gov (United States)

2010-07-01

... leave schedule is a leave schedule that reduces an employee's usual number of working hours per workweek, or hours per workday. A reduced leave schedule is a change in the employee's schedule for a period of... 29 Labor 3 2010-07-01 2010-07-01 false Intermittent leave or reduced leave schedule. 825.202...

Characterization of Genes Encoding Key Enzymes Involved in Anthocyanin Metabolism of Kiwifruit during Storage Period.

Science.gov (United States)

Li, Boqiang; Xia, Yongxiu; Wang, Yuying; Qin, Guozheng; Tian, Shiping

2017-01-01

'Hongyang' is a red fleshed kiwifruit with high anthocyanin content. In this study, we mainly investigated effects of different temperatures (25 and 0°C) on anthocyanin biosynthesis in harvested kiwifruit, and characterized the genes encoding key enzymes involved in anthocyanin metabolism, as well as evaluated the mode of the action, by which low temperature regulates anthocyanin accumulation in 'Hongyang' kiwifruit during storage period. The results showed that low temperature could effectively enhance the anthocyanin accumulation of kiwifruit in the end of storage period (90 days), which related to the increase in mRNA levels of ANS1, ANS2, DRF1, DRF2 , and UGFT2 . Moreover, the transcript abundance of MYBA1-1 and MYB5-1 , the genes encoding an important component of MYB-bHLH-WD40 (MBW) complex, was up-regulated, possibly contributing to the induction of specific anthocyanin biosynthesis genes under the low temperature. To further investigate the roles of AcMYB5-1/5-2/A1-1 in regulation of anthocyanin biosynthesis, genes encoding the three transcription factors were transiently transformed in Nicotiana benthamiana leaves. Overexpression of AcMYB5-1/5-2/A1-1 activated the gene expression of NtANS and NtDFR in tobacco. Our results suggested that low temperature storage could stimulate the anthocyanin accumulation in harvested kiwifruit via regulating several structural and regulatory genes involved in anthocyanin biosynthesis.

Genome-wide analysis of autophagy-related genes in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt.

Science.gov (United States)

Wei, Yunxie; Liu, Wen; Hu, Wei; Liu, Guoyin; Wu, Chunjie; Liu, Wei; Zeng, Hongqiu; He, Chaozu; Shi, Haitao

2017-08-01

MaATG8s play important roles in hypersensitive-like cell death and immune response, and autophagy is essential for disease resistance against Foc in banana. Autophagy is responsible for the degradation of damaged cytoplasmic constituents in the lysosomes or vacuoles. Although the effects of autophagy have been extensively revealed in model plants, the possible roles of autophagy-related gene in banana remain unknown. In this study, 32 MaATGs were identified in the draft genome, and the profiles of several MaATGs in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) were also revealled. We found that seven MaATG8s were commonly regulated by Foc. Through transient expression in Nicotiana benthamiana leaves, we highlight the novel roles of MaATG8s in conferring hypersensitive-like cell death, and MaATG8s-mediated hypersensitive response-like cell death is dependent on autophagy. Notablly, autophagy inhibitor 3-methyladenine (3-MA) treatment resulted in decreased disease resistance in response to Foc4, and the effect of 3-MA treatment could be rescued by exogenous salicylic acid, jasmonic acid and ethylene, indicating the involvement of autophagy-mediated plant hormones in banana resistance to Fusarium wilt. Taken together, this study may extend our understanding the putative role of MaATG8s in hypersensitive-like cell death and the essential role of autophagy in immune response against Foc in banana.

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

Science.gov (United States)

Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

2017-01-01

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

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Kiwamu Hyodo

2015-05-01

Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

Characterization and functional analysis of Calmodulin and Calmodulin-like genes in Fragaria vesca

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Kai Zhang

2016-12-01

Full Text Available Calcium is a universal messenger that is involved in the modulation of diverse developmental and adaptive processes in response to various stimuli. Calmodulin (CaM and calmodulin-like (CML proteins are major calcium sensors in all eukaryotes, and they have been extensively investigated for many years in plants and animals. However, little is known about CaMs and CMLs in woodland strawberry (Fragaria vesca. In this study, we performed a genome-wide analysis of the strawberry genome and identified 4 CaM and 36 CML genes. Bioinformatics analyses, including gene structure, phylogenetic tree, synteny and three-dimensional model assessments, revealed the conservation and divergence of FvCaMs and FvCMLs, thus providing insight regarding their functions. In addition, the transcript abundance of four FvCaM genes and the four most related FvCML genes were examined in different tissues and in response to multiple stress and hormone treatments. Moreover, we investigated the subcellular localization of several FvCaMs and FvCMLs, revealing their potential interactions based on the localizations and potential functions. Furthermore, overexpression of five FvCaM and FvCML genes could not induce a hypersensitive response, but four of the five genes could increase resistance to Agrobacterium tumefaciens in Nicotiana benthamiana leaves. This study provides evidence for the biological roles of FvCaM and CML genes, and the results lay the foundation for future functional studies of these genes.

Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum).

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Ono, Nadia N; Qin, Xiaoqiong; Wilson, Alexander E; Li, Gang; Tian, Li

2016-01-01

Hydrolyzable tannins (HTs) play important roles in plant herbivore deterrence and promotion of human health. A critical step in HT production is the formation of 1-O-galloyl-β-D-glucopyranoside (β-glucogallin, ester-linked gallic acid and glucose) by a UDP-glucosyltransferase (UGT) activity. We cloned and biochemically characterized four candidate UGTs from pomegranate (Punica granatum), of which only UGT84A23 and UGT84A24 exhibited β-glucogallin forming activities in enzyme assays. Although overexpression and single RNAi knockdown pomegranate hairy root lines of UGT84A23 or UGT84A24 did not lead to obvious alterations in punicalagin (the prevalent HT in pomegranate) accumulation, double knockdown lines of the two UGTs resulted in largely reduced levels of punicalagins and bis-hexahydroxydiphenyl glucose isomers. An unexpected accumulation of galloyl glucosides (ether-linked gallic acid and glucose) was also detected in the double knockdown lines, suggesting that gallic acid was utilized by an unidentified UGT activity for glucoside formation. Transient expression in Nicotiana benthamiana leaves and immunogold labeling in roots of pomegranate seedlings collectively indicated cytosolic localization of UGT84A23 and UGT84A24. Overall, functional characterization and localization of UGT84A23 and UGT84A24 open up opportunities for further understanding the regulatory control of HT metabolism in plants and its coordination with other biochemical pathways in the metabolic network.

(1,3;1,4)-β-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure.

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Dimitroff, George; Little, Alan; Lahnstein, Jelle; Schwerdt, Julian G; Srivastava, Vaibhav; Bulone, Vincent; Burton, Rachel A; Fincher, Geoffrey B

2016-04-05

Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-β-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4)-linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-β-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-β-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.

The molecular characterisation of a Sida-infecting begomovirus from Jamaica.

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Stewart, Cheryl; Kon, Tatsuya; Rojas, Maria; Graham, André; Martin, Darren; Gilbertson, Robert; Roye, Marcia

2014-02-01

The complete DNA sequence of both genome components of a new begomovirus (Sida golden mosaic Buckup virus-[Jamaica:St. Elizabeth:2004]; SiGMBuV-[JM:SE:04]) was determined from a field-infected Sida sp. sample from Buckup, St. Elizabeth, Jamaica. Phylogenetically, both genome components of SiGMBuV-[JM:SE:04] are most closely related to malvaceous weed-infecting Floridian and Mexican begomoviruses. Its DNA-B is a recombinant molecule, the majority of which was derived from a virus resembling Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005] (SiYMYuV-[MX:Yuc:05]), while nucleotides 43-342 were derived from a virus resembling Sida golden mosaic virus-[United States of America:Florida] (SiGMV-[US:Flo]). Symptomatic infectivity of our cloned SiGMBuV-[JM:SE:04] components was confirmed in Nicotiana benthamiana.

Diverse amino acid changes at specific positions in the N-terminal region of the coat protein allow Plum pox virus to adapt to new hosts.

Science.gov (United States)

Carbonell, Alberto; Maliogka, Varvara I; Pérez, José de Jesús; Salvador, Beatriz; León, David San; García, Juan Antonio; Simón-Mateo, Carmen

2013-10-01

Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3' region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana spp. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus spp.

High-efficiency Agrobacterium rhizogenes-mediated transformation of heat inducible sHSP18.2-GUS in Nicotiana tabacum.

Science.gov (United States)

Chen, Shih-Cheng; Liu, Hui-Wen; Lee, Kung-Ta; Yamakawa, Takashi

2007-01-01

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 microM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42 degrees C heat treatment, and the expressed GUS specific activity was 7-26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

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Lamotte, Olivier; Courtois, Cécile; Dobrowolska, Grazyna; Besson, Angélique; Pugin, Alain; Wendehenne, David

2006-04-15

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

Pollination ecology of the invasive tree tobacco Nicotiana glauca: comparisons across native and non-native ranges

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Jeff Ollerton

2012-10-01

Full Text Available Interactions with pollinators are thought to play a significant role in determining whether plant species become invasive, and ecologically generalised species are predicted to be more likely to invade than more specialised species. Using published and unpublished data we assessed the floral biology and pollination ecology of the South American native Nicotiana glauca (Solanaceae which has become a significant invasive of semi-arid parts of the world. In regions where specialised bird pollinators are available, for example hummingbirds in California and sunbirds in South Africa and Israel, N. glauca interacts with these local pollinators and sets seed by both out-crossing and selfing. In areas where there are no such birds, such as the Canary Islands and Greece, abundant viable seed is set by selfing, facilitated by the shorter stigma-anther distance compared to plants in native populations. Surprisingly, in these areas without pollinating birds, the considerable nectar resources are only rarely exploited by other flower visitors such as bees or butterflies, either legitimately or by nectar robbing. We conclude that Nicotiana glauca is a successful invasive species outside of its native range, despite its functionally specialised hummingbird pollination system, because it has evolved to become more frequently self pollinating in areas where it is introduced. Its invasion success is not predictable from what is known of its interactions with pollinators in its home range.

Functional Mitochondrial Complex I Is Required by Tobacco Leaves for Optimal Photosynthetic Performance in Photorespiratory Conditions and during Transients1

Science.gov (United States)

Dutilleul, Christelle; Driscoll, Simon; Cornic, Gabriel; De Paepe, Rosine; Foyer, Christine H.; Noctor, Graham

2003-01-01

The importance of the mitochondrial electron transport chain in photosynthesis was studied using the tobacco (Nicotiana sylvestris) mutant CMSII, which lacks functional complex I. Rubisco activities and oxygen evolution at saturating CO2 showed that photosynthetic capacity in the mutant was at least as high as in wild-type (WT) leaves. Despite this, steady-state photosynthesis in the mutant was reduced by 20% to 30% at atmospheric CO2 levels. The inhibition of photosynthesis was alleviated by high CO2 or low O2. The mutant showed a prolonged induction of photosynthesis, which was exacerbated in conditions favoring photorespiration and which was accompanied by increased extractable NADP-malate dehydrogenase activity. Feeding experiments with leaf discs demonstrated that CMSII had a lower capacity than the WT for glycine (Gly) oxidation in the dark. Analysis of the postillumination burst in CO2 evolution showed that this was not because of insufficient Gly decarboxylase capacity. Despite the lower rate of Gly metabolism in CMSII leaves in the dark, the Gly to Ser ratio in the light displayed a similar dependence on photosynthesis to the WT. It is concluded that: (a) Mitochondrial complex I is required for optimal photosynthetic performance, despite the operation of alternative dehydrogenases in CMSII; and (b) complex I is necessary to avoid redox disruption of photosynthesis in conditions where leaf mitochondria must oxidize both respiratory and photorespiratory substrates simultaneously. PMID:12529534

Propuesta de medio de cultivo para el estudio de Phytophthora Nicotianae Breda de Haan

OpenAIRE

Vaillant Flores, Daymara I; González García, Marleny; Ramírez Ochoa, Rebeca

2013-01-01

El género Phytophthora representa un grupo difícil de aislar y conservar. Por lo que se han desarrollado varios medios de cultivo para su estudio. Se propone al medio Agar-tabaco como alternativa para el crecimiento y desarrollo de P. nicotianae. Para realizar este trabajo se emplearon dos cepas: Pp1 y Pp6 aislados de tabaco y piña respectivamente. Ambas se sembraron en discos en agar-tabaco, y se comparó con agar tomate y agar harina de maíz. Se determinó el crecimiento lineal del hongo, las...

Negative leave balances

CERN Multimedia

Human Resources Department

2005-01-01

Members of the personnel entitled to annual leave and, where appropriate, saved leave and/or compensatory leave are requested to take note of the new arrangements described below, which were recommended by the Standing Concertation Committee (SCC) at its meeting on 1Â September 2005 and subsequently approved by the Director-General. The changes do not apply to members of the personnel participating in the Progressive Retirement Programme (PRP) or the Part-time Work as a pre-retirement measure, for whom the specific provisions communicated at the time of joining will continue to apply. Â Negative balances in annual leave, saved leave and/or compensatory leave accounts at the end of the leave year (30th September) and on the date on which bonuses are credited to the saved leave account (31st December): Where members of the personnel have a leave account with a negative balance on 30Â September and/or 31Â December, leave will automatically be transferred from one account to another on the relevant dates i...

Negative leave balances

CERN Multimedia

Human Resources Department

2005-01-01

Members of the personnel entitled to annual leave and, where appropriate, saved leave and/or compensatory leave are requested to take note of the new arrangements described below, which were recommended by the Standing Concertation Committee (SCC) at its meeting on 1 September 2005 and subsequently approved by the Director-General. The changes do not apply to members of the personnel participating in the Progressive Retirement Programme (PRP) or the Part-time Work as a pre-retirement measure, for whom the specific provisions communicated at the time of joining will continue to apply.  Negative balances in annual leave, saved leave and/or compensatory leave accounts at the end of the leave year (30th September) and on the date on which bonuses are credited to the saved leave account (31st December): Where members of the personnel have a leave account with a negative balance on 30 September and/or 31 December, leave will automatically be transferred from one account to another on the relevant dates in or...

Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis Flowers[W][OPEN

Science.gov (United States)

Ginglinger, Jean-François; Boachon, Benoit; Höfer, René; Paetz, Christian; Köllner, Tobias G.; Miesch, Laurence; Lugan, Raphael; Baltenweck, Raymonde; Mutterer, Jérôme; Ullmann, Pascaline; Beran, Franziska; Claudel, Patricia; Verstappen, Francel; Fischer, Marc J.C.; Karst, Francis; Bouwmeester, Harro; Miesch, Michel; Schneider, Bernd; Gershenzon, Jonathan; Ehlting, Jürgen; Werck-Reichhart, Danièle

2013-01-01

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (−)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined. PMID:24285789

Identification and characterisation of a highly divergent geminivirus: evolutionary and taxonomic implications.

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Bernardo, Pauline; Golden, Michael; Akram, Mohammad; Naimuddin; Nadarajan, Nagaswamy; Fernandez, Emmanuel; Granier, Martine; Rebelo, Anthony G; Peterschmitt, Michel; Martin, Darren P; Roumagnac, Philippe

2013-10-01

During a large scale "non a priori" survey in 2010 of South African plant-infecting single stranded DNA viruses, a highly divergent geminivirus genome was isolated from a wild spurge, Euphorbia caput-medusae. In addition to being infectious in E. caput-medusae, the cloned viral genome was also infectious in tomato and Nicotiana benthamiana. The virus, named Euphorbia caput-medusae latent virus (EcmLV) due to the absence of infection symptoms displayed by its natural host, caused severe symptoms in both tomato and N. benthamiana. The genome organisation of EcmLV is unique amongst geminiviruses and it likely expresses at least two proteins without any detectable homologues within public sequence databases. Although clearly a geminivirus, EcmLV is so divergent that we propose its placement within a new genus that we have tentatively named Capulavirus. Using a set of highly divergent geminiviruses genomes, it is apparent that recombination has likely been a primary process in the genus-level diversification of geminiviruses. It is also demonstrated how this insight, taken together with phylogenetic analyses of predicted coat protein and replication associated protein (Rep) amino acid sequences indicate that the most recent common ancestor of the geminiviruses was likely a dicot-infecting virus that, like modern day mastreviruses and becurtoviruses, expressed its Rep from a spliced complementary strand transcript. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

The 'PhenoBox', a flexible, automated, open-source plant phenotyping solution.

