Progress 12/23/02 to 04/22/07
Outputs
Progress Report Objectives (from AD-416) The overall objectives are to characterize the N disease resistance gene and understand the molecular nature of the N gene to elucidate how R gene products recognize pathogens, transduce signals, and how plant defense responses block pathogen growth. The specific objectives are to determine the functional relationship of the N encoded gene, determine the structural and functional relationship of the N encoded protein domains, and isolate NR resistance gene from N repanda. Approach (from AD-416) Characterization of N gene expression and structure function relationships will include construction of Ti-plasmid vectors bearing in- vitro constructed versions of the N gene, generation of transgenic plants bearing these N alleles using Agrobacterium tumefaciens-mediated transformation procedures, and challenge of transgenic plants and their progeny with TMV to assess the TMV resistance activity encoded in each N gene construct. The NR gene of N. repanda will be isolated using homology based screening of a lambda library and PRC approaches to amplify NR gene sequences. Once isolated the NR gene will be characterized. BSL-1; 7/1/05 REPLACES: 5335-22000-005-00D (12/02). Significant Activities that Support Special Target Populations This project ended in April 2007 and was replaced by CRIS #5335-22000-007- 00D. See that annual report for additional information. Accomplishments This project ended in April 2007 and was replaced by CRIS #5335-22000-007- 00D. See that annual report for additional information. 1. Cellular components of the tobacco mosaic virus (TMV) disease resistance N signaling pathway. The structurally conserved products of plant disease resistance genes can collectively mediate resistance to a diverse range of pathogens and suggest that plants may have evolved common resistance signal transduction mechanisms. Scientists in the PGEC, Albany, CA used classical genetic and reverse genetic: virus induced gene-silencing (VIGS) approaches and identified several genes encoding components of the N- mediated signaling pathway including genes encoding MAP kinases, NPK1, SIPK, WIPK, and MEKK1. A caspase-like protease and a predicted lipase- domain containing protein, EDS1 were also identified. These studies will lead to an understanding of how a limited number of structural classes of R proteins that cope with the potentially large array diverse of pathogens encountered by plants. NP Component: The research in this project falls under NP 303, Component 3, "Plant Disease Resistance." The problem addressed is relevant to Component 3 Plant Disease Resistance, Problem Statement 3A: Mechanisms of Plant Disease Resistance. 2. Role of resistance pathway components in multiple disease resistance pathways. Identification and understanding of common signal transduction mechanisms for resistance to multiple pathogens is needed for development of durable and broad-spectrum pathogen resistant crops. Sciedntists in the PGEC, Albany, CA used transgenic, genetic and pathogen challenge approaches and identified the role of N-mediated signaling pathway components described in Accomplishment 1 in the tomato Bs2, Pto, Ve, and Cf9-mediated resistance signaling pathways. Knowledge of the role of resistance signaling pathway components in several R-gene resistance pathways is being used for investigations of cellular mechanisms of plant disease resistance and development of novel strategies for broad-spectrum pathogen resistance for crop protection. NP Component: The research in this project falls under NP 303, Component 3, "Plant Disease Resistance." The problem addressed is relevant to Component 3 Plant Disease Resistance, Problem Statement 3A: Mechanisms of Plant Disease Resistance. 3. Role of cellular disease resistance signaling pathway components. The identification and characterization of cellular components that interact with R-genes and resistance signaling components that determine the events in pathogen defense is needed to understand key cellular proteins and processes functioning to mediate resistance and susceptibility. Scientists in PGEC Lab at Albany, CA used yeast two- hybrid and over-expression expression analysis of the tomato EDS1 and N protein, required for pathogen resistance and defense signaling, to identify, verify and characterize cellular resistance pathway proteins. Understanding the interactions of products of resistance signaling pathway genes and R-proteins with other cellular proteins to form functional protein complexes and impart plant immunity to pathogenic microbes is needed to improve plant health and to leverage natural plant innate immune responses for safer environmental practices for food production. NP Component: The research in this project falls under NP 303, Component 3, "Plant Disease Resistance." The problem addressed is relevant to Component 3 Plant Disease Resistance, Problem Statement 3A: Mechanisms of Plant Disease Resistance. Significant Activities that Support Special Target Populations We integrated outreach activities with our research, including training programs for underrepresented Native American and Hispanic students in hands-on, computer based approaches to explore the fundamental concepts gene structure-function and the genetic basis of diversity. We designed a website that presents information and resources from her outreach activities http://outreach.potatogenome.org/. Technology Transfer Number of U.S. Patents granted: 1 Number of Web Sites managed: 4 Number of Non-Peer Reviewed Presentations and Proceedings: 2 Number of Newspaper Articles,Presentations for NonScience Audiences: 3
