Progress 06/18/03 to 06/24/07
Outputs
Progress Report Objectives (from AD-416) Genetic analysis of the bean brown spot pathogen Pseudomonas syringae pv. syringae using functional genomics and molecular genetic techniques. Analysis of the tabtoxin microbial pathogenicity island via DNA sequencing and molecular genetics technologies. Develop a VIGS (virus induced gene silencing) approach to genetic analysis of tomato, potato and its wild relative Solanum bulbocastanum with the ultimate goal of identifying novel disease resistance genes. Approach (from AD-416) Our model organism of study is P. syringae pv. syringae, the causal agent of brown spot disease of bean. A novel TnlacZ reporter transposon delivery system will be used to identify genes regulated by the gacS/gacA two-component regulatory system. We have established that the gacS/gacA regulators are required for disease on bean, this approach will identify specific genes involved in the disease process. Analysis of the tabtoxin microbial pathogenicity island via DNA sequencing and molecular genetics technologies. We have cloned the tabtoxin biosynthetic cluster of Pseudomonas syringae. Tabtoxin biosynthetic genes will be identified through DNA sequencing, site-directed mutagenesis and testing for biological expression of tabtoxin. Develop a VIGS (virus induced gene silencing) approach to genetic analysis of potato and its wild relative Solanum bulbocastanum with the ultimate goal of identifying novel disease resistance genes. A novel system using plant RNA virus transient expression vectors allows us to analyze gene function by expressing RNA sequences (from cDNA or genomic clones) that will target and inactivate individual plant genes. Since this can be accomplished at the whole plant level, the phenotype resulting from the gene inactivation can be determined. Our goal is to expand the VIGS technology to potato and its wild relatives, and once developed, use gene silencing as a tool for the identification and isolation of disease resistance genes. Tomato will be used as an intermediate host for potato gene analysis due to its genetic relatedness to potato and the availability of the TRV VIGS system that functions well in tomato. Accomplishments Methods for effective real-time RT-PCR analysis of virus-induced gene silencing in tobacco and tomato. Virus-induced gene silencing (VIGS) is a method for gene inactivation that has a large potential for the identification of agriculturally important genes. We used real-time RT-PCR to quantitate the effectiveness of VIGS in two plant hosts. Our analysis of virus-induced gene silencing (VIGS) in tomato establishes the utility of real-time qRT-PCR for analysis of VIGS effectiveness and clearly indicates that successful VIGS will require improvement in viral vectors or selection of more permissive plant hosts. Our finding illustrates that VIGS can have a major impact in genetic studies of select crop plants, but will not easily be applied to all agronomically important plant species. This accomplishment is aligned with NP303, Component 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 5 Number of Newspaper Articles,Presentations for NonScience Audiences: 1
Impacts
(N/A)
Publications
- Rotenberg, D., Thompson, T.S., German, T.L., Willis, D.K. 2006. Methods for effective real-time RT-PCR analysis of virus-induced gene silencing. Journal of Virological Methods. 138:49-59.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Economic loss to agricultural crops due to disease-causing bacteria and plant viruses is a major problem in the U.S. Currently, adequate chemical or biological control of bacterial disease is not economically feasible or simply not available. There is no effective chemical control of plant viruses and the efficacy of insecticides that control insect virus vectors is decreasing as new pesticide resistance insects emerge. In order to more effectively protect our crops, we need to understand and identify at a molecular level those genetic traits within phytopathogenic bacteria that lead to disease. We also need to develop new approaches toward understanding virus-plant interactions. This will include the identification of novel plant resistance genes and identification of plant genes required
for virus replication. This program falls within Component IVB (Pathogen Biology, Genetics, Population Dynamics, Spread, and relationship with Host and Vectors-70%) and Component V (Host Plant Resistance to Disease-30%) of NP303, Plant Diseases. This research has identified numerous Pseudomonas syringae genes required for growth in the plant environment and the elicitation of disease on plants. The data generated in this work has been essential to research within other ARS units and academics world-wide. Gene discovery for viral diseases in tomato and potato is in collaboration through a SCA with the Department of Entomology at UW-Madison. Bacterial and viral diseases cost billions of dollars annually to U.S. agriculture. Breeding for resistance has been successful in a limited number of crop species but this resistance does not persist. Clearly, alternatives for the control of bacterial diseases need to be developed. Our mission has been to identify those genetic factors within