Science.gov (United States)

Czedik-Eysenberg, Angelika; Seitner, Sebastian; Güldener, Ulrich; Koemeda, Stefanie; Jez, Jakub; Colombini, Martin; Djamei, Armin

2018-04-05

There is a need for flexible and affordable plant phenotyping solutions for basic research and plant breeding. We demonstrate our open source plant imaging and processing solution ('PhenoBox'/'PhenoPipe') and provide construction plans, source code and documentation to rebuild the system. Use of the PhenoBox is exemplified by studying infection of the model grass Brachypodium distachyon by the head smut fungus Ustilago bromivora, comparing phenotypic responses of maize to infection with a solopathogenic Ustilago maydis (corn smut) strain and effector deletion strains, and studying salt stress response in Nicotiana benthamiana. In U. bromivora-infected grass, phenotypic differences between infected and uninfected plants were detectable weeks before qualitative head smut symptoms. Based on this, we could predict the infection outcome for individual plants with high accuracy. Using a PhenoPipe module for calculation of multi-dimensional distances from phenotyping data, we observe a time after infection-dependent impact of U. maydis effector deletion strains on phenotypic response in maize. The PhenoBox/PhenoPipe system is able to detect established salt stress responses in N. benthamiana. We have developed an affordable, automated, open source imaging and data processing solution that can be adapted to various phenotyping applications in plant biology and beyond. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death.

Science.gov (United States)

Yu, Xiaoli; Tang, Junli; Wang, Qunqing; Ye, Wenwu; Tao, Kai; Duan, Shuyi; Lu, Chenchen; Yang, Xinyu; Dong, Suomeng; Zheng, Xiaobo; Wang, Yuanchao

2012-10-01

• The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen's full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway

DEFF Research Database (Denmark)

Andersen-Ranberg, Johan; Kongstad, Kenneth Thermann; Nafisi, Majse

2017-01-01

unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway...

Genetic and Biochemical Analysis of Intragenic Complementation Events among Nitrate Reductase Apoenzyme-Deficient Mutants of Nicotiana Plumbaginifolia

OpenAIRE

Pelsy, F.; Gonneau, M.

1991-01-01

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These obser...

Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

Science.gov (United States)

Moriwaki, M; Yamakawa, T; Washino, T; Kodama, T; Igarashi, Y

1999-01-01

The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae.

Science.gov (United States)

Lim, K Yoong; Kovarik, Ales; Matyasek, Roman; Chase, Mark W; Knapp, Sandra; McCarthy, Elizabeth; Clarkson, James J; Leitch, Andrew R

2006-12-01

Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.

Synchronous high-resolution phenotyping of leaf and root growth in Nicotiana tabacum over 24-h periods with GROWMAP-plant

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Ruts Tom

2013-01-01

Full Text Available Abstract Background Root growth is highly responsive to temporal changes in the environment. On the contrary, diel (24 h leaf expansion in dicot plants is governed by endogenous control and therefore its temporal pattern does not strictly follow diel changes in the environment. Nevertheless, root and shoot are connected with each other through resource partitioning and changing environments for one organ could affect growth of the other organ, and hence overall plant growth. Results We developed a new technique, GROWMAP-plant, to monitor growth processes synchronously in leaf and root of the same plant with a high resolution over the diel period. This allowed us to quantify treatment effects on the growth rates of the treated and non-treated organ and the possible interaction between them. We subjected the root system of Nicotiana tabacum seedlings to three different conditions: constant darkness at 22°C (control, constant darkness at 10°C (root cooling, and 12 h/12 h light–dark cycles at 22°C (root illumination. In all treatments the shoot was kept under the same 12 h/12 h light–dark cycles at 22°C. Root growth rates were found to be constant when the root-zone environment was kept constant, although the root cooling treatment significantly reduced root growth. Root velocity was decreased after light-on and light-off events of the root illumination treatment, resulting in diel root growth rhythmicity. Despite these changes in root growth, leaf growth was not affected substantially by the root-zone treatments, persistently showing up to three times higher nocturnal growth than diurnal growth. Conclusion GROWMAP-plant allows detailed synchronous growth phenotyping of leaf and root in the same plant. Root growth was very responsive to the root cooling and root illumination, while these treatments altered neither relative growth rate nor diel growth pattern in the seedling leaf. Our results that were obtained simultaneously in growing

Ionomic profiling of Nicotiana langsdorffii wild-type and mutant genotypes exposed to abiotic stresses.

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Ardini, Francisco; Soggia, Francesco; Abelmoschi, Maria Luisa; Magi, Emanuele; Grotti, Marco

2013-01-01

To provide a new insight into the response of plants to abiotic stresses, the ionomic profiles of Nicotiana langsdorffii specimens have been determined before and after exposure to toxic metals (chromium) or drought conditions. The plants were genetically transformed with the rat glucocorticoid receptor (GR) or the gene for Agrobacterium rhizogenes rolC, because these modifications are known to produce an imbalance in phytohormone equilibria and a significant change in the defence response of the plant. Elemental profiles were obtained by developing and applying analytical procedures based on inductively coupled plasma atomic emission and mass spectrometry (ICP-AES/MS). In particular, the removal of isobaric interferences affecting the determination of Cr and V by ICP-MS was accomplished by use of a dynamic reaction cell, after optimization of the relevant conditions. The combined use of ICP atomic emission and mass spectrometry enabled the determination of 29 major and trace elements (Ba, Bi, Ca, Cd, Co, Cr, Cu, Eu, Fe, Ga, K, Li, Mg, Mn, Mo, Na, P, Pb, Pt, Rb, S, Sb, Sn, Sr, Te, V, W, Y, and Zn) in different parts of the plants (roots, stems, and leaves), with high accuracy and precision. Multivariate data processing and study of element distribution patterns provided new information about the ionomic response of the target organism to chemical treatment or water stress. Genetic modification mainly affected the distribution of Bi, Cr, Mo, Na, and S, indicating that these elements were involved in biochemical processes controlled by the GR or rolC genes. Chemical stress strongly affected accumulation of several elements (Ba, Ca, Fe, Ga, K, Li, Mn, Mo, Na, P, Pb, Rb, S, Sn, Te, V, and Zn) in different ways; for Ca, Fe, K, Mn, Na, and P the effect was quite similar to that observed in other studies after treatment with other transition elements, for example Cu and Cd. The effect of water deficit was less evident, mainly consisting in a decrease of Ba, Cr, Na, and Sr

Evolution of rDNA in Nicotiana Allopolyploids: A Potential Link between rDNA Homogenization and Epigenetics

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Kovarik, Ales; Dadejova, Martina; Lim, Yoong K.; Chase, Mark W.; Clarkson, James J.; Knapp, Sandra; Leitch, Andrew R.

2008-01-01

Background The evolution and biology of rDNA have interested biologists for many years, in part, because of two intriguing processes: (1) nucleolar dominance and (2) sequence homogenization. We review patterns of evolution in rDNA in the angiosperm genus Nicotiana to determine consequences of allopolyploidy on these processes. Scope Allopolyploid species of Nicotiana are ideal for studying rDNA evolution because phylogenetic reconstruction of DNA sequences has revealed patterns of species divergence and their parents. From these studies we also know that polyploids formed over widely different timeframes (thousands to millions of years), enabling comparative and temporal studies of rDNA structure, activity and chromosomal distribution. In addition studies on synthetic polyploids enable the consequences of de novo polyploidy on rDNA activity to be determined. Conclusions We propose that rDNA epigenetic expression patterns established even in F1 hybrids have a material influence on the likely patterns of divergence of rDNA. It is the active rDNA units that are vulnerable to homogenization, which probably acts to reduce mutational load across the active array. Those rDNA units that are epigenetically silenced may be less vulnerable to sequence homogenization. Selection cannot act on these silenced genes, and they are likely to accumulate mutations and eventually be eliminated from the genome. It is likely that whole silenced arrays will be deleted in polyploids of 1 million years of age and older. PMID:18310159

Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco.

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Cameron, Kimberly D; Teece, Mark A; Smart, Lawrence B

2006-01-01

Cuticular wax deposition and composition affects drought tolerance and yield in plants. We examined the relationship between wax and dehydration stress by characterizing the leaf cuticular wax of tree tobacco (Nicotiana glauca L. Graham) grown under periodic dehydration stress. Total leaf cuticular wax load increased after each of three periods of dehydration stress using a CH2Cl2 extraction process. Overall, total wax load increased 1.5- to 2.5-fold, but composition of the wax was not altered. Homologous series of wax components were classified into organic groups; n-hentriacontane was the largest component (>75%) with alcohols and fatty acids representing drying event. Leaves excised from plants subjected to multiple drying events were more resistant to water loss compared to leaves excised from well-watered plants, indicating that there is a negative relationship between total wax load and epidermal conductance. Lipid transfer proteins (LTPs) are thought to be involved in the transfer of lipids through the extracellular matrix for the formation of cuticular wax. Using northern analysis, a 6-fold increase of tree tobacco LTP gene transcripts was observed after three drying events, providing further evidence that LTP is involved in cuticle deposition. The simplicity of wax composition and the dramatic wax bloom displayed by tree tobacco make this an excellent species in which to study the relationship between leaf wax deposition and drought tolerance.

Solanum americanum: reservoir for Potato virus Y and Cucumber mosaic virus in sweet pepper crops

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Monika Fecury Moura

2014-03-01

Full Text Available Weeds can act as important reservoirs for viruses. Solanum americanum (Black nightshade is a common weed in Brazil and samples showing mosaic were collected from sweet pepper crops to verify the presence of viruses. One sample showed mixed infection between Cucumber mosaic virus (CMV and Potato virus Y (PVY and one sample showed simple infection by PVY. Both virus species were transmitted by plant extract and caused mosaic in tomato (Solanum lycopersicum cv. Santa Clara, sweet pepper (Capsicum annuum cv. Magda, Nicotiana benthamiana and N. tabaccum TNN, and local lesions on Chenopodium quinoa, C. murale and C. amaranticolor. The coat protein sequences for CMV and PVY found in S. americanum are phylogenetically more related to isolates from tomato. We conclude that S. americanum can act as a reservoir for different viruses during and between sweet pepper crop seasons.

Multivariate optimization of a headspace solid-phase microextraction method followed by gas chromatography with mass spectrometry for the determination of terpenes in Nicotiana langsdorffii.

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Ardini, Francisco; Carro, Marina Di; Abelmoschi, Maria Luisa; Grotti, Marco; Magi, Emanuele

2014-07-01

A simple and sensitive procedure based on headspace solid-phase microextraction and gas chromatography with mass spectrometry was developed for the determination of five terpenes (α-pinene, limonene, linalool, α-terpineol, and geraniol) in the leaves of Nicotiana langsdorffii. The microextraction conditions (extraction temperature, equilibration time, and extraction time) were optimized by means of a Doehlert design. The experimental design showed that, for α-pinene and limonene, a low temperature and a long extraction time were needed for optimal extraction, while linalool, α-terpineol, and geraniol required a high temperature and a long extraction time. The chosen compromise conditions were temperature 60°C, equilibration time 15 min and extraction time 50 min. The main analytical figures of the optimized method were evaluated; LODs ranged from 0.07 ng/g (α-pinene) to 8.0 ng/g (geraniol), while intraday and interday repeatability were in the range 10-17% and 9-13%, respectively. Finally, the procedure was applied to in vitro wild-type and transgenic specimens of N. langsdorffii subjected to abiotic stresses (chemical and heat stress). With the exception of geraniol (75-374 ng/g), low concentration levels of terpenes were measured (ng/g level or lower); some interesting variations in terpene concentration induced by abiotic stress were observed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA Sequencing Analysis Reveals Transcriptomic Variations in Tobacco (Nicotiana tabacum Leaves Affected by Climate, Soil, and Tillage Factors

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Bo Lei

2014-04-01

Full Text Available The growth and development of plants are sensitive to their surroundings. Although numerous studies have analyzed plant transcriptomic variation, few have quantified the effect of combinations of factors or identified factor-specific effects. In this study, we performed RNA sequencing (RNA-seq analysis on tobacco leaves derived from 10 treatment combinations of three groups of ecological factors, i.e., climate factors (CFs, soil factors (SFs, and tillage factors (TFs. We detected 4980, 2916, and 1605 differentially expressed genes (DEGs that were affected by CFs, SFs, and TFs, which included 2703, 768, and 507 specific and 703 common DEGs (simultaneously regulated by CFs, SFs, and TFs, respectively. GO and KEGG enrichment analyses showed that genes involved in abiotic stress responses and secondary metabolic pathways were overrepresented in the common and CF-specific DEGs. In addition, we noted enrichment in CF-specific DEGs related to the circadian rhythm, SF-specific DEGs involved in mineral nutrient absorption and transport, and SF- and TF-specific DEGs associated with photosynthesis. Based on these results, we propose a model that explains how plants adapt to various ecological factors at the transcriptomic level. Additionally, the identified DEGs lay the foundation for future investigations of stress resistance, circadian rhythm and photosynthesis in tobacco.

Maternal synthesis of abscisic acid controls seed development and yield in Nicotiana plumbaginifolia.

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Frey, Anne; Godin, Béatrice; Bonnet, Magda; Sotta, Bruno; Marion-Poll, Annie

2004-04-01

The role of maternally derived abscisic acid (ABA) during seed development has been studied using ABA-deficient mutants of Nicotiana plumbaginifolia Viviani. ABA deficiency induced seed abortion, resulting in reduced seed yield, and delayed growth of the remaining embryos. Mutant grafting onto wild-type stocks and reciprocal crosses indicated that maternal ABA, synthesized in maternal vegetative tissues and translocated to the seed, promoted early seed development and growth. Moreover ABA deficiency delayed both seed coat pigmentation and capsule dehiscence. Mutant grafting did not restore these phenotypes, indicating that ABA synthesized in the seed coat and capsule envelope may have a positive effect on capsule and testa maturation. Together these results shed light on the positive role of maternal ABA during N. plumbaginifolia seed development.

Pengaruh Pemberian Fungi Mikoriza Arbuskular (Fma) Terhadap Pertumbuhan Dan Produksi Beberapa Varietas Tembakau (Nicotiana Tabaccum L.) Di Lapangan

OpenAIRE

Sinaga, Parulian; Purba, Edison; Ginting, Jonis

2014-01-01

The growth and yield of a selected tobacco varieties (Nicotiana tabaccum L) treated withmycorhiza fungi arbuskular were evaluated in a field experiment. The aimed of the research was todetermine the effect of mycorhiza fungi on the growth and yield of several varieties of tobacco. Theresearch was conducted outdoor in the field at Balai Benih Penelitian Tembakau Deli Medan withaltitude of about 25 meters above sea level at the beginning of February until May 2012, with twotreatment factors. Th...

The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

Czech Academy of Sciences Publication Activity Database

Doubnerová, V.; Potůčková, L.; Müller, Karel; Ryšlavá, H.