Impacts
(N/A)
Publications
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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? Each year billions of dollars are lost by farmers worldwide, due to plant disease caused by viruses, bacteria fungi, oomycetes, and nematodes. Infectious plant diseases are a serious problem, have caused devastating crop losses resulting in human suffering. Protection of crops from disease can significantly improve agricultural production, and protect resources. Our objectives are to understand the mechanism of disease resistance to aid in development of integrated management strategies to combat plant diseases. The current knowledge of the mechanism of disease resistance is inadequate to develop integrated management strategies to combat plant diseases using natural and environmentally friendly mechanisms. Mechanisms to control major economically important diseases of plants are not available and the
need to develop environmentally safe and effective methods is critical. The 'gene-for-gene' hypothesis was proposed to explain the observation that in plants, resistance to a specified pathogen is frequently correlated with a genetically defined interaction between a plant resistance gene (R gene) and a corresponding pathogen avirulence gene (Avr gene). All plants harbor many R genes each specifying resistance to a different pathogen or group of pathogens. The guard hypothesis explains these gene-for-gene interactions: Pathogen avirulence gene products trigger host resistance by interacting with cellular complexes composed of R gene products that are complexed with and 'guarding' cellular proteins that serve as virulence targets. In the absence of an active resistance protein the pathogen avirulence protein(s) interact with cellular virulence targets and modify cellular signaling, metabolism and suppress host defenses resulting in disease. The cloning and characterization of dozens of
plant disease resistance genes has overcome a major stumbling block in the elucidation of the molecular basis of disease resistance to a wide range of phytopathogens. Future challenges include determining how R gene products recognize pathogen avr products and how the plant defense response blocks pathogen growth. The basic knowledge obtained from this research will aid the quest to produce novel forms of genetically based durable disease resistance that can withstand pathogen attack and reduce the use of environmentally damaging pesticides. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1: Conduct genetic screens of FN N-tomato seedlings Yrs: 1-2 Positional cloning of suppressor mutant gene. Yrs 2-3 Characterize tomato mutants. Yr 3 Publish characterization of tomato mutant. Yr 3 Microarray analysis of mutant tomato and silenced plants. Yr 3 Objective 2: Screen yeast two-hybrid library with baits. Yr 3-5 Objective 3: Silence genes identified by
yeast two hybrid analysis. Yr 4-5 Submit manuscript on silencing in the N pathway. Yr 2 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. Positional cloning of suppressor mutant gene. Milestone Fully Met 2. Publish characterization of tomato mutant. Milestone Fully Met 3. Further characterize mutant. Milestone Substantially Met 4. Microarray analyses. Milestone Substantially Met 5. Yeast two hybrid screen. Milestone Substantially Met 6. Submit manuscript on silencing in the N pathway. Milestone Fully 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? FY 2006 1. Functional analysis of R gene signaling component, Le-EDS1: Identify tomato cellular proteins that interact with EDS1. Construct yeast two hybrid EDS1 "bait"
vectors for yeast two hybrid screens of tomato yeast two-hybrid cDNA library. Begin library screens. 2. Role Le-EDS1 mediated signaling in non-TIR R gene pathways: Cross Le-eds1-1 mutant to different tomato cultivars each bearing either Pto, Bs2, or Cf4 R genes. 3. Role of EDS1 interacting proteins in disease resistance: Vectors construction initiated for testing candidate Eds1 interacting protein genes, identified by yeast two hybrid analysis, for silencing and over-expression in wild-type and transgenic Solanaceae plants. FY 2007 1. Functional analysis of R gene signaling component, Le-EDS1: Conduct yeast two hybrid screens using EDS1 "bait" vector, identify and verify clones. 2. Test the role of Le-EDS1 gene in other R gene signaling pathways: determine the genotype (Eds1/eds/R) of progeny derived from crosses between Le-eds1-1 mutant and tomato cultivars for the Pto, Bs2, or Cf4 R genes. 3. Role of EDS1 interacting proteins in disease resistance: Genes encoding candidate Eds1
interacting proteins, identified by yeast two hybrid analysis, will be silenced using transient gene silencing method, virus induced gene silencing: VIGS. FY 2008 1. Functional analysis of R gene signaling component, Le-EDS1: Conduct additional yeast two hybrid screens using EDS1 "bait" vectors, identify, verify and characterize clones. 2. Test the role of Le-EDS1 gene in other R gene signaling pathways: Challenge progeny (Eds1/eds/R/r) of progeny derived from crosses between Le-eds1-1 mutant and tomato cultivars with panel of pathogens and evaluate levels of resistance and systemic acquired resistance. 3. Role of EDS1 interacting proteins in disease resistance: Genes encoding candidate Eds1 interacting proteins, identified by yeast two hybrid analysis, will stably silenced using RNAi and over-expression in Solanaceae. 4a What was the single most significant accomplishment this past year? Defining Disease Resistance Pathways In Plants. Gongshe Hu and Amy deHart, postdoctoral