bacteria that lead to the development of disease on the plant. One of our model organisms is P. syringae pv. syringae - the causal agent of brown spot disease that costs bean growers in Wisconsin millions of dollars annually. Better knowledge of the biological mechanisms of virulence will enable us to develop unique forms of disease control. The analysis of the gacS/gacA regulatory net that is required for lesion formation and toxin production in P. syringae will directly impact research in a number of USDA and academic laboratories. Homologs of gacS and gacA have been found in a number of members of the genus Pseudomonas including the biocontrol agent P. fluorescens and the human pathogen P. aeruginosa. In an extension of this research, we have recently begun investigating the use of virus-induced gene silencing (VIGS) for the identification and analysis of genes in tomato and potato that encode disease resistance. In order to improve this technique in tomato and potato, we are
investigating plant genes that are required for virus replication and the development of new viral vectors that provide enhanced VIGS effectiveness. Successful completion of this project will enable a reduction in the amount of insecticide applied to agricultural fields for the control of virus diseases with a saving of millions of dollars to the American farmer. It will also provide information important to plant breeders for the control of plant pathogens. 2. List by year the currently approved milestones (indicators of research progress) FY2004 Continue isolation and preliminary characterization of TnlacZ fusions in genes regulated by gacS/gacA regulatory loci. Finish sequencing of the tabtoxin biosynthetic cluster. Establish the utility of PVX and TRV-based VIGS in potato and S. bulbocastanum and begin analysis of R1 gene. FY2005 Exchange insertions of interest into wild-type and mutant backgrounds to determine regulation of insertions (i.e. are the genes of interest regulated by
salA and or gidA). Finish analysis of the carnosinase gene and publish result. Continue R1 gene and begin analysis of RB locus. FY2006 Determine role of regulated genes in lesion formation on bean. Continue analysis of RB locus and begin analysis of cDNA generated by subtractive hybridization. FY2007 Finish studies, transfer technology. Finish studies, transfer technology. 4a List the single most significant research accomplishment during FY 2006. Accomplishment is aligned with NP303, Component 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Sequencing of the biosynthetic gene cluster for the beta-lactam antibiotic tabtoxin in Pseudomonas syringae. Tabtoxin is a unique antibiotic that affects bacteria, fungi, plants, and animals. We established that the tabtoxin biosynthetic region contains 20 putative genes. We located the resistance to the tabtoxin antibiotic within a gene cluster on one end of the region. Genes at other locations in the region appear
to encode the ability of the biosynthetic cluster to move from bacteria to bacteria. This work identifies a novel antibiotic synthesis pathway and a novel resistance mechanism. It also illustrates a potential mechanism for the movement of this powerful plant toxin between plant pathogenic bacteria. Identification of the bacterial resistance mechanism to tabtoxin may enable the engineering of resistant crop plants. Tabtoxin also has potential use in preventing damage resulting from stroke in humans. 5. Describe the major accomplishments to date and their predicted or actual impact. Accomplishment is aligned with NP303, Component 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen and Component 2B: Plant-Microbe-Vector Interactions Our analysis of lesion formation and toxin production in Pseudomonas syringae led to the discovery of the gacS/gacA transcriptional regulatory network (Milestone 1 & 2). These genes are highly conserved within the genus Pseudomonas.
Our work has impacted directly on other USDA labs analyzing the genetics of biocontrol by Pseudomonas fluorescens, in addition to laboratories investigating the genetics of pathogenicity in P. viridiflava, P. tolaasii, P. syringae pv. tabaci and Erwinia carotovora. This work has also impacted the genetic analysis of the human pathogens Pseudomonas aeruginosa and Vibrio cholerae. Our analysis of virus-induced gene silencing (VIGS) in tomato establishes the utility of real-time qRT- PCR for analysis of VIGS effectiveness and clearly indicates that successful VIGS will require improvement in viral vectors or selection of more permissive plant hosts (Milestone #3). This work impacts gene discovery and gene function analysis in plants. This work addressed the National Program Action Plan for genome characterization and genetic improvement. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user
(industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Genetic sequences were deposited in GenBank, making them available to the global research community free of charge. Techniques for real-time qRT-PCR analysis of transcript amounts in plants have been transferred to several labs at UW-Madison.