2009-01-01

Roč. 14, 8-9 (2009), s. 893-906 ISSN 0352-5139 Institutional research plan: CEZ:AV0Z50380511 Keywords : NADP-malic enzyme * macroergic compounds * Nicotiana tabacum L. Subject RIV: ED - Physiology Impact factor: 0.820, year: 2009

Trophic relations of Opatrum sabulosum (Coleoptera, Tenebrionidae with leaves of cultivated and uncultivated species of herbaceous plants under laboratory conditions

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Viktor Brygadyrenko

2015-02-01

Full Text Available We carried out a quantitative assessment of the consumption of herbaceous plants by Opatrum sabulosum (Linnaeus, 1761 – a highly significant agricultural pest species. We researched the feeding preferences of this pest species with respect to 33 uncultivated and 22 cultivated plant species. This species of darkling beetle feeds on many uncultivated plant species, including those with hairy leaves and bitter milky sap, such as Scabiosa ucrainca (5.21 mg/specimen/24 hours, Euphorbia virgata (3.45, Solanum nigrum (3.32, Centauria scabiosa (2.47, Lamium album (2.41, Aristolochia clematitis (1.76, Chenopodium album (1.73, Arctium lappa (1.51, Asperula odorata (1.20. A high rate of leaf consumption is also characteristic for cultivated species, for example, Perilla nankinensis (5.05 mg/specimen/24 hours, Lycopersicon esculentum (3.75, Tropaeolum majus (3.29, Nicotiana tabacum (2.66, Rumex acetosa (1.96, Beta vulgaris (1.27. O. sabulosum is capable of feeding on plants which are poisonous to cattle. This species of darkling beetle consumes 95.5% of the cultivated and 48.5% of the uncultivated herbaceous plants researched.

Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves.

Science.gov (United States)

Ji, Xia-Jie; Mao, Xue; Hao, Qing-Ting; Liu, Bao-Ling; Xue, Jin-Ai; Li, Run-Zhi

2018-01-05

The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean ( Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1 , designated as RcWRI1-A and RcWRI1-B . The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues "VYL" that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsis wri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3-4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A . Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves

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Xia-Jie Ji

2018-01-01

Full Text Available The plant-specific WRINKLED1 (WRI1 is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean (Ricinus connunis L., an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1, designated as RcWRI1-A and RcWRI1-B. The open reading frames of RcWRI1-A (1341 bp and RcWRI1-B (1332 bp differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues “VYL” that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsis wri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3–4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A. Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

REMINDER: Saved Leave Scheme (SLS)

CERN Multimedia

2003-01-01

Transfer of leave to saved leave accounts Under the provisions of the voluntary saved leave scheme (SLS), a maximum total of 10 days'* annual and compensatory leave (excluding saved leave accumulated in accordance with the provisions of Administrative Circular No 22B) can be transferred to the saved leave account at the end of the leave year (30 September). We remind you that unused leave of all those taking part in the saved leave scheme at the closure of the leave year accounts is transferred automatically to the saved leave account on that date. Therefore, staff members have no administrative steps to take. In addition, the transfer, which eliminates the risk of omitting to request leave transfers and rules out calculation errors in transfer requests, will be clearly shown in the list of leave transactions that can be consulted in EDH from October 2003 onwards. Furthermore, this automatic leave transfer optimizes staff members' chances of benefiting from a saved leave bonus provided that they ar...

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

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Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

2017-01-01

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia.

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Meyer, C; Pouteau, S; Rouzé, P; Caboche, M

1994-01-01

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after gamma-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 bp insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 bp terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5' and 3' ends of dTnp1 together with a perfect palindrome located after the 5' inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.

Radiosensitivity of Nicotiana protoplasts. Action on cell; cycle effects of low dose and fractionated irradiations; biological repair

International Nuclear Information System (INIS)

Magnien, E.

1981-10-01

Leaf protoplasts of Nicotiana plumbaginifolia and Nicotiana sylvestris demonstrate five main qualities: they can be maintained as haploid lines; they constitute starting populations with a remarkable cytological homogeneity; they show a transient initial lag-phase; they yield very high plating efficiencies and retain permanently a complete differentiation capacity; being derived of a cell wall, they appear well adapted for fusion experiments or enzymatic dosages. The resumption of mitotic activity was followed by cytophotometric measurements, labelling experiments, nuclear sizing and enzymatic assays. The action of 5 Gy gamma-ray irradiations delayed entrance in the S-phase, provoked an otherwise not verified dependency between transcription, translation and protein synthesis, increased nuclear volumes in the G2-phase, and slightly stimulated the activity of a repair enzyme. The plating efficiency was a sensitive end-point which allowed the evaluation of the biological effectiveness of low to medium radiation-doses after gamma-ray and fast neutron irradiations. The neutron dose-RBE relationship increased from 3 to 25 when the dose decreased from 5 Gy to 5 mGy. When fractionated into low single doses only, a neutron dose of 300 mGy markedly increased its biological effectiveness: this phenomenon could not be explained by cell progression, and necessitated additional hypotheses involving other mechanisms in the specific action of low radiation doses. Radiation-induced UDS was measured in presence of aphidicolin. A beta-like DNA-polymerase was shown to be definitely involved in nuclear repair synthesis [fr

Assessment of natural radionuclides concentration from 238U and 232Th series in Virginia and Burley varieties of Nicotiana tabacum L

International Nuclear Information System (INIS)

Silva, Carolina Fernanda da

2015-01-01

Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop production of 2013/2014. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco products varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation (compression, filter and paper) and the temperature variations resulting from the incomplete combustion of tobacco. Tobacco products are extensively used throughout the world, and the most consumed are cigarettes, cigars and narghile. The damaging effects that these products cause to human health are discussed globally, and many surveys are performed with the aim of relating the use of these products with various illnesses. There is a lack of information about the radiological characterization of the tobacco plant both in international and Brazilian literature. The objective of this study was to determine the concentration of radionuclides 238 U, 234 U, 230 Th, 22 '6Ra, 210 Pb and 210 Po, members from the 238 U decay series, and the radionuclides 232 Th and 228 Ra members of the 232 Th decay series in the varieties Burley and Virginia, which are the most cultivated in Brazil. Plants from these varieties were cultivated in pots with organic substrate and fertilizer and also acquired from the producers and analyzed by alpha spectrometry for U and Th isotopes and 210 Po determination, and gross alpha and beta counting, 228 Ra, 226 Ra and 210 Pb determination. The whole plant, from both places, was analyzed; root, stem, leaves, as well as the organic substrate, the fertilizers, and the soil. The results for U and Th isotopes presented values below the detection limits of the methods to the leaves and stems of all plants analyzed, with measurable results only in roots, soil, and substrate. The radionuclides 226 Ra, 228 Ra, 210 Pb, and 210 Po, were determined in most

The Potato Aphid Salivary Effector Me47 Is a Glutathione-S-Transferase Involved in Modifying Plant Responses to Aphid Infestation.

Science.gov (United States)

Kettles, Graeme J; Kaloshian, Isgouhi

2016-01-01

Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae) is a notable pest of solanaceous crops, however, the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here, we describe a novel aphid effector (Me47) which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST). Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae). Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum) by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant-aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs), compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues.

Cloning and expression analysis of cinnamoyl-CoA reductase (CCR) genes in sorghum.

Science.gov (United States)

Li, Jieqin; Fan, Feifei; Wang, Lihua; Zhan, Qiuwen; Wu, Peijin; Du, Junli; Yang, Xiaocui; Liu, Yanlong

2016-01-01

Cinnamoyl-CoA reductase (CCR) is the first enzyme in the monolignol-specific branch of the lignin biosynthetic pathway. In this research, three sorghum CCR genes including SbCCR1, SbCCR2-1 and SbCCR2-2 were cloned and characterized. Analyses of the structure and phylogeny of the three CCR genes showed evolutionary conservation of the functional domains and divergence of function. Transient expression assays in Nicotiana benthamiana leaves demonstrated that the three CCR proteins were localized in the cytoplasm. The expression analysis showed that the three CCR genes were induced by drought. But in 48 h, the expression levels of SbCCR1 and SbCCR2-2 did not differ between CK and the drought treatment; while the expression level of SbCCR2-1 in the drought treatment was higher than in CK. The expression of the SbCCR1 and SbCCR2-1 genes was not induced by sorghum aphid [Melanaphis sacchari (Zehntner)] attack, but SbCCR2-2 was significantly induced by sorghum aphid attack. It is suggested that SbCCR2-2 is involved in the process of pest defense. Absolute quantitative real-time PCR revealed that the three CCR genes were mainly expressed in lignin deposition organs. The gene copy number of SbCCR1 was significantly higher than those of SbCCR2-1 and SbCCR2-2 in the tested tissues, especially in stem. The results provide new insight into the functions of the three CCR genes in sorghum.

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd.

Science.gov (United States)

Bolognesi, A; Polito, L; Olivieri, F; Valbonesi, P; Barbieri, L; Battelli, M G; Carusi, M V; Benvenuto, E; Del Vecchio Blanco, F; Di Maro, A; Parente, A; Di Loreto, M; Stirpe, F

1997-12-01

New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) Bougainvillea spectabilis had an LD50 > 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

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Ouyang Shu

2005-09-01

Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

Expression and biological activity of the cystine knot bioinsecticide PA1b (Pea Albumin 1 Subunit b.

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Vanessa Eyraud

Full Text Available The PA1b (Pea Albumin 1, subunit b peptide is an entomotoxin extract from Legume seeds with lethal activity on several insect pests, such as mosquitoes, some aphids and cereal weevils. This 37 amino-acid cysteine-rich peptide has been, until now, obtained by biochemical purification or chemical synthesis. In this paper, we present our results for the transient production of the peptide in Nicotiana benthamiana by agro-infiltration, with a yield of about 35 µg/g of fresh leaves and maximum production 8 days after infiltration. PA1b is part of the PA1 gene which, after post-translational modifications, encodes two peptides (PA1b and PA1a. We show that transforming tobacco with the PA1b cDNA alone does not result in production of the toxin and, in fact, the entire cDNA is necessary, raising the question of the role of PA1a. We constructed a PA1-cassette, allowing for the quick "cut/paste" of different PA1b mutants within a conserved PA1 cDNA. This cassette enabled us to produce the six isoforms of PA1b which exist in pea seeds. Biological tests revealed that all the isoforms display similar activity, with the exception of one which is inactive. The lack of activity in this isoform led us to conclude that the amphiphilic nature of the peptide is necessary for activity. The possible applications of this expression system for other cysteine-rich biomolecules are discussed.

A plant EPF-type zinc-finger protein, CaPIF1, involved in defence against pathogens.

Science.gov (United States)

Oh, Sang-Keun; Park, Jeong Mee; Joung, Young Hee; Lee, Sanghyeob; Chung, Eunsook; Kim, Soo-Yong; Yu, Seung Hun; Choi, Doil

2005-05-01

SUMMARY To understand better the defence responses of plants to pathogen attack, we challenged hot pepper plants with bacterial pathogens and identified transcription factor-encoding genes whose expression patterns were altered during the subsequent hypersensitive response. One of these genes, CaPIF1 (Capsicum annuum Pathogen-Induced Factor 1), was characterized further. This gene encodes a plant-specific EPF-type protein that contains two Cys(2)/His(2) zinc fingers. CaPIF1 expression was rapidly and specifically induced when pepper plants were challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generated weak CaPIF1 expression. CaPIF1 expression was also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene-releasing compound, and salicylic acid, whereas methyl jasmonate had only moderate effects. CaPIF1 localized to the nuclei of onion epidermis when expressed as a CaPIF1-smGFP fusion protein. Transgenic tobacco plants over-expressing CaPIF1 driven by the CaMV 35S promoter showed increased resistance to challenge with a tobacco-specific pathogen or non-host bacterial pathogens. These plants also showed constitutive up-regulation of multiple defence-related genes. Moreover, virus-induced silencing of the CaPIF1 orthologue in Nicotiana benthamiana enhanced susceptibility to the same host or non-host bacterial pathogens. These observations provide evidence that an EPF-type Cys(2)/His(2) zinc-finger protein plays a crucial role in the activation of the pathogen defence response in plants.

Leaving home in Denmark

DEFF Research Database (Denmark)

Nielsen, Rikke Skovgaard

2015-01-01

The paper focuses on ethnic differences in the timing and patterns of leaving the parental home. Leaving home is a key transition in the life course of the individual, and extensive research has been conducted on the timing and patterns of leaving it. However, ethnic differences in these patterns...... of leaving home. Results showed that while some differences disappeared when controlling for covariates, others persisted, thus indicating ethnic differences in home-leaving patterns. A strong link between leaving home and marriage was substantiated for Turks, but not for Somalis. The home-leaving patterns...... of Somalis were much more similar to those of Danes. Overall, Turkish descendants were similar to Turkish immigrants but with some differentiation. The analyses identified the existence of ethnic differences in home-leaving patterns but also found evidence of a shift towards less traditional patterns, i...

Symptoms on apple and pear indicators after back-transmission from Nicotiana occidentalis confirm the identity of apple stem pitting virus with pear vein yellows virus

NARCIS (Netherlands)

Leone, G.; Lindner, J.L.; Meer, van der F.A.; Schoen, C.D.; Jongedijk, G.

1998-01-01

Isolates of apple stem pitting virus (ASPV) from diseased apple trees were maintained in Nicotiana occidentalis then back-transmitted mechanically from the herbaceous host to apple seedlings and indexed by double budding on apple and pear indicators for the following syndromes: apple stem pitting,

Parental Leave in Denmark

DEFF Research Database (Denmark)

Rostgaard, Tine; Christoffersen, Mogens; Weise, Hanne

This artcle considders the political aims for different leave schemes and reviews studies af these schemes. The use of parental leave is sensitive to the financial loss involved in taking leave: a decrease in the benefit payments has had a significant influence on take-up, while, in general, fami......, families'' loss of income is less if leave is taken up by the mothers. Only few fathers participate in parental leave....

Regular article

African Journals Online (AJOL)

手塚　孝弘

2013-04-10

Apr 10, 2013 ... sionally on the cotyledons and first and second leaves, but generally ... Kanagawa, Japan) and vermiculite (Nittai Co., Osaka, Japan) and were grown in a ..... cDNA clone from genetic tumors formed on a Nicotiana glauca-N.

Management of chili pepper root rot and wilt (caused by Phytophthora nicotianae) by grafting onto resistant rootstock

OpenAIRE

Mourad SAADOUN; Mohamed Bechir ALLAGUI

2013-01-01

Root rot and plant wilting caused by Phytophthora nicotianae is a severe disease of chili pepper (Capsicum annuum L.) in open fields and under greenhouse production in Tunisia. Chili pepper grafting for disease manage- ment is attracting increased interest in recent years. Using the tube grafting technique, different compatible scion/rootstock combinations were obtained with the wild-type pepper SCM334 and the local chili pepper cultivars ‘Beldi’ and ‘Baker’. SCM334 was resistant to P. nicoti...

Regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, and hexose sugars.