fellows, identified a tomato mutant, sun1-1, that is defective in the N-TMV signaling pathway and determined that the sun1-1 mutation occurred in the Eds1 gene. We determined that tomato EDS1 lies upstream of salicylic acid production and is required for resistance mediated by TIR class resistance genes and the receptor-like resistance gene Ve. sun1-1 plants show enhanced susceptibility to TMV, providing evidence that the intersection of general and resistance gene-mediated pathways is conserved a Solanaceous species. Understanding the mechanism of disease resistance is vital for developing integrated management strategies to combat plant diseases. 4b List other significant accomplishments, if any. Evolutionary Studies of Disease Resistance Hotspots in Solanaaceae Physical maps were produced by postdoctoral fellow, Hanhui Kuang for the major late blight resistance locus on chromosome 5 of the wild potato species Solanum demissum, the origin of most of the known late blight resistance
genes. Three distinct resistance gene families were identified at the chromosome 5 resistance locus. We determined that the R1 homologues form three independent groups of fast-evolving Type I resistance genes. Understanding the evolutionary processes leading to generation of new R gene recognitional specificity of genetically polymorphic pathogen strains is important for understanding R gene diversity. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This work will provide the foundation for establishment of a plant disease resistance database to support broad based research and establishment of genetic resources to address and reduce crop losses caused by pathogen diseases. This work will also provide a framework for establishment of plant disease resistance resources and database to support development of phyto-sensors for detection of pathogens that threaten the quality and safety of food and human health. The genetic
manipulation of genes for disease resistance by plant breeders has been employed as a major method to control plant disease since the turn of the century. However, breeders are in need of additional and robust genetics for crop protection. The recent molecular cloning of several plant genes that confer disease resistance to a diverse range of pathogens has revealed that the encoded proteins have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms for expressing disease resistance to a wide range of unrelated pathogens. The characterization of the molecular signals involved in pathogen recognition and the elucidation of the molecular events specifying the expression of resistance may lead to the development of novel strategies for disease control. Attaining these objectives will provide benefits to farmers, and the public and will result in increased economic growth by helping to overcome the impact of plant disease.
New natural genetic strategies expected to control diseases and reduce dependency on pesticides and chemicals. The identification of genes for plant immunity to pathogenic microbes and improved understanding of natural innate immune responses and plant health is expected to lead to safer environmental practices for food production. Moreover this effort will provide the foundation of a disease resistance database providing a comprehensive resource for researchers in ARS and national and international research labs. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). News Focus Article. "Taking the Bite Out of Potato Blight" 2002. Science, 298:1702-1704. November 29, 2002 Publications Kuang, H., Wei, F., Marano, M. R., Wirtz, U., Wang, X., Liu, J., Shum, W. P., Zaborsky, J., Tallon, L., Rensink, W., Iobst, S., Zhang, P., Tornqvist, C. E., Tek, A.,
Bamberg, J., Helgeson, J., Fry, W., You, F., Luo, M. C., Jiang, J., Buell, R. C. and Baker, B. 2005. The R1 disease resistance gene cluster contains three groups of independently evolving, Type I R1 homologues and shows substantial structural variation among haplotypes of Solanum demissum. (The Plant Journal) PRESENTATIONS 09/30/2004 "Genomic and Molecular Genetic Studies of Plant Innate Immunity." ARS Symposium on Scientific Excellence with Impact: The Future of Food and Agricultural Research in the West. Treasure Island, San Francisco, CA. 10/23/2004 "Genomic and Molecular Genetic Studies of Plant Innate Immunity." Department of Plant Pathology, University of Minnesota, St. Paul, MN. 01/23/20005 "Genomic and Molecular Genetic Studies of Plant Innate Immunity." Gordon Research Conference Chemical & Biological Terrorism Defense, Santa Barbara CA. 03/16/2005 "Genomic and Molecular Genetic Studies of Plant Innate Immunity." University of California , Riverside, CA. 04/08/2005
"Genomic and Molecular Genetic Studies of Plant Innate Immunity." Williams College, Williamstown, MA. 04/23/2005 "Genomic and Molecular Genetic Studies of Plant Innate Immunity." NSF Potato Genome Annual Meeting, St. Paul, MN.