Impacts
(N/A)
Publications
- Tsitsigiannis, D.I., Kunze, S., Willis, D.K., Feussner, I., Keller, N.P. 2005. Aspergillus infection inhibits the expression of peanut 13s-hpode seed lipoxygenases. Molecular Plant Microbe Interactions. 18:1081-1089.
- Simon, H. M., C. E. Jahn, L. T. Bergerud, M. K. Sliwinski, P. J. Weimer, D. K. Willis, and R. M. Goodman. 2005. Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots. Appl. Environ. Microbiol. 71:4751-4760.
- Kinscherf, T.G., Willis, D.K. 2005. The biosynthetic gene cluster for the beta-lactam antibiotic tabtoxin in Pseudomonas syringae. Journal of Antibiotics. 58:817-821.
- Barta, T.M., Willis, D.K. 2005. Biological and molecular evidence that pseudomonas syringae pathovars coronafaciens, striafaciens, and garcae are likely the same pathovar. Journal of Phytopathology. 153:492-499.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Economic loss to agricultural crops due to disease-causing bacteria and plant viruses is a major problem in the U.S. Currently, adequate chemical or biological control of bacterial disease is not economically feasible or simply not available. There is no effective chemical control of plant viruses and the efficacy of insecticides that control insect virus vectors is decreasing as new pesticide resistance insects emerge. In order to more effectively protect our crops, we need to understand and identify at a molecular level those genetic traits within phytopathogenic bacteria that lead to disease. We also need to develop new approaches toward understanding virus-plant interactions. This will include the identification of novel plant resistance genes and identification of plant genes required for virus
replication. This program falls within Component IVB (Pathogen Biology, Genetics, Population Dynamics, Spread, and relationship with Host and Vectors-70%) and Component V (Host Plant Resistance to Disease-30%) of NS 303. This research has identified numerous Pseudomonas syringae genes required for growth in the plant environment and the elicitation of disease on plants. The data generated in this work has been essential to research within other ARS units and academics world-wide. Gene discovery for viral diseases in tomato and potato is in collaboration through a SCA with Dr. Tom German in the Department of Entomology at UW-Madison. Bacterial and viral diseases cost billions of dollars annually to U.S. agriculture. Breeding for resistance has been successful in a limited number of crop species but this resistance does not persist. Clearly, alternatives for the control of bacterial diseases need to be developed. Our mission has been to identify those genetic factors within bacteria
that lead to the development of disease on the plant. One of our model organisms is P. syringae pv. syringae - the causal agent of brown spot disease that costs bean growers in Wisconsin millions of dollars annually. Better knowledge of the biological mechanisms of virulence will enable us to develop unique forms of disease control. The analysis of the gacS/gacA regulatory net that is required for lesion formation and toxin production in P. syringae will directly impact research in number of USDA and academic laboratories. Homologs of gacS and gacA have been found in a number of members of the genus Pseudomonas including the biocontrol agent P. fluorescens and the human pathogen P. aeruginosa. In an extension of this research, we have recently begun investigating the use of virus- induced gene silencing (VIGS) for the identification and analysis of genes in tomato and potato that encode disease resistance. In order to improve this technique in tomato and potato, we are investigating
plant genes that are required for virus replication and the development of new viral vectors that provide enhanced VIGS effectiveness. Successful completion of this project will enable a reduction in the amount of insecticide applied to agricultural fields for the control of virus diseases with a saving of millions of dollars to the American farmer. It will also provide information important to plant breeders for the control of plant pathogens. 2. List the milestones (indicators of progress) from your Project Plan. FY2004 Continue isolation and preliminary characterization of TnlacZ fusions in genes regulated by gacS/gacA regulatory loci. Finish sequencing of the tabtoxin biosynthetic cluster. Establish the utility of PVX and TRV-based VIGS in potato and S. bulbocastanum and begin analyis of R1 gene. FY2005 Exchange insertions of interest into wild-type and mutant backgrounds to determine regulation of insertions (i.e. are the genes of interest regulated by salA and or gidA). Finish
analysis of the carnosinase gene and publish result. Continue R1 gene and begin analysis of RB locus. FY2006 Determine role of regulated genes in lesion formation on bean. Continue analysis of RB locus and begin analysis of cDNA generated by subtractive hybridization. FY2007 Finish studies, transfer technology. Finish studies, transfer technology. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Exchange insertions of interest into wild-type and mutant backgrounds to determine regulation of insertions (i.e. are the genes of interest regulated by salA and or gidA). Milestone Fully Met 2. Finish analysis of the carnosinase gene and publish result. Milestone Substantially Met 3. Continue R1 gene analysis and begin analysis of RB locus. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008).