Science.gov (United States)

Goldschmidt, E E; Huber, S C

1992-08-01

In the present study, leaves of different plant species were girdled by the hot wax collar method to prevent export of assimilates. Photosynthetic activity of girdled and control leaves was evaluated 3 to 7 days later by two methods: (a) carbon exchange rate (CER) of attached leaves was determined under ambient CO(2) concentrations using a closed gas system, and (b) maximum photosynthetic capacity (A(max)) was determined under 3% CO(2) with a leaf disc O(2) electrode. Starch, hexoses, and sucrose were determined enzymically. Typical starch storers like soybean (Glycine max L.) (up to 87.5 milligrams of starch per square decimeter in girdled leaves), cotton (Gossypium hirsutum L.), and cucumber (Cucumis sativus L.) responded to 7 days of girdling by increased (80-100%) stomatal resistance (r(s)) and decreased A(max) (>50%). On the other hand, spinach (Spinacia oleracea L.), a typical sucrose storer (up to 160 milligrams of sucrose per square decimeter in girdled leaves), showed only a slight reduction in CER and almost no change in A(max). Intermediate plants like tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuus L.), broad bean (Vicia faba L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.), which upon girdling store both starch and sucrose, responded to the girdle by a considerable reduction in CER but only moderate inhibition of A(max), indicating that the observed reduction in CER was primarily a stomatal response. Both the wild-type tobacco (Nicotiana sylvestris) (which upon girdling stored starch and hexoses) and the starchless mutant (which stored only hexoses, up to 90 milligrams per square decimeter) showed 90 to 100% inhibition of CER and approximately 50% inhibition of A(max). In general, excised leaves (6 days) behaved like girdled leaves of the respective species, showing 50% reduction of A(max) in wild-type and starchless N. sylvestris but only slight decline of A(max) in spinach. The results of the present study

MORFOMETRÍA COMPARADA DE SEMILLAS DE NICOTIANA (SOLANACEAE E IDENTIFICACIÓN DE SEMILLAS CARBONIZADAS PROVENIENTES DE UN SITIO ARQUEOLÓGICO EN CHILE CENTRAL

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M. Teresa Planella

2012-01-01

Full Text Available La presencia frecuente de pipas para fumar en sitios arqueológicos del Período Alfarero Temprano deChile central y las evidencias en relación con la costumbre de fumar especies de Nicotiana halladas en sitios prehispánicos de otros lugares de las Américas, muestran la necesidad de contar con una metodología para identificar las especies de este género usadas en Chile. En este trabajo se ha realizado un estudio morfométrico en semillas de especies de Nicotiana que sirve de referencia para comparaciones con semillas de origen arqueológico. La forma y tamaño de la semilla, el patrón de ornamentación dado por las células epidérmicas y la ubicación del hilum resultaron ser caracteres relevantes para identificaciones confiables. Utilizando estos caracteres, se determinaron como N. corymbosa a las semillas recuperadas en el sitio arqueológico Las Morrenas 1, ubicado en Chile central.

Impact of herbicides on some agronomic and chemical characteristics of flue-cured virginia (FCV tobacco (Nicotiana tabacum L.

Directory of Open Access Journals (Sweden)

Muhammad Azim Khan

2006-09-01

Full Text Available A field experiment was carried out at Tobacco Research Station Khan Ghari, Mardan, (NWFP- Pakistan during spring 2003 to study the impact of herbicides on some agronomic and chemical characteristics of flue-cured virginia (FCV tobacco (Nicotiana tabacum L.. The experiment was laid out in RCB design, replicated four times with ten treatments, comprising hand weeding, weedy check, pre-transplanting herbicides; S-metalocholar @ 1.92, pendimethalin (EC @ 1.00, pendimethalin (CS @ 1.00, and butralin @ 1.44 kg a.i ha-1 and the post-transplanting herbicides include; clodinafop @ 0.04, fenoxaprop-p-ethyl @ 1.00, acetochlor @ 0.125 and glyphosate @ 0.95 kg a.i ha-1. None of the herbicides except S-metalocholar had a phytotoxic effect on tobacco. All the parameters except the number of leaves plant-1 were significantly affected by different treatments. The highest (228.3 weeds density m-2 was observed in weedy check while minimum (69 was recorded in pendimethalin (EC treatment. The maximum grade index of 74.60% was recorded in acetochlor and minimum grade index of 53.88% was recorded in S-metalocholar treatments. Nicotine (% was higher in pendimethalin (EC treated plots with 2.362%; however it was comparable to all other treatments. The maximum percent reducing sugar of 18.22% was recorded in pendimethalin (CS treatment, while minimum of 12.42% reducing sugar was recorded in weedy check. Similarly maximum yield of 2465 kg ha-1 was recorded in pendimethalin (EC treatment and minimum yield of 1703 kg ha-1 was recorded in weedy check (control treatment. Thus it can be concluded from the experiment that herbicides proved effective against weeds and their growth and promoted tobacco quality and yield. Hence the use of herbicides not only increases the net income of the farmers but also will make the weed seed bank poorer.

Rpi-blb2-Mediated Hypersensitive Cell Death Caused by Phytophthora infestans AVRblb2 Requires SGT1, but not EDS1, NDR1, Salicylic Acid-, Jasmonic Acid-, or Ethylene-Mediated Signaling

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Sang-Keun Oh

2014-09-01

Full Text Available Potato Rpi-blb2 encodes a protein with a coiled-coil-nucleotide binding site and leucine-rich repeat (CC-NBS-LRR motif that recognizes the Phytophthora infestans AVRblb2 effector and triggers hypersensitive cell death (HCD. To better understand the components required for Rpi-blb2-mediated HCD in plants, we used virus-induced gene silencing to repress candidate genes in Rpi-blb2-transgenic Nicotiana benthamiana plants and assayed the plants for AVRblb2 effector. Rpi-blb2 triggers HCD through NbSGT1-mediated pathways, but not NbEDS1- or NbNDR1-mediated pathways. In addition, the role of salicylic acid (SA, jasmonic acid (JA, and ethylene (ET in Rpi-blb2-mediated HCD were analyzed by monitoring of the responses of NbICS1-, NbCOI1-, or NbEIN2-silenced or Rpi-blb2::NahG-transgenic plants. Rpi-blb2-mediated HCD in response to AVRblb2 was not associated with SA accumulation. Thus, SA affects Rpi-blb2-mediated resistance against P. infestans, but not Rpi-blb2-mediated HCD in response to AVRblb2. Additionally, JA and ET signaling were not required for Rpi-blb2-mediated HCD in N. benthamiana. Taken together, these findings suggest that NbSGT1 is a unique positive regulator of Rpi-blb2-mediated HCD in response to AVRblb2, but EDS1, NDR1, SA, JA, and ET are not required.

Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

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Ying Wen Huang

Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fungal infections.

Science.gov (United States)

Zhang, Lisha; Ni, Hao; Du, Xuan; Wang, Sheng; Ma, Xiao-Wei; Nürnberger, Thorsten; Guo, Hui-Shan; Hua, Chenlei

2017-07-01

Fungal pathogens secrete effector proteins to suppress plant basal defense for successful colonization. Resistant plants, however, can recognize effectors by cognate R proteins to induce effector-triggered immunity (ETI). By analyzing secretomes of the vascular fungal pathogen Verticillium dahliae, we identified a novel secreted protein VdSCP7 that targets the plant nucleus. The green fluorescent protein (GFP)-tagged VdSCP7 gene with either a mutated nuclear localization signal motif or with additional nuclear export signal was transiently expressed in Nicotiana benthamiana, and investigated for induction of plant immunity. The role of VdSCP7 in V. dahliae pathogenicity was characterized by gene knockout and complementation, and GFP labeling. Expression of the VdSCP7 gene in N. benthamiana activated both salicylic acid and jasmonate signaling, and altered the plant's susceptibility to the pathogens Botrytis cinerea and Phytophthora capsici. The immune response activated by VdSCP7 was highly dependent on its initial extracellular secretion and subsequent nuclear localization in plants. Knockout of the VdSCP7 gene significantly enhanced V. dahliae aggressiveness on cotton. GFP-labeled VdSCP7 is secreted by V. dahliae and accumulates in the plant nucleus. We conclude that VdSCP7 is a novel effector protein that targets the host nucleus to modulate plant immunity, and suggest that plants can recognize VdSCP7 to activate ETI during fungal infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Assessment of natural radionuclides concentration from {sup 238}U and {sup 232}Th series in Virginia and Burley varieties of Nicotiana tabacum L; Avaliacao da concentracao dos radionuclideos naturais das series do {sup 238}U e {sup 232}Th nas variedades Burley e Virginia da Nicotiana tabacum L.

Energy Technology Data Exchange (ETDEWEB)

Silva, Carolina Fernanda da

2015-07-01

Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop production of 2013/2014. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco products varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation (compression, filter and paper) and the temperature variations resulting from the incomplete combustion of tobacco. Tobacco products are extensively used throughout the world, and the most consumed are cigarettes, cigars and narghile. The damaging effects that these products cause to human health are discussed globally, and many surveys are performed with the aim of relating the use of these products with various illnesses. There is a lack of information about the radiological characterization of the tobacco plant both in international and Brazilian literature. The objective of this study was to determine the concentration of radionuclides {sup 238}U, {sup 234}U, {sup 230}Th, {sup 22}'6Ra, {sup 210}Pb and {sup 210}Po, members from the {sup 238}U decay series, and the radionuclides {sup 232}Th and {sup 228}Ra members of the {sup 232}Th decay series in the varieties Burley and Virginia, which are the most cultivated in Brazil. Plants from these varieties were cultivated in pots with organic substrate and fertilizer and also acquired from the producers and analyzed by alpha spectrometry for U and Th isotopes and {sup 210}Po determination, and gross alpha and beta counting, {sup 228}Ra, {sup 226}Ra and {sup 210}Pb determination. The whole plant, from both places, was analyzed; root, stem, leaves, as well as the organic substrate, the fertilizers, and the soil. The results for U and Th isotopes presented values below the detection limits of the methods to the leaves and stems of all plants analyzed, with measurable results only in roots, soil, and substrate. The

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

OpenAIRE

Donini, Marcello; Lico, Chiara; Baschieri, Selene; Conti, Stefania; Magliani, Walter; Polonelli, Luciano; Benvenuto, Eugenio

2005-01-01

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial acti...

5 CFR 630.1015 - Movement between voluntary leave bank and leave transfer programs.

Science.gov (United States)

2010-01-01

... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Movement between voluntary leave bank and leave transfer programs. 630.1015 Section 630.1015 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS ABSENCE AND LEAVE Voluntary Leave Bank Program § 630.1015 Movement...

The Lettuce infectious yellows virus (LIYV)-encoded P26 is associated with plasmalemma deposits within LIYV-infected cells

International Nuclear Information System (INIS)

Medina, V.; Sudarshana, M.R.; Tian, T.; Ralston, K.S.; Yeh, H.-H.; Falk, B.W.

2005-01-01

Cytological, immunological, and mutagenesis approaches were used to identify the viral factors associated with the formation of plasmalemma deposits (PLDs) in whole plants and protoplasts infected by Lettuce infectious yellows virus (LIYV). Transmission electron microscopy and immunogold labeling using polyclonal antibodies to four of the five LIYV RNA 2-encoded large proteins, capsid protein (CP), minor capsid protein (CPm), HSP70 homolog (HSP70h), and P59, showed specific labeling of LIYV virions or virion aggregates around the vesiculated membranous inclusions, but not PLDs in LIYV-infected Nicotiana benthamiana, Nicotiana clevelandii, Lactuca sativa, and Chenopodium murale plants, and Nicotiana tabacum protoplasts. In contrast, antibodies to the RNA 2-encoded P26 showed specific labeling of PLDs but not virions in both LIYV-infected plants and protoplasts. Virion-like particles (VLPs) were seen in protoplasts infected by all LIYV RNA 2 mutants except for the CP (major capsid protein) mutant. PLDs were more difficult to find in protoplasts, but were seen in protoplasts infected by the CP and CPm mutants, but not in protoplasts infected by the P26, HSP70h, or P59 mutants. Interestingly, although the CPm mutant showed VLPs and PLDs, the PLDs did not show associated virions/virion-like particles as was always observed for PLDs seen in protoplasts infected by wild-type LIYV. Immunoblot analyses performed on purified LIYV virions showed that P26 was not detected with purified virions, but was detected in the cell wall, 1000 g and 30,000 g pellet fractions of LIYV-infected plants. These data suggest that P26 is associated with the LIYV-induced PLDs, and in contrast to the other RNA 2-encoded large proteins, P26 is not a virion protein

Two tandemly repeated telomere-associated sequences in Nicotiana plumbaginifolia.

Science.gov (United States)

Chen, C M; Wang, C T; Wang, C J; Ho, C H; Kao, Y Y; Chen, C C

1997-12-01

Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 x 10(4), 6 x 10(3) and 1.5 x 10(6) copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.

Evaluation of some varieties and breeding lines of tomato (Lycopersison sp) against tomato yellow leaf curl disease in the Greater Accra Region (Ghana)

International Nuclear Information System (INIS)

Kusi-Adjei, R.

2011-01-01

. Comparison of natural whitefly inoculation in the field to controlled whitefly inoculation in insect-proof cages showed there was a higher disease incidence in the field than in the cage. Wild Tomato and the hybrids did not show any symptoms of TYLCVD after controlled whitefly inoculation in insect-proof cages. Mechanically (sap) inoculated Nicotiana benthamiana, Nicotiana glutinosa and Phaseolus vulgaris showed characteristic TTLCVD symptoms such as yellowing and leaf distortion. TAS-ELISA and PCR confirmed the presence of TYLCV in Wild Tomato, Roma x Wild Tomato and Wosowoso x Wild Tomato plants evaluated in the field indicating that they were symptomless carriers of TYLCV and in Roma, Wosowoso and Navrongo indicating that TYLCV-like symptoms observed on the plants were due to transmission of TYLCV by whiteflies in the field and in the cage. PCR detected TYLCV in mechanically inoculated Nicotiana nethamiana test plants showing the TYLCV is sap transmissible to Nicotiana benthamiana. This study has shown that the hybrids Cherry Red x Wild Tomato and Wosowoso x Wild Tomato are resistant to TYLCV while Roma x Wild Tomato was only mildly susceptible in the dry season. These three hybrids could therefore be selected for improvement in future breeding programmes, aimed at breeding high-yielding and TYLCVD resistant varieties. (au)

Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

Science.gov (United States)

Murfett, J; McClure, B A

1998-06-01

Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

Endomembrane Ca2+-AtPases play a significant role in virus-induced adaptation to oxidative stress

DEFF Research Database (Denmark)

Shabala, Sergey; Bækgaard, Lone; Shabala, Lana

2011-01-01

Although the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca2+ efflux systems in this process. In our recent paper,17 we reported Potato Virus X (PVX)-induced acquired resistance to oxidative...... in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects...... stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P2A and P2B Ca2+-ATPases play significant roles...

In silico design and functional assessment of semisynthetic genes that confer tolerance to phosphinothricin

Directory of Open Access Journals (Sweden)

Jenny Paola Jiménez

2016-07-01

Full Text Available Herbicide tolerance is one of the features most used in GM crops, which has shown positive results for farmers and the environment. The starting point is the development of expression cassettes that express the characteristic of interest, they are initially constructed by standard molecular biology techniques. Currently, by bioinformatics and synthetic biology tools, it is possible to design and test the construct in silico, and then hire their synthesis. This approach allows optimizing expression by modifying the codon usage. In this work there were designed and evaluated semi-synthetic versions of genes in Nicotiana benthamiana, these genes confer tolerance to the herbicide phosphinothricin. It was made an analysis of freedom to operate in order to ensure that the designed constructs not violate intellectual property in Colombia. There were obtained two expression cassettes with freedom to operate, which express versions of the bar gene.

In vitro growth response of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to antibiotics and fungicides.

Science.gov (United States)

Pánek, M; Tomšovský, M

2017-07-01

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.

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.

Phenylpropanoids accumulation in eggplant fruit: characterization of biosynthetic genes and regulation by a MYB transcription factor

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Teresa eDocimo

2016-01-01

Full Text Available Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena fruits. Chlorogenic acid (CGA accounts for 70 to 90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena.Higher contents of CGA, Delphinidin 3-rutinoside and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group 6 MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties.In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation.Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9 resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of

Polymethoxyflavones from Nicotiana plumbaginifolia (Solanaceae) Exert Antinociceptive and Neuropharmacological Effects in Mice.