Impacts
(N/A)
Publications
- Rojo, E., Martin, R., Carter, C., Zouhar, J., Pan, S., Plotnikova, J., Jin, H., Paneque, M., Sanchez-Serrano, J., Baker, B.J., Ausubel, F.M., Raikhel, N.V. 2004. VPEy Exhibits a Caspase-like Activity that Contributes to Defense against Pathogens. Current Biology, 14:1897-1906.
- Hu, G., Dehart, A.K., Li, Y., Ustach, C., Handley, V., Navarre, R., Hwang, C.F., Aegerter, B.J., Williamson, V., Baker, B.J. 2005. EDS1 in tomato is required for resistance mediated by TIR-class R genes and the receptor- like R gene Ve. Plant Journal 42(3):376-91.
- Huang, S., Van Der Vossen, E.A., Kuang, H., Vleeshouwers, V.G., Zhang, N., Borm, T.J., Van Eck, H.J., Baker, B.J., Jacobsen, E., Visser, R.G. 2005. Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. Plant Journal 42(2):251-61.
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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? The current knowledge of the mechanism of disease resistance is inadequate to develop integrated management strategies to combat plant diseases using natural and environmentally friendly mechanisms. Mechanisms to control major economically important diseases of plants are not available and the need to develop environmentally safe and effective methods is critical. The 'gene-for-gene' hypothesis was proposed to explain the observation that in plants, resistance to a specified pathogen is frequently correlated with a genetically defined interaction between a plant resistance gene (R gene) and a corresponding pathogen avirulence gene (Avr gene). All plants harbor many R genes each specifying resistance to a different pathogen or group of pathogens. The guard hypothesis explains these gene-for-gene
interactions: Pathogen avirulence gene products trigger host resistance by interacting with cellular complexes composed of R gene products that are complexed with and 'guarding' cellular proteins that serve as virulence targets. In the absence of an active resistance protein the pathogen avirulence protein(s) interact with cellular virulence targets and modify cellular signaling, metabolism and suppress host defenses resulting in disease. The cloning and characterization of dozens of plant disease resistance genes has overcome a major stumbling block in the elucidation of the molecular basis of disease resistance to a wide range of phytopathogens. Future challenges include determining how R gene products recognize pathogen avr products and how the plant defense response blocks pathogen growth. The basic knowledge obtained from this research will aid the quest to produce novel forms of genetically based durable disease resistance that can withstand pathogen attack and reduce the use
of environmentally damaging pesticides. 2. List the milestones (indicators of progress) from your Project Plan. Each year billions of dollars are lost by farmers worldwide, due to plant disease caused by viruses, bacteria fungi, oomycetes, and nematodes. Infectious plant diseases have caused devastating crop losses resulting in human suffering. Protection of crops from disease can significantly improve agricultural production. This is a very serious problem. 3. Milestones: This project contributes to the goals of the National Program 303 (60%) and 302 (40%). This project focuses primarily on plant disease resistance mechanisms, falling within component 5 (Host Plant Resistance to Disease) of NP 303. 4. What were the most significant accomplishments this past year? A. Understanding the mechanism of disease resistance is vital for developing integrated management strategies to combat plant diseases. Scientists at the Plant Gene Expression Center, Albany, CA in collaboration with UC
Berkeley scientists characterized the sun1-1 mutation that was identified by a suppressor screen of N gene mediated TMV resistance. The work is important as SUN1 appears to mediate resistance signaling for a number of pathogens, including bacterial, fungal and viral pathogens in addition, the results show that R gene- mediated pathways and general resistance pathways intersect or overlap. The results are extremely important as SUN1 is in a pivotal position between pathogens and the ability to respond to disease. Research will lead to the develpment of disease resistance crops. B. The Bs4 gene, which provides resistance against Xanthomonas campestris pv. vesicatoria (Xcv), was cloned. The results are important as the Bs4 gene could be used for providing resistance to other tomato varieties. The results are also important for increasing our knowledge of disease resistance. The work was carried out in collaboration with the Lahaye lab. The identification of disease resistance genes