What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY2006 Determine role of regulated genes in lesion formation on bean. We will mutate genes of interest in wild-type bacteria and establish the effect (if any) on lesion formation. Continue analysis of RB locus and begin analysis of cDNA generated by subtractive hybridization. Continue the Real-Time qrt-PCR analysis of the gacS/gacA regulon in Pseudomonas syringae. Continue efforts to improve VIGS of tomato by silencing RDR1 gene of tomato and investigate use of altenate viral vectors. FY2007 Finish studies, transfer technology. Finish analysis of select gacS/gacA regulated genes, transfer technology (i.e. publish results. Finish studies, transfer technology. Publish efforts to improve VIGS of tomato by silencing RDR1 gene or using alternate viral vectors. FY2008 Project will end in 2007. 4a What was the single most significant accomplishment this past year? Real-time qRT-PCR analysis of
virus-induced gene silencing in Nicotiana benthamiana and tomato VIGS is a powerful technique with tremendous potential for use in gene discovery and gene function analysis in plants. We established that the variability of VIGS effectiveness in tomato is likely due to limited viral spread in the plant host. The problem for VIGS analysis is that the technique works very well in a single host (N. benthamiana) but is highly variable in agronomically important crop plants. Using real-time qRT-PCR, we established that VIGS variability in tomato was not due to the degree of transcript reduction or instability of the VIGS vector. This work was accomplished through a SCA between the labs of D. K. Willis (ARS Vegetable Crops Research Unit) and T. L. German (Department of Entomology, UW-Madison). The impact of this accomplishment is to quatitatively demostrate that VIGS using current technology is too variable to use in a fully effective manner in plant hosts other than N. benthamiana. 5.
Describe the major accomplishments over the life of the project, including their predicted or actual impact. Our analysis of lesion formation and toxin production in Pseudomonas syringae led to the discovery of the gacS/gacA transcriptional regulatory network (Milestone 1 & 2). These genes are highly conserved within the genus Pseudomonas. Our work has impacted directly on other USDA labs analyzing the genetics of biocontrol by Pseudomonas fluorescens, in addition to laboratories investigating the genetics of pathogenicity in P. viridiflava, P. tolaasii, P. syringae pv. tabaci and Erwinia carotovora. This work has also impacted the genetic analysis of the human pathogens Pseudomonas aeruginosa and Vibrio cholerae. Our analysis of VIGS in tomato establishes the utility of real-time qRT-PCR for analysis of VIGS effectiveness and clearly idicates that successful VIGS will require improvement in viral vectors or selection of more permissive plant hosts (Milestone #3). This work impacts
gene discovery and gene function analysis in plants. This work addressed the National Program Action Plan for genome characterization and genetic improvement. Actual impact/potential impact: None 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Genetic sequences were deposited in GenBank, making them available to the global research community free of charge. Techniques for real-time qRT-PCR analysis of transcript amounts in plants have been transferred to several labs at UW-Madison.
Impacts
(N/A)
Publications
- Rotenberg, D., Thompson, T.S., German, T.L., Willis, D.K. 2005. Real-time qRT-PCR analysis of virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato [abstract]. American Society for Virology Meeting. p. 211-212.