Science.gov (United States)

Shajib, Md Shafiullah; Rashid, Ridwan B; Ming, Long C; Islam, Shanta; Sarker, Md Moklesur R; Nahar, Lutfun; Sarker, Satyajit D; Datta, Bidyut K; Rashid, Mohammad A

2018-01-01

Polymethoxylavones (PMFs) are known to exhibit significant anti-inflammatory and neuroprotective properties. Nicotiana plumbaginifolia , an annual Bangladeshi herb, is rich in polymethoxyflavones that possess significant analgesic and anxiolytic activities. The present study aimed to determine the antinociceptive and neuropharmacological activities of polyoxygenated flavonoids namely- 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone ( 1 ), 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin) ( 2 ), 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone ( 3 ), and 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone ( 4 ), isolated and identified from N. plumbaginifolia . Antinociceptive activity was assessed using the acetic-acid induced writhing, hot plate, tail immersion, formalin and carrageenan-induced paw edema tests, whereas neuropharmacological effects were evaluated in the hole cross, open field and elevated plus maze test. Oral treatment of compounds 1 , 3 , and 4 (12.5-25 mg/kg b.w.) exhibited dose-dependent and significant ( p Plumbaginifolia could be considered as suitable candidates for the development of analgesic and anxiolytic agents.

Deciphering the complex leaf transcriptome of the allotetraploid species Nicotiana tabacum: a phylogenomic perspective

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Bombarely Aureliano

2012-08-01

Full Text Available Abstract Background Polyploidization is an important mechanism in plant evolution. By analyzing the leaf transcriptomes taken from the allotetraploid Nicotiana tabacum (tobacco and parental genome donors, N. sylvesteris (S-Genome and N. tomentosiformis (T-Genome, a phylogenomic approach was taken to map the fate of homeologous gene pairs in this plant. Results A comparison between the genes present in the leaf transcriptomes of N. tabacum and modern day representatives of its progenitor species demonstrated that only 33% of assembled transcripts could be distinguished based on their sequences. A large majority of the genes (83.6% of the non parent distinguishable and 87.2% of the phylogenetic topology analyzed clusters expressed above background level (more than 5 reads showed similar overall expression levels. Homeologous sequences could be identified for 968 gene clusters, and 90% (6% of all genes of the set maintained expression of only one of the tobacco homeologs. When both homeologs were expressed, only 15% (0.5% of the total showed evidence of differential expression, providing limited evidence of subfunctionalization. Comparing the rate of synonymous nucleotide substitution (Ks and non-synonymous nucleotide substitution (Kn provided limited evidence for positive selection during the evolution of tobacco since the polyploidization event took place. Conclusions Polyploidization is a powerful mechanism for plant speciation that can occur during one generation; however millions of generations may be necessary for duplicate genes to acquire a new function. Analysis of the tobacco leaf transcriptome reveals that polyploidization, even in a young tetraploid such as tobacco, can lead to complex changes in gene expression. Gene loss and gene silencing, or subfunctionalization may explain why both homeologs are not expressed by the associated genes. With Whole Genome Duplication (WGD events, polyploid genomes usually maintain a high percentage of

Jasmonate mediates salt-induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.

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Xiaodong Chen

2016-04-01

Full Text Available Jasmonate (JA, as an important signal, plays a key role in multiple processes of plant growth, development and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L. are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA-responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a-recognized G-box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti-COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.

The Temporary Leave Dilemma -

DEFF Research Database (Denmark)

Amilon, Anna

2010-01-01

Lone mothers have to take care of a sick child with little or no help from the child’s other parent and have to carry all costs connected to leave-taking. This paper empirically tests whether lone mothers take more temporary parental leave to care for sick children than partnered mothers...... and whether parental leave is associated with a signaling cost. The results from this study of Swedish mothers show that lone mothers use more temporary parental leave than partnered mothers. Further, within the group of lone mothers, those with higher socioeconomic status take less temporary parental leave...... than those with lower socioeconomic status, whereas no such differences are found within the group of partnered mothers. One possible interpretation is that signaling costs negatively influence the utilization of temporary parental leave for lone mothers....

["Paternity leave"? Retrospective view on a delayed reform of maternity leave in Austria].

Science.gov (United States)

Munz, R

1984-01-01

Only 1 of 3 Austrian fathers involves himself daily in child rearing, and the younger the children, the less likely he is to be involved. Austria is among those European countries with the greatest pregnancy benefits. New mothers may take up to 1 year of paid maternity leave without fear of losing their jobs. This article uses 1982 Institute of Demography survey data to determine support for similar paternity leave for fathers. In the last few years, both Social Democrat and Conservative women have worked for this leave, although the movement has also found opposition by women in trade unions, as well as from conservative groups. Survey results show that 46% of married Austrian women, under age 40, favor paternity leave; 1 or 4 women can imagine their husbands taking such leave. Among husbands, 34% favored the leave option, and 1 of 4 could imagine taking the leave for a least part of the baby's first year. The study attempts to identify those husbands most likely to take advantage of paternity leave. At present, most men will not choose to stay with their children at the expense of earnings reduction. Compensation reforms for both mothers and fathers must first occur before men and women in a position to make real decisions on maternity and paternity leave.

The pepper Bs4C proteins are localized to the endoplasmic reticulum (ER) membrane and confer disease resistance to bacterial blight in transgenic rice.

Science.gov (United States)

Wang, Jun; Zeng, Xuan; Tian, Dongsheng; Yang, Xiaobei; Wang, Lanlan; Yin, Zhongchao

2018-03-30

Transcription activator-like effector (TALE)-dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced on infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C-R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C-R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C-R and two other Bs4C-like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C-S) and the CaBs4C-R homologue gene in the cultivar 'CM334' of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C-eCFP fusion proteins were localized to the endoplasmic reticulum (ER) membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter-Bs4C fusion genes in transgenic rice conferred strain-specific disease resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight in rice, and were specifically induced by the Xa10-incompatible Xoo strain PXO99 A (pHM1avrXa10). The results indicate that the Bs4C proteins from pepper species function broadly in rice and the Bs4C protein-mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. © 2018 TEMASEK LIFE SCIENCES LABORATORY. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

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 ...

[Disability leave and sick leave in Spain. 2016 legislative update].

Science.gov (United States)

Vicente-Herrero, María Teófila; Terradillos-García, María Jesús; Capdevila-García, Luisa M; Ramírez-Íñiguez de la Torre, María Victoria; Aguilar-Jiménez, Encarna; Aguado-Benedí, María José; López-González, Angel Arturo; Torres-Alberich, José Ignacio

2018-01-01

In Spanish, the concepts of discapacidad (disability leave) and incapacidad (sick leave) jointly refer to the impairment of a person due to injuries, diseases or deficiencies that limit their activity in a social, personal or occupational field. However, this common link does not imply that both concepts are the same. Statistical data from INE (Instituto Nacional de Estadística: Statistic National Institute) show that Spain had in 2015 3.85 million persons with a disability (59.8% were women). Statistical data from 2015 from INSS (Instituto Nacional de Seguridad Social: Social Security National Institute) show high levels in the number of processes and in workers affected by temporary sick leave, with social costs to the social security system. Both concepts have been updated: about disability leave, Law 39/2006 adjusted terminology by avoiding the use of concepts with discriminating or pejorative connotation. Regarding sick leave, the Ley General de Seguridad Social (General Social Security Law)has been amended and came into effect in January, 2016. It is necessary to know and distinguish these aspects for a better administrative management, and a more oriented information to the affected patient.

Gene transfer in Nicotiana rustica by means of irradiated pollen II. Cytogenetical consequences

International Nuclear Information System (INIS)

Werner, C.P.; Dunkin, I.M.; Cornish, M.A.; Jones, G.H.

1984-01-01

Pollen from Nicotiana paniculata and the V12 variety of N. rustica was irradiated with a range of high doses of gamma-rays up to 100 Krads. Both kinds of pollen were used to pollinate the V27 variety of N. rustica. Radiation treatments above 30 Krads gave no viable seed. A cytological examination of the M 1 progeny from the 20 Krad treatments of both crosses revealed conventional radiation damage in the form of losses of whole chromosomes and parts of chromosomes, and rearrangements. The plants possessed hybrid or aberrantly hybrid phenotypes. It was concluded that they were the products of a conventional fertilisation mechanism rather than the gene transfer mechanism proposed by Pandey (1980). The expression of mutational damage can probably account for most of the maternal trends observed in the intervarietal M 2 of N. rustica examined previously, although post-meiotic selection may also play a role. (author)

Parental Leave Policies and Parents' Employment and Leave-Taking

Science.gov (United States)

Han, Wen-Jui; Ruhm, Christopher; Waldfogel, Jane

2009-01-01

We describe trends in maternal employment and leave-taking after birth of a newborn and analyze the extent to which these behaviors are influenced by parental leave policies. Data are from the June Current Population Survey (CPS) Fertility Supplements, merged with other months of the CPS, and cover the period 1987 to 1994. This time span is one…

Effet comparé des poudres de Nicotiana tabacum L, Cymbopogon citratus (D.C. Stapf et de l'huile de Ricinus communis L sur la conservation des graines de Vigna unguiculata (L Walp

Directory of Open Access Journals (Sweden)

Gakuru, S.

1995-01-01

Full Text Available Compared Effect of Nicotiana tabacum L, Cymbopogon citratus (D.C. Stapf Powders and Castor Oil Ricinus communis L. on Conservation of Cowpea Vigna Unguiculata (L. Walp Grains. The effect of powder of tobacco Nicotiana tabacum L. and citronella grass Cymbopogon citratus (D.C. Stapf and castor oil Ricinus communis L. on conservation of cowpea Vigna unguiculata (L. Walp. grains was investigated in Kisangani, Zaire. After 5 months of conservation, infestation rates by bean weevil Acanthoscelides obtectus Say were 72.5 %, 74.5 %, 49.5 % and 5 % respectively for the check, the samples treated by 1 % of citronella grass and tobacco powder and 1 % of castor oil. The powder dose of 7.5 % did not give more interesting results.

Purification and characterisation of an antifungal protein, MCha-Pr, from the intercellular fluid of bitter gourd (Momordica charantia) leaves.

Science.gov (United States)

Zhang, Beibei; Xie, Chengjian; Wei, Yunming; Li, Jing; Yang, Xingyong

2015-03-01

An antifungal protein, designated MCha-Pr, was isolated from the intercellular fluid of bitter gourd (Momordica charantia) leaves during a screen for potent antimicrobial proteins from plants. The isolation procedure involved a combination of extraction, ammonium sulphate precipitation, gel filtration on Bio-Gel P-6, ion exchange chromatography on CM-Sephadex, an additional gel filtration on HiLoad 16/60 Superdex 30, and finally, HPLC on a SOURCE 5RPC column. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that the protein had a molecular mass of 25733.46Da. Automated Edman degradation was used to determine the N-terminal sequence of MCha-Pr, and the amino acid sequence was identified as V-E-Y-T-I-T-G-N-A-G-N-T-P-G-G. The MCha-Pr protein has some similarity to the pathogenesis-related proteins from Atropa belladonna (deadly nightshade), Solanum tuberosum (potato), Ricinus communis (castor bean), and Nicotiana tabacum (tobacco). Analysis of the circular dichroism spectra indicated that MCha-Pr predominantly contains α-helix and β-sheet structures. MCha-Pr had inhibitory effects towards a variety of fungal species and the 50% inhibition of fungal growth (IC50) for Alternaria brassicae, Cercospora personata, Fusarium oxysporum, Mucor sp., and Rhizoctonia solani are 33 μM, 42 μM, 37 μM, 40 μM, and 48 μM, respectively. In addition, this antifungal protein can inhibit the germination of A. brassicae spores at 12.5 μM. These results suggest that MCha-Pr in bitter gourd leaves plays a protective role against phytopathogens and has a wide antimicrobial spectrum. Copyright © 2014 Elsevier Inc. All rights reserved.

29 CFR 825.205 - Increments of FMLA leave for intermittent or reduced schedule leave.

Science.gov (United States)

2010-07-01

... intermittent leave or working a reduced leave schedule to commence or end work mid-way through a shift, such as... per week, but works only 20 hours a week under a reduced leave schedule, the employee's ten hours of... 29 Labor 3 2010-07-01 2010-07-01 false Increments of FMLA leave for intermittent or reduced...

The Cucumber vein yellowing virus silencing suppressor P1b can functionally replace HCPro in Plum pox virus infection in a host-specific manner.

Science.gov (United States)

Carbonell, Alberto; Dujovny, Gabriela; García, Juan Antonio; Valli, Adrian

2012-02-01

Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumber, CVYV's natural host. The deleterious effect of P1a on PPV infection is host dependent, because the P1aP1b-expressing PPV chimera accumulated in cucumber to higher levels than PPV expressing P1b alone. These results demonstrate that a potyvirus can use different RSS, and that particular RSS and upstream P1-like proteins contribute to defining the virus host range.

A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis.

Science.gov (United States)

Levy, Amit; Erlanger, Michael; Rosenthal, Michal; Epel, Bernard L

2007-02-01

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribe AtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced, and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions in the gating of Pd.

Maternity Leave Policies

Science.gov (United States)

Strang, Lucy; Broeks, Miriam

2017-01-01

Abstract Over recent years many European Union countries have made changes to the design of the maternity leave provision. These policy developments reflect calls for greater gender equality in the workforce and more equal share of childcare responsibilities. However, while research shows that long period of leave can have negative effects on women's labour market attachment and career advancements, early return to work can be seen as a factor preventing exclusive breastfeeding, and therefore, potentially having negative health impacts for babies. Indeed, the World Health Organisation recommends exclusive breastfeeding up to 6 months of age to provide babies with the nutrition for healthy growth and brain development, protection from life-threatening ailments, obesity and non-communicable diseases such as asthma and diabetes. Therefore, labour market demands on women may be at odds with the health benefits for children gained by longer periods of maternity leave. The aim of this article is to examine the relationship between leave provision and health benefits for children. We examine maternity and parental leave provision across European countries and its potential impact on the breastfeeding of very young babies (up to 6-months of age). We also consider economic factors of potential extension of maternity leave provision to 6 months, such as costs to businesses, effects on the female labour market attachment, and wider consequences (benefits and costs) for individuals, families, employers and the wider society. PMID:28983432

5 CFR 630.1104 - Donations from a leave bank to an emergency leave transfer program.

Science.gov (United States)

2010-01-01

... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Donations from a leave bank to an emergency leave transfer program. 630.1104 Section 630.1104 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS ABSENCE AND LEAVE Emergency Leave Transfer Program § 630.1104 Donations...

RNA virus interference via CRISPR/Cas13a system in plants

KAUST Repository

Aman, Rashid

2017-11-04

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

Plant growth-promoting traits of yeasts isolated from the phyllosphere and rhizosphere of Drosera spatulata Lab.

Science.gov (United States)

Fu, Shih-Feng; Sun, Pei-Feng; Lu, Hsueh-Yu; Wei, Jyuan-Yu; Xiao, Hong-Su; Fang, Wei-Ta; Cheng, Bai-You; Chou, Jui-Yu

2016-03-01

Microorganisms can promote plant growth through direct and indirect mechanisms. Compared with the use of bacteria and mycorrhizal fungi, the use of yeasts as plant growth-promoting (PGP) agents has not been extensively investigated. In this study, yeast isolates from the phyllosphere and rhizosphere of the medicinally important plant Drosera spatulata Lab. were assessed for their PGP traits. All isolates were tested for indole-3-acetic acid-, ammonia-, and polyamine-producing abilities, calcium phosphate and zinc oxide solubilizing ability, and catalase activity. Furthermore, the activities of siderophore, 1-aminocyclopropane-1-carboxylate deaminase, and fungal cell wall-degrading enzymes were assessed. The antagonistic action of yeasts against pathogenic Glomerella cingulata was evaluated. The cocultivation of Nicotiana benthamiana with yeast isolates enhanced plant growth, indicating a potential yeast-plant interaction. Our study results highlight the potential use of yeasts as plant biofertilizers under controlled and field conditions. Copyright © 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Nuclear targeting by fragmentation of the Potato spindle tuber viroid genome

International Nuclear Information System (INIS)

Abraitiene, Asta; Zhao Yan; Hammond, Rosemarie

2008-01-01

Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequent GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus

Complete nucleotide sequence of watermelon chlorotic stunt virus originating from Oman.

Science.gov (United States)

Khan, Akhtar J; Akhtar, Sohail; Briddon, Rob W; Ammara, Um; Al-Matrooshi, Abdulrahman M; Mansoor, Shahid

2012-07-01

Watermelon chlorotic stunt virus (WmCSV) is a bipartite begomovirus (genus Begomovirus, family Geminiviridae) that causes economic losses to cucurbits, particularly watermelon, across the Middle East and North Africa. Recently squash (Cucurbita moschata) grown in an experimental field in Oman was found to display symptoms such as leaf curling, yellowing and stunting, typical of a begomovirus infection. Sequence analysis of the virus isolated from squash showed 97.6-99.9% nucleotide sequence identity to previously described WmCSV isolates for the DNA A component and 93-98% identity for the DNA B component. Agrobacterium-mediated inoculation to Nicotiana benthamiana resulted in the development of symptoms fifteen days post inoculation. This is the first bipartite begomovirus identified in Oman. Overall the Oman isolate showed the highest levels of sequence identity to a WmCSV isolate originating from Iran, which was confirmed by phylogenetic analysis. This suggests that WmCSV present in Oman has been introduced from Iran. The significance of this finding is discussed.

Functional analysis of bipartite begomovirus coat protein promoter sequences

International Nuclear Information System (INIS)

Lacatus, Gabriela; Sunter, Garry

2008-01-01

We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters

A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium

Science.gov (United States)

Hoshi, Ayaka; Oshima, Kenro; Kakizawa, Shigeyuki; Ishii, Yoshiko; Ozeki, Johji; Hashimoto, Masayoshi; Komatsu, Ken; Kagiwada, Satoshi; Yamaji, Yasuyuki; Namba, Shigetou

2009-01-01

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development. PMID:19329488

F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function

Science.gov (United States)

Pazhouhandeh, Maghsoud; Dieterle, Monika; Marrocco, Katia; Lechner, Esther; Berry, Bassam; Brault, Véronique; Hemmer, Odile; Kretsch, Thomas; Richards, Kenneth E.; Genschik, Pascal; Ziegler-Graff, Véronique

2006-01-01

Plants employ small RNA-mediated posttranscriptional gene silencing as a virus defense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode of action of most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 of two Arabidopsis-infecting poleroviruses interacts by means of a conserved minimal F-box motif with Arabidopsis thaliana orthologs of S-phase kinase-related protein 1 (SKP1), a component of the SCF family of ubiquitin E3 ligases. Point mutations in the F-box-like motif abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity of P0. Knockdown of expression of a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus infection. Together, the results support a model in which P0 acts as an F-box protein that targets an essential component of the host posttranscriptional gene silencing machinery. PMID:16446454

Influence of decreased level of endogenous cytokinins on antioxidative mechanisms in tobacco leaves during ageing

Czech Academy of Sciences Publication Activity Database

Mýtinová, Zuzana; Wilhelmová, Naděžda; Haisel, Daniel; Motyka, Václav

2005-01-01

Roč. 49, Supplement 1 (2005), S24-S24 ISSN 0006-3134 R&D Projects: GA ČR GA522/03/0312 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * senescence * Nicotiana tabacum L. Subject RIV: CE - Biochemistry Impact factor: 0.792, year: 2005

Development of SCAR markers and PCR assays for single or simultaneous species-specific detection of Phytophthora nicotianae and Pythium helicoides in ebb-and-flow irrigated kalanchoe.

Science.gov (United States)

Ahonsi, Monday O; Ling, Yin; Kageyama, Koji

2010-11-01

Phytophthora nicotianae and Pythium helicoides are important water-borne oomycete pathogens of irrigated ornamentals particularly ebb-and-flow irrigated kalanchoe in Japan. We developed novel PCR-based sequence characterized amplified region markers and assays for rapid identification and species-specific detection of both pathogens in separate PCR reactions or simultaneously in a duplex PCR.

Work-family balance after childbirth: the association between employer-offered leave characteristics and maternity leave duration.

Science.gov (United States)

Guendelman, Sylvia; Goodman, Julia; Kharrazi, Martin; Lahiff, Maureen

2014-01-01

Early return to work after childbirth has been increasing among working mothers in the US. We assessed the relationship between access to employer-offered maternity leave (EOML) (both paid and unpaid) and uptake and duration of maternity leave following childbirth in a socio-economically diverse sample of full-time working women. We focus on California, a state that has long provided more generous maternity leave benefits than those offered by federal maternity leave policies through the State Disability Insurance program. The sample included 691 mothers who gave birth in Southern California in 2002-2003. Using weighted logistic regression, we examined the EOML-maternity leave duration relationship, controlling for whether the leave was paid, as well as other occupational, personality and health-related covariates. Compared with mothers who were offered more than 12 weeks of maternity leave, mothers with leave had six times higher odds of an early return. These relationships were similar after controlling for whether the leave was paid and after controlling for other occupational and health characteristics. Access to and duration of employer-offered maternity leave significantly determine timing of return to work following childbirth, potentially affecting work-family balance. Policy makers should recognize the pivotal role of employers in offering job security during and after maternity leave and consider widening the eligibility criteria of the Family and Medical Leave Act.

Water and Salt Stresses, Kinetin and Protein Synthesis in Tobacco Leaves 1

Science.gov (United States)

Ben-Zioni, Aliza; Itai, C.; Vaadia, Y.

1967-01-01

The capacity of tobacco (Nicotiana rustica) leaf discs to incorporate l-leucine 14C into proteins was measured. Leaf discs were obtained from plants which experienced soil water depletion, or which were exposed to a saline or osmotic stress in the root medium. The stresses were brief of relatively short duration and water potential did not decrease below 4 bars in the root media. Leaf discs were sampled 2 hours after stress removal, achieved by reirrigation, or replacement of saline and osmotic solutions with normal nutrient solution. Plants were always turgid when leaves were sampled. All stressed tissues showed reduced capacity to incorporate l-leucine 14C into protein. The reduction was about 50% and could not be attributed either to reduced uptake into the discs, or to possible isotopic dilution. Incorporation decreased progressively with leaf age in control discs as well as in stressed leaf discs. At all ages tested, incorporation in stressed discs was lower than that of the control. Full recovery of incorporation capacity in stressed discs was obtained when discs were sampled 72 hours after stress removal but not earlier. Kinetin pretreatment prior to incubation with labelled leucine partially restored incorporation in stressed discs. The differences in response to kinetin of stressed and control discs suggest a lower endogenous level of cytokinins in the stressed discs. The results were qualitatively similar regardless of the kind of stress given to the plants during pretreatment. This supports the hypothesis that the normal supply of root cytokinins is important in shoot metabolism. PMID:16656515

Maternity leave duration and postpartum mental and physical health: implications for leave policies.

Science.gov (United States)

Dagher, Rada K; McGovern, Patricia M; Dowd, Bryan E

2014-04-01

This study examines the association of leave duration with depressive symptoms, mental health, physical health, and maternal symptoms in the first postpartum year, using a prospective cohort design. Eligible employed women, eighteen years or older, were interviewed in person at three Minnesota hospitals while hospitalized for childbirth in 2001. Telephone interviews were conducted at six weeks (N = 716), twelve weeks (N = 661), six months (N = 625), and twelve months (N = 575) after delivery. Depressive symptoms (Edinburgh Postnatal Depression Scale), mental and physical health (SF-12 Health Survey), and maternal childbirth-related symptoms were measured at each time period. Two-stage least squares analysis showed that the relationship between leave duration and postpartum depressive symptoms is U-shaped, with a minimum at six months. In the first postpartum year, an increase in leave duration is associated with a decrease in depressive symptoms until six months postpartum. Moreover, ordinary least squares analysis showed a marginally significant linear positive association between leave duration and physical health. Taking leave from work provides time for mothers to rest and recover from pregnancy and childbirth. Findings indicate that the current leave duration provided by the Family and Medical Leave Act, twelve weeks, may not be sufficient for mothers at risk for or experiencing postpartum depression.

The Effector SPRYSEC-19 of Globodera rostochiensis Suppresses CC-NB-LRR-Mediated Disease Resistance in Plants1[C][W][OA

Science.gov (United States)

Postma, Wiebe J.; Slootweg, Erik J.; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O.G.; van Gelderen, Kasper; Lozano-Torres, Jose L.; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

2012-01-01

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants. PMID:22904163

The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR-mediated disease resistance in plants.

Science.gov (United States)

Postma, Wiebe J; Slootweg, Erik J; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O G; van Gelderen, Kasper; Lozano-Torres, Jose L; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

2012-10-01

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.

Transcription profiling and identification of infection-related genes in Phytophthora cactorum.

Science.gov (United States)

Chen, Xiao-Ren; Huang, Shen-Xin; Zhang, Ye; Sheng, Gui-Lin; Zhang, Bo-Yue; Li, Qi-Yuan; Zhu, Feng; Xu, Jing-You

2018-04-01

Phytophthora cactorum, an oomycete pathogen, infects more than 200 plant species within several plant families. To gain insight into the repertoire of the infection-related genes of P. cactorum, Illumina RNA-Seq was used to perform a global transcriptome analysis of three life cycle stages of the pathogen, mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). From over 9.8 million Illumina reads for each library, 18,402, 18,569 and 19,443 distinct genes were identified for MY, ZO and GC libraries, respectively. Furthermore, the transcriptome difference among MY, ZO and GC stages was investigated. Gene ontology (GO) and KEGG pathway enrichment analyses revealed diverse biological functions and processes. Comparative analysis identified a large number of genes that are associated with specific stages and pathogenicity, including 166 effector genes. Of them, most of RXLR and NLP genes showed induction while the majority of CRN genes were down-regulated in GC, the important pre-infection stage, compared to either MY or ZO. And 14 genes encoding small cysteine-rich (SCR) secretory proteins showed differential expression during the developmental stages and in planta. Ectopic expression in the Solanaceae indicated that SCR113 and one elicitin PcINF1 can trigger cell death on Nicotiana benthamiana, tobacco (N. tabacum) and tomato (Solanum lycopersicum) leaves. Neither conserved domain nor homologues of SCR113 in other organisms can be identified. Collectively, our study provides a comprehensive examination of gene expression across three P. cactorum developmental stages and describes pathogenicity-related genes, all of which will help elucidate the pathogenicity mechanism of this destructive pathogen.

Genome-Wide Identification and Analysis of Drought-Responsive Genes and MicroRNAs in Tobacco

Directory of Open Access Journals (Sweden)

Fuqiang Yin

2015-03-01

Full Text Available Drought stress response is a complex trait regulated at transcriptional and post-transcriptional levels in tobacco. Since the 1990s, many studies have shown that miRNAs act in many ways to regulate target expression in plant growth, development and stress response. The recent draft genome sequence of Nicotiana benthamiana has provided a framework for Digital Gene Expression (DGE and small RNA sequencing to understand patterns of transcription in the context of plant response to environmental stress. We sequenced and analyzed three Digital Gene Expression (DGE libraries from roots of normal and drought-stressed tobacco plants, and four small RNA populations from roots, stems and leaves of control or drought-treated tobacco plants, respectively. We identified 276 candidate drought responsive genes (DRGs with sequence similarities to 64 known DRGs from other model plant crops, 82 were transcription factors (TFs including WRKY, NAC, ERF and bZIP families. Of these tobacco DRGs, 54 differentially expressed DRGs included 21 TFs, which belonged to 4 TF families such as NAC (6, MYB (4, ERF (10, and bZIP (1. Additionally, we confirmed expression of 39 known miRNA families (122 members and five conserved miRNA families, which showed differential regulation under drought stress. Targets of miRNAs were further surveyed based on a recently published study, of which ten targets were DRGs. An integrated gene regulatory network is proposed for the molecular mechanisms of tobacco root response to drought stress using differentially expressed DRGs, the changed expression profiles of miRNAs and their target transcripts. This network analysis serves as a reference for future studies on tobacco response stresses such as drought, cold and heavy metals.

The potato aphid salivary effector Me47 is a glutathione-S-transferase involved in modifying plant responses to aphid infestation

Directory of Open Access Journals (Sweden)

Graeme James Kettles

2016-08-01

Full Text Available Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae is a notable pest of solanaceous crops, however the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here we describe a novel aphid effector (Me47 which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST. Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae. Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant-aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs, compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues.

Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[OPEN

Science.gov (United States)

2015-01-01

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets. PMID:26362606

The prevalence of sick leave

DEFF Research Database (Denmark)

Backhausen, Mette; Damm, Peter; Bendix, Jane

2018-01-01

of long-term sick leave. Method Data from 508 employed pregnant women seeking antenatal care was collected by questionnaires from August 2015 to March 2016. The questionnaires, which were filled in at 20 and 32 weeks of gestation, provided information on maternal characteristics, the number of days spent...... on sick leave and the associated reasons. Descriptive statistics and logistic regression analysis were applied. Results The prevalence of sick leave was 56% of employed pregnant women in the first 32 weeks of gestation and more than one in four reported long-term sick leave (>20 days, continuous...... was a negative predictor. Conclusions The prevalence of sick leave was 56% in the first 32 weeks of gestation and more than one in four women reported long-term sick leave. The majority of reasons for sick leave were pregnancy-related and low back pain was the most frequently given reason....

Natural variation in floral nectar proteins of two Nicotiana attenuata accessions.

Science.gov (United States)

Seo, Pil Joon; Wielsch, Natalie; Kessler, Danny; Svatos, Ales; Park, Chung-Mo; Baldwin, Ian T; Kim, Sang-Gyu

2013-07-13

Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata. We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary. Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.

Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.

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James R Petrie

Full Text Available BACKGROUND: Monoacylglycerol acyltransferases (MGATs are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG pathway by acylating MAG to form diacylglycerol (DAG. Typical plant triacylglycerol (TAG biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT. Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.

Molecular and biological characterisation of two novel pomo-like viruses associated with potato (Solanum tuberosum) fields in Colombia.

Science.gov (United States)

Gil, Jose Fernando; Adams, Ian; Boonham, Neil; Nielsen, Steen Lykke; Nicolaisen, Mogens

2016-06-01

Potato is the fourth most important crop worldwide that is used as a staple food, after rice, wheat and maize. The crop can be affected by a large number of pathogens, including fungi, oomycetes, bacteria and viruses. Diseases caused by viruses are among the most important factors contributing to reduced quality and yield of the crop. Potato mop-top virus (genus Pomovirus) induces necrotic flecks in the tuber flesh and skin of potato in temperate countries. Spongospora subterranea is the vector of PMTV. Both the virus and its vector cause disease in potato. In Colombia, PMTV has been detected throughout the country together with a novel pomo-like virus in the centre (Cundinamarca and Boyacá) and south west (Nariño) of the country. We studied the molecular and biological characteristics of this novel virus. Its genome resembles those of members of the genus Pomovirus, and it is closely related to PMTV. It induces mild systemic symptoms in Nicotiana benthamiana (mosaic, branch curling), but no symptoms in N. tabacum, N. debneyi and Chenopodium amaranticolor. The proposed name for the virus is "Colombian potato soil-borne virus" (CPSbV). Additionally, another pomo-like virus was identified in Nariño. This virus induces severe systemic stem declining and mild mosaic in N. benthamiana. The tentative name "soil-borne virus 2" (SbV2) is proposed for this virus. No vectors have been identified for these viruses despite several attempts. This work focused on the characterisation of CPSbV. The risk posed by these viruses if they are introduced into new territories is discussed.