- Willis, D.K., Whitfield, A.E., Kumar, K.K., Ullman, D.E., Rotenberg, D., German, T.L. 2005. Tomato spotted wilt virus glycoprotein GN inhibits virus acquisition and transmission by thrips [abstract]. American Society for Virology Meeting. p. 111.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Economic loss of agricultural crops due to disease-causing bacteria and plant viruses is a major problem in the U.S. Currently, adequate chemical or biological control of bacterial disease is not economically feasible or simply not available. There is no effective chemical control of plant viruses and the efficacy of insecticides that control insect virus vectors is decreasing as new pesticide resistance insects emerge. In order to more effectively protect our crops, we need to understand and identify at a molecular level those genetic traits within phytopathogenic bacteria that lead to disease. We also need to develop new approaches toward understanding virus-plant interactions. This will include the identification of novel plant resistance genes and identification of plant genes required for virus
replication. This program falls within Component IVB (Pathogen Biology, Genetics, Population Dynamics, Spread, and relationship with Host and Vectors-70%) and Component V (Host Plant Resistance to Disease-30%) of NP 303. This research has identified numerous Pseudomonas syringae genes required for growth in the plant environment and the elicitation of disease on plants. The data generated in this work has been essential to research within other ARS units and academics world-wide. For the analysis of virulence in P. syringae, SCAs are in place with Drs. Susan Hirano and Walt Stevenson in the Department of Plant Pathology at UW-Madison. Gene discovery for viral diseases in tomato and potato is in collaboration through a SCA with Dr. Tom German in the Department of Entomology at UW- Madison. Bacterial and viral diseases cost billions of dollars annually to U.S. agriculture. Breeding for resistance has been successful in a limited number of crop species but this resistance does not
persist. Clearly, alternatives for the control of bacterial diseases need to be developed. Our mission has been to identify those genetic factors within bacteria that lead to the development of disease on the plant. One of our model organisms is P. syringae pv. syringae - the causal agent of brown spot disease that costs bean growers in Wisconsin millions of dollars annually. Better knowledge of the biological mechanisms of virulence will enable us to develop unique forms of disease control. The analysis of the gacS/gacA regulatory net that is required for lesion formation and toxin production in P. syringae will directly impact research in number of USDA and academic laboratories. Homologs of gacS and gacA have been found in a number of members of the genus Pseudomonas including the biocontrol agent P. fluorescens and the human pathogen P. aeruginosa. In an extension of this research, we have recently begun the identification and analysis of genes in tomato that encode resistance to
bacterial disease. We are using virus induced gene silencing (VIGS) for this research. In order to improve this technique in tomato, we are investigating plant genes that are required for virus replication. Successful completion of this project will enable a reduction in the amount of insecticide applied to agricultural fields for the control of virus diseases with a saving of millions of dollars to the American farmer. It will also provide information important to plant breeders for the control of bacterial pathogens. 2. List the milestones (indicators of progress) from your Project Plan. MILESTONES 2004: Continue isolation and preliminary characterization of TnlacZ fusions in genes regulated by gacS/gacA regulatory loci. Finish sequencing of the tabtoxin biosynthetic cluster and publish results. Virus induced gene silencing (VIGS) driven gene discovery in potato and tomato. Establish the utility of PVX based VIGS in potato and S. bulbocastanum and begin analysis of potato R genes.
2005: Exchange insertions of interest in putative regulated genes into wild- type and mutant backgrounds and determine regulation of insertions (i.e. are the genes of interest regulated by salA and or gidA. Finnish analysis of the carnosinase gene that regulates tabtoxin production and results Continue resistance gene and viral interaction gene analysis in tomato and potato using VIGS. 2006: Determine role of gacS/gacA regulated genes in lesion formation on bean. Continue analysis of RB locus and begin analysis of cDNA generated by subtractive hybridization 2007: Finish analysis of select gacS/gacA regulated genes, transfer technology (i.e. publish results. Finish VIGS analysis of bacterial resistance and viral replication genes in tomato and transfer technology (i.e. publish results). 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate which ones were not fully or
substantially met, briefly explain why not, and your plans to do so. Milestone: Continue isolation and preliminary characterization of TnlacZ fusions in genes regulated by gacS/gacA regulatory loci. Progress: Milestone complete. Finish sequencing of the tabtoxin biosynthetic cluster and submit results for publication. Progress: Milestone complete. VIGS driven gene discovery in potato and tomato Establish the utility of PVX based VIGS in potato and S. bulbocastanum and begin analysis of potato R genes. Progress: PVX found to be unsuitable for VIGS analysis in potato or tomato. Switched to tobacco rattle virus (TRV) based VIGS system that works well in tomato but poorly in potato. Attempt to introduce potato R genes into tomato failed as the RB gene caused sterility in tomato. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2005: Begin the