An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation

KAUST Repository

Idris, Ali

2010-11-17

The Oman strain of Tomato yellow leaf curl virus (TYLCV-OM) and its associated betasatellite, an isolate of Tomato leaf curl betasatellite (ToLCB), were previously reported from Oman. Here we report the isolation of a second, previously undescribed, begomovirus [Tomato leaf curl Oman virus (ToLCOMV)] and an alphasatellite from that same plant sample. This alphasatellite is closely related (90% shared nucleotide identity) to an unusual DNA-2-type Ageratum yellow vein Singapore alphasatellite (AYVSGA), thus far identified only in Singapore. ToLCOMV was found to have a recombinant genome comprising sequences derived from two extant parents, TYLCV-OM, which is indigenous to Oman, and Papaya leaf curl virus from the Indian subcontinent. All possible combinations of ToLCOMV, TYLCV-OM, ToLCB and AYVSGA were used to agro-inoculate tomato and Nicotiana benthamiana. Infection with ToLCOMV yielded mild leaf-curl symptoms in both hosts; however, plants inoculated with TYLCV-OM developed more severe symptoms. Plants infected with ToLCB in the presence of either helper begomovirus resulted in more severe symptoms. Surprisingly, symptoms in N. benthamiana infected with the alphasatellite together with either of the helper viruses and the betasatellite were attenuated and betasatellite DNA accumulation was substantially reduced. However, in the latter plants no concomitant reduction in the accumulation of helper virus DNA was observed. This is the first example of an attenuation of begomovirus-betasatellite symptoms by this unusual class of alphasatellites. This observation suggests that some DNA-2 alphasatellites encode a pathogenicity determinant that may modulate begomovirus-betasatellite infection by reducing betasatellite DNA accumulation. © 2011 SGM.

Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL domain effector of Rhizobium sp. strain NGR234.

Directory of Open Access Journals (Sweden)

Da-Wei Xin

Full Text Available Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.

An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation

KAUST Repository

Idris, Ali; Shahid, Muhammad Shafiq; Briddon, Rob William; Khan, Akhtarjamal; Zhu, Jian-Kang; Brown, Judith K.

2010-01-01

The Oman strain of Tomato yellow leaf curl virus (TYLCV-OM) and its associated betasatellite, an isolate of Tomato leaf curl betasatellite (ToLCB), were previously reported from Oman. Here we report the isolation of a second, previously undescribed, begomovirus [Tomato leaf curl Oman virus (ToLCOMV)] and an alphasatellite from that same plant sample. This alphasatellite is closely related (90% shared nucleotide identity) to an unusual DNA-2-type Ageratum yellow vein Singapore alphasatellite (AYVSGA), thus far identified only in Singapore. ToLCOMV was found to have a recombinant genome comprising sequences derived from two extant parents, TYLCV-OM, which is indigenous to Oman, and Papaya leaf curl virus from the Indian subcontinent. All possible combinations of ToLCOMV, TYLCV-OM, ToLCB and AYVSGA were used to agro-inoculate tomato and Nicotiana benthamiana. Infection with ToLCOMV yielded mild leaf-curl symptoms in both hosts; however, plants inoculated with TYLCV-OM developed more severe symptoms. Plants infected with ToLCB in the presence of either helper begomovirus resulted in more severe symptoms. Surprisingly, symptoms in N. benthamiana infected with the alphasatellite together with either of the helper viruses and the betasatellite were attenuated and betasatellite DNA accumulation was substantially reduced. However, in the latter plants no concomitant reduction in the accumulation of helper virus DNA was observed. This is the first example of an attenuation of begomovirus-betasatellite symptoms by this unusual class of alphasatellites. This observation suggests that some DNA-2 alphasatellites encode a pathogenicity determinant that may modulate begomovirus-betasatellite infection by reducing betasatellite DNA accumulation. © 2011 SGM.

Pollination with heavily irradiated pollen in Nicotiana: induced parthenogenesis and embryological study

International Nuclear Information System (INIS)

Musial, K.; Przywara, L.

1999-01-01

Nicotiana crosses were pollinated in situ and in vitro with heavily irradiated pollen (500, 700, 1000 Gy) to induce parthenogenesis and to study the development of embryo and endosperm. Haploids were obtained after in situ pollination only; however, parthenogenetic proembryos occurred also after in vitro pollination. It was demonstrated that ovule culture following pollination offers a better chance to produce haploids than undisturbed pollination does. Pollination with irradiated pollen (PwIP) stron gly decreased the number of endosperm cells and the size of embryo sacs, and it affected the development of embryos; no significant differences between applied irradiation doses were found. Ovules with endosperm only, embryo only, and with both embryo and endosperm were observed. The most frequent were the ovules with endosperm only, the rarest with embryo only. A small amount of storage products occurred in the endosperm cells. The diploid chromosome number counted in the endosperm produced after PwIP points to their origin without fertilization. An interesting phenomenon observed after PwIP was vigorous growth of endothelium. (author)

Cre recombinase expression can result in phenotypic aberrations in plants

NARCIS (Netherlands)

Coppoolse, E.; Vroomen, de M.J.; Roelofs, D.; Smit, J.; Gennip, van F.; Hersmus, B.J.M.; Nijkamp, H.J.J.; Haaren, van M.J.

2003-01-01

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between

Cre recombinase expression can result in phenotypic aberrations in plants

NARCIS (Netherlands)

Coppoolse, Eric R; de Vroomen, Marianne J; Roelofs, Dick; Smit, Jaap; van Gennip, Femke; Hersmus, Bart J M; Nijkamp, H John J; van Haaren, Mark J J

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between

Phosphorus acquisition by citrate- and phytase-exuding Nicotiana tabacum plant mixtures depends on soil phosphorus availability and root intermingling.

Science.gov (United States)

Giles, Courtney D; Richardson, Alan E; Cade-Menun, Barbara J; Mezeli, Malika M; Brown, Lawrie K; Menezes-Blackburn, Daniel; Darch, Tegan; Blackwell, Martin Sa; Shand, Charles A; Stutter, Marc I; Wendler, Renate; Cooper, Patricia; Lumsdon, David G; Wearing, Catherine; Zhang, Hao; Haygarth, Philip M; George, Timothy S

2018-03-02

Citrate and phytase root exudates contribute to improved phosphorus (P) acquisition efficiency in Nicotiana tabacum (tobacco) when both exudates are produced in a P deficient soil. To test the importance of root intermingling in the interaction of citrate and phytase exudates, Nicotiana tabacum plant-lines with constitutive expression of heterologous citrate (Cit) or fungal phytase (Phy) exudation traits were grown under two root treatments (roots separated or intermingled) and in two soils with contrasting soil P availability. Complementarity of plant mixtures varying in citrate efflux rate and mobility of the expressed phytase in soil was determined based on plant biomass and P accumulation. Soil P composition was evaluated using solution 31 P NMR spectroscopy. In the soil with limited available P, positive complementarity occurred in Cit+Phy mixtures with roots intermingled. Root separation eliminated positive interactions in mixtures expressing the less mobile phytase (Aspergillus niger PhyA) whereas positive complementarity persisted in mixtures that expressed the more mobile phytase (Peniophora lycii PhyA). Soils from Cit+Phy mixtures contained less inorganic P and more organic P compared to monocultures. Exudate-specific strategies for the acquisition of soil P were most effective in P-limited soil and depended on citrate efflux rate and the relative mobility of the expressed phytase in soil. Plant growth and soil P utilization in plant systems with complementary exudation strategies are expected to be greatest where exudates persist in soil and are expressed synchronously in space and time. This article is protected by copyright. All rights reserved.

[Precautionary maternity leave in Tirol].

Science.gov (United States)

Ludescher, K; Baumgartner, E; Roner, A; Brezinka, C

1998-01-01

Under Austrian law, precautionary maternity leave is a decree issued by the district public health physician. It forbids a pregnant woman to work and mandates immediate maternity leave. Regular maternity leave for all women employed in all jobs begins at 32 weeks of gestation. Women who work in workplaces deemed dangerous and women with a history of obstetric problems such as premature or growth-retarded babies from previous pregnancies are regularly 'sent' into precautionary maternity leave. The public health physicians of Tirol's nine administrative districts were interviewed and supplied data on precautionary maternity leave from their districts. In 100 women who attended the clinic for pregnancies at risk of the Obstetrics/Gynecology Department of Innsbruck University Hospital and who had already obtained precautionary maternity leave, the medical/administrative procedure was studied in each case and correlated with pregnancy outcome. The town district of Innsbruck and the district that comprises the suburbs of the provincial capital had the highest rates of precautionary maternity leave. The town district of Innsbruck had a rate of 24.3% of all pregnant women (employed and not employed) in precautionary maternity leave in 1997, whereas the whole province of Tirol had 13.4%. More than 80% of decrees for precautionary maternity leave are issued by district public health physicians on the basis of written recommendations from gynecologists. One third of women who are sent into precautionary maternity leave are issued the decree prior to 12 weeks of gestation - mostly cases of multiple pregnancies and women with previous miscarriages. The present system of precautionary maternity leave appears to work in the sense that most working pregnant women with risk factors are correctly identified - with most errors on the side of caution. As the system also helps employers - the employee's pay is paid from the federal family support fund and state insurance once she is in

Parental leave and child health.

Science.gov (United States)

Ruhm, C J

2000-11-01

This study investigates whether rights to parental leave improve pediatric health. Aggregate data are used for 16 European countries over the 1969 through 1994 period. More generous paid leave is found to reduce deaths of infants and young children. The magnitudes of the estimated effects are substantial, especially where a causal effect of leave is most plausible. In particular, there is a much stronger negative relationship between leave durations and post-neonatal or child fatalities than for perinatal mortality, neonatal deaths, or low birth weight. The evidence further suggests that parental leave may be a cost-effective method of bettering child health.

Leaves of Absence. School Law Summary.

Science.gov (United States)

National Education Association, Washington, DC. Research Div.

This report contains State-by-State statutory summaries on three types of leaves of absence relating to teachers -- sick leave, maternity leave, and sabbatical leave. Only State laws that have specific reference to one of these three types of leaves of absence are included. Not included are those statutes granting boards of education the general…

PET imaging of thin objects: measuring the effects of positron range and partial-volume averaging in the leaf of Nicotiana tabacum

Energy Technology Data Exchange (ETDEWEB)

Alexoff, David L., E-mail: alexoff@bnl.gov; Dewey, Stephen L.; Vaska, Paul; Krishnamoorthy, Srilalan; Ferrieri, Richard; Schueller, Michael; Schlyer, David J.; Fowler, Joanna S.

2011-02-15

Introduction: PET imaging in plants is receiving increased interest as a new strategy to measure plant responses to environmental stimuli and as a tool for phenotyping genetically engineered plants. PET imaging in plants, however, poses new challenges. In particular, the leaves of most plants are so thin that a large fraction of positrons emitted from PET isotopes ({sup 18}F, {sup 11}C, {sup 13}N) escape while even state-of-the-art PET cameras have significant partial-volume errors for such thin objects. Although these limitations are acknowledged by researchers, little data have been published on them. Methods: Here we measured the magnitude and distribution of escaping positrons from the leaf of Nicotiana tabacum for the radionuclides {sup 18}F, {sup 11}C and {sup 13}N using a commercial small-animal PET scanner. Imaging results were compared to radionuclide concentrations measured from dissection and counting and to a Monte Carlo simulation using GATE (Geant4 Application for Tomographic Emission). Results: Simulated and experimentally determined escape fractions were consistent. The fractions of positrons (mean{+-}S.D.) escaping the leaf parenchyma were measured to be 59{+-}1.1%, 64{+-}4.4% and 67{+-}1.9% for {sup 18}F, {sup 11}C and {sup 13}N, respectively. Escape fractions were lower in thicker leaf areas like the midrib. Partial-volume averaging underestimated activity concentrations in the leaf blade by a factor of 10 to 15. Conclusions: The foregoing effects combine to yield PET images whose contrast does not reflect the actual activity concentrations. These errors can be largely corrected by integrating activity along the PET axis perpendicular to the leaf surface, including detection of escaped positrons, and calculating concentration using a measured leaf thickness.

Characterization of cDNA for PMT: a Partial Nicotine Biosynthesis-Related Gene Isolated from Indonesian Local Tobacco (Nicotiana tabacum cv. Sindoro1

Directory of Open Access Journals (Sweden)

SESANTI BASUKI

2013-12-01

Full Text Available Nicotine is the major alkaloid compound in cultivated tobacco (Nicotiana tabacum that could potentially be converted into carcinogenic compound (nor-nicotine. The PMT gene encoding putrescine N-methyltransferase (PMT is one of the two key genes that play a prominent role in nicotine biosynthesis. The aimed of this study was to isolate and characterize the cDNA sequence originated from Indonesian local tobacco cv. Sindoro1 (Ntpmt_Sindoro1. The results showed that the Ntpmt_Sindoro1 was 1124 bp in length. This cDNA fragment encodes for 374 amino acid residues. The predicted polypeptide from the cDNA is a hidrophilic protein, and has a predicted molecular weight of 40.95 kDa. The predicted amino acids sequence also showed high similarity to the PMT gene product Nicotiana sp. available in the GenBank data base. The amino acid sequences also exert conserved residues specifically exhibited only by PMT gene originated from N. tabacum. Clustering analysis revealed that Ntpmt_Sindoro1 belongs to the same clade as the PMT3 gene, a member of the N. tabacum PMT gene family. The Ntpmt_Sindoro1 cDNA sequence covering exon1-exon8 of the PMT gene fragment has been registered in the GenBank data base, under the accession number JX978277.

Ectopic expression of class 1 KNOX genes induce and adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

Science.gov (United States)

Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...

Nectar sugars and amino acids in day- and night-flowering Nicotiana species are more strongly shaped by pollinators' preferences than organic acids and inorganic ions.

Science.gov (United States)

Tiedge, Kira; Lohaus, Gertrud

2017-01-01

Floral nectar contains mainly sugars but also amino acids, organic acids, inorganic ions and secondary compounds to attract pollinators. The genus Nicotiana exhibits great diversity among species in floral morphology, flowering time, nectar compositions, and predominant pollinators. We studied nectar samples of 20 Nicotiana species, composed equally of day- and night-flowering plants and attracting different groups of pollinators (e.g. hummingbirds, moths or bats) to investigate whether sugars, amino acids, organic acids and inorganic ions are influenced by pollinator preferences. Glucose, fructose and sucrose were the only sugars found in the nectar of all examined species. Sugar concentration of the nectar of day-flowering species was 20% higher and amino acid concentration was 2-3-fold higher compared to the nectar of night-flowering species. The sucrose-to-hexose ratio was significantly higher in night-flowering species and the relative share of sucrose based on the total sugar correlated with the flower tube length in the nocturnal species. Flowers of different tobacco species contained varying volumes of nectar which led to about 150-fold higher amounts of total sugar per flower in bat- or sunbird-pollinated species than in bee-pollinated or autogamous species. This difference was even higher for total amino acids per flower (up to 1000-fold). As a consequence, some Nicotiana species invest large amounts of organic nitrogen for certain pollinators. Higher concentrations of inorganic ions, predominantly anions, were found in nectar of night-flowering species. Therefore, higher anion concentrations were also associated with pollinator types active at night. Malate, the main organic acid, was present in all nectar samples but the concentration was not correlated with pollinator type. In conclusion, statistical analyses revealed that pollinator types have a stronger effect on nectar composition than phylogenetic relations. In this context, nectar sugars and amino

Nectar sugars and amino acids in day- and night-flowering Nicotiana species are more strongly shaped by pollinators’ preferences than organic acids and inorganic ions

Science.gov (United States)

Tiedge, Kira; Lohaus, Gertrud

2017-01-01

Floral nectar contains mainly sugars but also amino acids, organic acids, inorganic ions and secondary compounds to attract pollinators. The genus Nicotiana exhibits great diversity among species in floral morphology, flowering time, nectar compositions, and predominant pollinators. We studied nectar samples of 20 Nicotiana species, composed equally of day- and night-flowering plants and attracting different groups of pollinators (e.g. hummingbirds, moths or bats) to investigate whether sugars, amino acids, organic acids and inorganic ions are influenced by pollinator preferences. Glucose, fructose and sucrose were the only sugars found in the nectar of all examined species. Sugar concentration of the nectar of day-flowering species was 20% higher and amino acid concentration was 2-3-fold higher compared to the nectar of night-flowering species. The sucrose-to-hexose ratio was significantly higher in night-flowering species and the relative share of sucrose based on the total sugar correlated with the flower tube length in the nocturnal species. Flowers of different tobacco species contained varying volumes of nectar which led to about 150-fold higher amounts of total sugar per flower in bat- or sunbird-pollinated species than in bee-pollinated or autogamous species. This difference was even higher for total amino acids per flower (up to 1000-fold). As a consequence, some Nicotiana species invest large amounts of organic nitrogen for certain pollinators. Higher concentrations of inorganic ions, predominantly anions, were found in nectar of night-flowering species. Therefore, higher anion concentrations were also associated with pollinator types active at night. Malate, the main organic acid, was present in all nectar samples but the concentration was not correlated with pollinator type. In conclusion, statistical analyses revealed that pollinator types have a stronger effect on nectar composition than phylogenetic relations. In this context, nectar sugars and amino

Nectar sugars and amino acids in day- and night-flowering Nicotiana species are more strongly shaped by pollinators' preferences than organic acids and inorganic ions.

Directory of Open Access Journals (Sweden)

Kira Tiedge

Full Text Available Floral nectar contains mainly sugars but also amino acids, organic acids, inorganic ions and secondary compounds to attract pollinators. The genus Nicotiana exhibits great diversity among species in floral morphology, flowering time, nectar compositions, and predominant pollinators. We studied nectar samples of 20 Nicotiana species, composed equally of day- and night-flowering plants and attracting different groups of pollinators (e.g. hummingbirds, moths or bats to investigate whether sugars, amino acids, organic acids and inorganic ions are influenced by pollinator preferences. Glucose, fructose and sucrose were the only sugars found in the nectar of all examined species. Sugar concentration of the nectar of day-flowering species was 20% higher and amino acid concentration was 2-3-fold higher compared to the nectar of night-flowering species. The sucrose-to-hexose ratio was significantly higher in night-flowering species and the relative share of sucrose based on the total sugar correlated with the flower tube length in the nocturnal species. Flowers of different tobacco species contained varying volumes of nectar which led to about 150-fold higher amounts of total sugar per flower in bat- or sunbird-pollinated species than in bee-pollinated or autogamous species. This difference was even higher for total amino acids per flower (up to 1000-fold. As a consequence, some Nicotiana species invest large amounts of organic nitrogen for certain pollinators. Higher concentrations of inorganic ions, predominantly anions, were found in nectar of night-flowering species. Therefore, higher anion concentrations were also associated with pollinator types active at night. Malate, the main organic acid, was present in all nectar samples but the concentration was not correlated with pollinator type. In conclusion, statistical analyses revealed that pollinator types have a stronger effect on nectar composition than phylogenetic relations. In this context

Short-Term Saved Leave Scheme

CERN Multimedia

2007-01-01

As announced at the meeting of the Standing Concertation Committee (SCC) on 26 June 2007 and in http://Bulletin No. 28/2007, the existing Saved Leave Scheme will be discontinued as of 31 December 2007. Staff participating in the Scheme will shortly receive a contract amendment stipulating the end of financial contributions compensated by save leave. Leave already accumulated on saved leave accounts can continue to be taken in accordance with the rules applicable to the current scheme. A new system of saved leave will enter into force on 1 January 2008 and will be the subject of a new implementation procedure entitled "Short-term saved leave scheme" dated 1 January 2008. At its meeting on 4 December 2007, the SCC agreed to recommend the Director-General to approve this procedure, which can be consulted on the HR Department’s website at the following address: https://cern.ch/hr-services/services-Ben/sls_shortterm.asp All staff wishing to participate in the new scheme a...

Short-Term Saved Leave Scheme

CERN Multimedia

HR Department

2007-01-01

As announced at the meeting of the Standing Concertation Committee (SCC) on 26 June 2007 and in http://Bulletin No. 28/2007, the existing Saved Leave Scheme will be discontinued as of 31 December 2007. Staff participating in the Scheme will shortly receive a contract amendment stipulating the end of financial contributions compensated by save leave. Leave already accumulated on saved leave accounts can continue to be taken in accordance with the rules applicable to the current scheme. A new system of saved leave will enter into force on 1 January 2008 and will be the subject of a new im-plementation procedure entitled "Short-term saved leave scheme" dated 1 January 2008. At its meeting on 4 December 2007, the SCC agreed to recommend the Director-General to approve this procedure, which can be consulted on the HR Department’s website at the following address: https://cern.ch/hr-services/services-Ben/sls_shortterm.asp All staff wishing to participate in the new scheme ...

5 CFR 630.1111 - Limitation on the amount of donated annual leave received by an emergency leave recipient.

Science.gov (United States)

2010-01-01

... needs of individual emergency leave recipients, an employing agency may allow an employee to receive... annual leave received by an emergency leave recipient. 630.1111 Section 630.1111 Administrative Personnel... recipient. An emergency leave recipient may receive a maximum of 240 hours of donated annual leave at any...

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

Science.gov (United States)

Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

2016-11-01

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures.

Science.gov (United States)

Bartholomeusz, Trixie Ann; Molinié, Roland; Roscher, Albrecht; Felpin, François-Xavier; Gillet, Françoise; Lebreton, Jacques; Mesnard, François; Robins, Richard J

2005-08-01

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.

Wheat bran soil inoculant of sumateran nematode-trapping fungi as biocontrol agents of the root-knot nematode meloidogyne incognita on deli tobacco (nicotiana tabaccum l) cv. deli 4

Science.gov (United States)

Dwi Sri Hastuti, Liana; Faull, Jane

2018-03-01

A pot experiment was carried out to test the effectiveness of nematode-trapping fungi (NTF) isolated from Sumatera for controlling infection by the root-knot nematode (RKN) on Deli tobacco plant. Wheat bran soil containing 109 conidia of Arthrobotrys. oligospora, Candellabrella musiformis and Dactylella eudermata was added to the soil as a dry inoculum. Carbofuran was also applied as chemical agent and comparison treatment. Seedling tobacco (Nicotiana tabacum L.) cv. Deli 4 was inoculated with root knot (Meloidogyne incognita Chitwood.) seven days after the plant were transplanted to the pots. A. oligospora, C. musiformis and D. eudermata were found to be reliable as biocontrol agents, reducing the number of vermiform nematodes, swollen root, sausage shaped and galls in tobacco plant after 7, 15 and 30 days of infection with M. incognita. Treatment with NTF produced results that were comparable with Carbofuran® as a control agent in the reduction of the number of infections in tobacco plant caused by M. incognita in Nicotiana tabacum var. Deli 4. They also optimize the growth of the tobacco plants especially up to 15 days after infection.

The rate of nitrite reduction in leaves as indicated by O₂ and CO₂ exchange during photosynthesis.

Science.gov (United States)

Eichelmann, H; Oja, V; Peterson, R B; Laisk, A

2011-03-01

Light response (at 300 ppm CO(2) and 10-50 ppm O(2) in N(2)) and CO(2) response curves [at absorbed photon fluence rate (PAD) of 550 μmol m(-2) s(-1)] of O(2) evolution and CO(2) uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO(3)(-) or NH(4)(+) as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH(4)NO(3). Photosynthetic O(2) evolution in excess of CO(2) uptake was measured with a stabilized zirconia O(2) electrode and an infrared CO(2) analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO(2), mainly NO(2)(-), SO(4)(2-), and oxaloacetate. In NO(3)(-)-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O(2)-CO(2) flux difference rapidly increased to about 1 μmol m(-2) s(-1) at very low PADs and the process was saturated at 50 μmol quanta m(-2) s(-1). At higher PADs the O(2)-CO(2) flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m(-2) s(-1). In NH(4)(+)-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O(2) evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO(2)(-) which successfully competes with CO(2) reduction and saturates at a rate of about 1 μmol O(2) m(-2) s(-1) (9% of the maximum O(2) evolution rate). The high-PAD component of about 1 μmol O(2) m(-2) s(-1), superimposed on NO(2)(-) reduction, may represent oxaloacetate reduction. The roles of NO(2)(-), oxaloacetate, and O(2) reduction in the regulation of ATP/NADPH balance are discussed.

Silencing Nicotiana attenuata LHY and ZTL alters circadian rhythms in flowers.

Science.gov (United States)

Yon, Felipe; Joo, Youngsung; Cortés Llorca, Lucas; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

2016-02-01

The rhythmic opening/closing and volatile emissions of flowers are known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach has led to the identification of core circadian clock components in Arabidopsis thaliana, the involvement of these clock components in floral rhythms has remained untested, probably because of the weak diurnal rhythms in A. thaliana flowers. Here, we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents and move vertically through a 140° arc. We first measured N. attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission and pedicel movement, but not flower closing. We generated transgenic N. attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known to be core clock components. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N. attenuata coordinate these floral rhythms. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupinus, Conium and Nicotiana species.

Science.gov (United States)

Panter, K E; Keeler, R F; Bunch, T D; Callan, R J

1990-01-01

Three piperidine alkaloid containing plants, Conium maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupinus formosus (lunara lupine), induced multiple congenital contractures (MCC) and palatoschisis in goat kids when their dams were gavaged with the plant during gestation days 30-60. The skeletal abnormalities included fixed extension or flexure of the carpal, tarsal, and fetlock joints, scoliosis, lordosis, torticollis and rib cage abnormalities. Clinical signs of toxicity included those reported in sheep, cattle and pigs--ataxia, incoordination, muscular weakness, prostration and death. One quinolizidine alkaloid containing plant, Lupinus caudatus (tailcup lupine), on the other hand, which is also known to cause MCC in cows, caused only slight signs of toxicity in pregnant goats and no teratogenic effects in their offspring.

Genome-wide identification, subcellular localization and gene expression analysis of the members of CESA gene family in common tobacco (Nicotiana tabacum L.).

Science.gov (United States)

Xu, Zong-Chang; Kong, Yingzhen

2017-06-20

Cellulose-synthase proteins (CESAs) are membrane localized proteins and they form protein complexes to produce cellulose in the plasma membrane. CESA proteins play very important roles in cell wall construction during plant growth and development. In this study, a total of 21 NtCESA gene sequences were identified by using PF03552 conserved protein sequence and 10 AtCESA protein sequences of Arabidopsis thaliana to blast against the common tobacco (Nicotiana tabacum L.) genome database with TBLASTN protocol. We analyzed the physical and chemical properties of protein sequences based on some software or on-line analysis tools. The results showed that there were no significant variances in terms of the physical and chemical properties of the 21 NtCESA proteins. First, phylogenetic tree analysis showed that 21 NtCESA genes and 10 AtCESA genes were clustered into five groups, and the gene structures were similar among the genes that are clustered into the same group. Second, in all of the 21 NtCESA proteins the conserved zinc finger domain was identified in the N-terminus, transmembrane domains were identified in the C-terminus and the DDD-QXXRW conserved domains were also identified. Third, gene expression analysis results indicated that most NtCESA genes were expressed in roots and leaves of seedling or mature tissues of tobacco, seeds and callus tissues. The genes that clustered into the same group share similar expression patterns. Importantly, NtCESA proteins that are involved in secondary cell wall cellulose synthesis have two extra transmembrane domains compared with that involved in primary cell wall cellulose biosynthesis. In addition, subcellular localization results showed that NtCESA9 and NtCESA14 were two plasma membrane anchored proteins. This study will lay a foundation for further functional characterization of these NtCESA genes.

Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana.

Science.gov (United States)

Fernández, Aurora Piñas; Gil, Patricia; Valkai, Ildiko; Nagy, Ferenc; Schäfer, Eberhard

2005-05-01

To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.

Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia

Science.gov (United States)

Santoso; Thornburg

1998-02-01

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

Paid Maternity Leave and Breastfeeding Outcomes.

Science.gov (United States)

Mirkovic, Kelsey R; Perrine, Cria G; Scanlon, Kelley S

2016-09-01

Despite the benefits of breastfeeding, rates in the United States are low. Shorter maternity leave is associated with lower initiation and shorter durations of breastfeeding; however, little is known about how paid maternity leave may influence breastfeeding rates. We used data from the 2006-2010 U.S. National Survey of Family Growth on the most recent birth to employed women who delivered a child within the previous 5 years. Separate multivariable logistic regression models were used to describe the associations between paid leave duration (0, 1-5, 6-11, ≥ 12 weeks, maternity leave not taken) and three outcomes: 1) breastfeeding initiation, 2) 6-month duration, and 3) 6-month duration among initiators. Twenty-eight percent of prenatally employed women received no paid leave. Women who received 12 or more weeks of paid leave were more likely to initiate breastfeeding compared to women with no paid leave (87.3% vs 66.7%, adjusted odds ratio [aOR] 2.83 [95% confidence interval {CI} 1.23-6.48]). Similarly, women with 12 or more weeks of paid leave were more likely to breastfeed at 6 months, compared to women with no paid leave (24.9% vs 50.1%, aOR 2.26 [95% CI 1.20-4.26]). Among women who initiated breastfeeding, having received 12 or more weeks' paid leave increased the odds of breastfeeding for 6 or more months; however, the association was not statistically significant in the adjusted model (aOR 1.81 [95% CI 0.93-3.52]). Employed women who received 12 or more weeks of paid maternity leave were more likely to initiate breastfeeding and be breastfeeding their child at 6 months than those without paid leave. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Goodbye, Mandatory Maternity Leaves

Science.gov (United States)

Nation's Schools, 1972

1972-01-01

In precedent-setting decrees, courts and federal and State authorities have branded compulsory maternity leaves either unconstitutional or illegal. School administrators are urged to prod boards of education to adopt more lenient maternity leave policies -- now. (Author)

Retrotransposons of the Tnt1B family are mobile in Nicotiana plumbaginifolia and can induce alternative splicing of the host gene upon insertion.

Science.gov (United States)

Leprinc, A S; Grandbastien, M A; Christian, M

2001-11-01

Active retrotransposons have been identified in Nicotiana plumbaginifolia by their ability to disrupt the nitrate reductase gene in chlorate-resistant mutants selected from protoplast-derived cultures. In mutants E23 and F97, two independent insertions of Tnp2, a new retrotransposon closely related to the tobacco Tnt1 elements, were detected in the nitrate reductase gene. These two Tnp2 elements are members of the Tnt1B subfamily which shows that Tnt1B elements can be active and mutagenic in the N. plumbaginifolia genome. Furthermore, these results suggest that Tnt1B is the most active family of Tntl elements in N. plumbaginifolia, whereas in tobacco only members of the Tnt1A subfamily were found inserted in the nitrate reductase gene. The transcriptional regulations of Tnp2 and Tnt1A elements are most probably different due to non-conserved U3 regions. Our results thus support the hypothesis that different Nicotiana species contain different active Tntl subfamilies and that only one active Tntl subfamily might be maintained in each of these species. The Tnp2 insertion found in the F97 mutant was found to be spliced out of the nitrate reductase mRNA by activation of cryptic donor and acceptor sites in the nitrate reductase and the Tnp2 sequences respectively.