exchange of insertions of interest in putative gacS/gacA regulated genes into wild-type and mutant backgrounds and determine regulation of insertions (i.e. are the genes of interest regulated by salA and or gidA.) Begin the analysis of the gacS/gacA regulon in Pseudomonas syringae using Real-Time qrt-PCR. We have recently developed methods for using Real-Time quantitative reverse-transcription PCR to analyze transcript levels in Pseudomonas syringae. Finish analysis of the carnosinase gene which regulates tabtoxin production and prepare results for publication. Begin efforts to improve VIGS of tomato by silencing RdRp1 gene of tomato. This gene has recently been identified as defective in Nicotiana benthamiana and this plant is an excellent host for VIGS silencing. Improvement of VIGS in tomato would bring a very useful scientific technique to an agronomically important crop. Begin the analysis of the effect of the tomato Prf gene resistance on bacterial populations on tomato using
VIGS. Through our SCA with Susan Hirano in the Department of Plant Pathology at UW-Madison, we have found an effect of the Prf/Pto resistance locus on bacterial populations in the field. We will use VIGS to silence Prf and examine if this gene is responsible for the reduction in bacterial growth. Begin the analysis of putative tomato genes required for viral replication using VIGS with quatitation of effectiveness using Real-Time qrt-PCR. Through our SCA with Tom German in the Department of Entomology at UW-Madison, we will analyze tomato genes that physically interact with tomato spotted wilt virus for a role in viral replication. 2006: Determine role of gacS/gacA-regulated genes in lesion formation on bean. We will mutate genes of interest in wild-type bacteria and establish the effect (if any) on lesion formation. Continue the Real-Time qrt-PCR analysis of the gacS/gacA regulon in Pseudomonas syringae. Publish results of analysis from previous year. Complete analysis of the tomato
Prf gene effect on bacterial populations and prepare results for publication. Finish efforts to improve VIGS of tomato by silencing RdRp1 gene of tomato and analyze results. Continue analysis of putative tomato genes required for viral replication using VIGS with quatitation of effectiveness using Real-Time qrt-PCR. 2007: Finish analysis of select gacS/gacA regulated genes, transfer technology (i.e. publish results. Continue the Real-Time qrt-PCR analysis of the gacS/gacA regulon in Pseudomonas syringae. Publish results of analysis from previous year. If successful, publish efforts to improve VIGS of tomato by silencing RdRp1 gene of tomato. Finish VIGS analysis of viral replication genes in tomato and transfer technology (i.e. publish results). 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004. We submitted our analysis of the 27 kb tabtoxin biosynthetic cluster of P. syringae biosynthetic cluster for
publication. The product of this gene cluster is tabtoxinine-beta-lactam, a potent inhibitor of glutamine synthetase. Through our work, we have identified genes required for synthesis of and resistance to this potent toxin. This is new information and a finding that will impact research on plant-bacterial interactions. This toxin has potential medical uses in the treatment of stroke. B. Other Significant Accomplishment(s), if any: We developed techniques for the use of Real-Time qrt-PCR for the analysis of transcription in both plants and bacteria. This is a relatively new technique with tremendous potential for the quantitation of DNA and RNA. We used this method to quantitate the effect of VIGS silencing in both N. benthamiana and tomato. Our results show that high level of endogenous gene silencing produced in N. benthamiana compared to tomato is consistent with the dramatically increased expression of viral genes (viral replication) in the former. We now have a basis to pursue the
improvement of VIGS technology in tomato and potato. C. Significant activities that support special target populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Describe the major accomplishments over the life of the project, including their predicted or actual impact.NB Our analysis of lesion formation and toxin production in Pseudomonas syringae led tothe discovery of the gacS/gacA transcriptional regulatory network. These genes are highly conserved within the genus Pseudomonas. Our work has impacted directly on other USDA labs analyzing the genetics of biocontrol by Pseudomonas fluorescens, in addition to laboratories investigating the genetics of pathogenicity in P. viridiflava, P. tolaasii, P. syringae pv. tabaci and Erwinia carotovora. This work has also impacted the genetic analysis of the human pathogens Pseudomonas aeruginosa and Vibrio cholerae. This work addressed the National Program Action Plan
for genome characterization and genetic improvement. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Genetic sequences were deposited in GenBank, making them available to the global research community free of charge. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Results of our field survey of viral disease in Wisconsin snap beans was presented at the 2004 Wisconsin Fertilizer, Aglime and Pest Management Conference. German,T.L., Thompson, A., Willis, D.K., 2004. Statewide distribution of virus problems on processing beans. In: Proceedings 2004 Wisconsin Fertilizer, Aglime, & Pest Management Conference. Madison, WI, 291-293.
Impacts
(N/A)
Publications
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