increases our ability to improve crops. C. Significant Accomplishments/Activities that Support Target Populations: None to report. D. Progress Report: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This work will provide the foundation for establishment of a plant disease resistance database to support broad based research and establishment of genetic resources to address and reduce crop losses caused by pathogen diseases. This work will also provide a framework for establishment of plant disease resistance resources and database to support development of phyto-sensors for detection of pathogens that threaten the quality and safety of food and human health. The genetic manipulation of genes for disease resistance by plant breeders has been employed as a major method to control plant disease since the turn of the century. However, breeders are in need of additional and robust genetics for crop protection. The
recent molecular cloning of several plant genes that confer disease resistance to a diverse range of pathogens has revealed that the encoded proteins have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms for expressing disease resistance to a wide range of unrelated pathogens. The characterization of the molecular signals involved in pathogen recognition and the elucidation of the molecular events specifying the expression of resistance may lead to the development of novel strategies for disease control. Attaining these objectives will provide benefits to farmers, and the public and will result in increased economic growth by helping to overcome the impact of plant disease. New natural genetic strategies expected to control diseases and reduce dependency on pesticides and chemicals. The identification of genes for plant immunity to pathogenic microbes and improved understanding of natural innate immune responses
and plant health is expected to lead to safer environmental practices for food production. Moreover this effort will provide the foundation of a disease resistance database providing a comprehensive resource for researchers in ARS and national and international research labs. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. PATENTS We have filed five patents and all have been issued. 1. U.S. Patent Issued, 11/05/96, Patent Number 5,571,706 'Plant Virus Resistance Gene and Methods.' 2. U.S. Patent Issued, 11/09/99, Patent Number 5,981,730 'RPS Gene Family, Primers, Probes. 3. U. S. Patent Issued, 07/17/01, Patent Number US 6,262,248 B1 'RPS Gene Family, Primers, Probes, and Detection Methods.' 4. U. S. Patent Issued, 04/16/02, Patent Number US 6,372,962 B1 'Pathogen Resistance in Plants Using cDNA-N/Intron Constructs.' 5. U.S. Patent Issued, 10/07/03, Patent Number US 6,630,618 B2 'Transgenic Plants
Having Non-Pathogen Induced Systemic Acquired Resistance (SAR).' Together these technologies provide the means for design of environmentally friendly crop protection strategies. PUBLICATIONS 1. Liu, Y., Jin, H., Yang, K., Kim, C. Y., Baker, B. and Zhang, S. 2003. Interaction between two mitogen-activated protein kinases during tobacco defense signaling. The Plant Journal 34, 149-60. 2. Schornack, S., Ballvora, A., Gurlebeck, D., Peart, J., Baulcombe, D. , Ganal, M., Baker, B., Bonas, U. and Lahaye, T. 2004. The tomato TIR-NB- LRR resistance protein Bs4 discriminates between highly homologous, type III-secreted AvrBs3-like effector proteins. The Plant Journal. 37:46-60. 3. Brigneti, G. , Martin-Hernandex, A.M. , Jin,H. , Chen, J. , Baulcombe, D.C., Baker, B. and Jones, J.D.G. (2004) Virus-induced gene silencing in Solanum species The Plant Journal, 39: 264-271 PRESENTATIONS 1. 10/30/2003 'Molecular Mechanisms of N-Mediated Resistance'. National Citrus Genomic Workshop, ARS, Ft.
Pierce, CA. 2. 06/23/2003 US-EC Workshop on Biotechnological Challenges to Disease Resistance in Plants and Animals, Washington DC. 3. 08/27/2003 'Molecular Mechanisms of N-Mediated Resistance'. Application of Plant Gene Discovery-Host-Pathogen Interaction Discussion, Washington DC. 4. 11/02/2003 'Genetic and Physical Maps of Disease Resistance Regions of the Potato Genome Reveal Multiple NBS-LRR Gene Clusters.' ASA-CSSA- SSSA Annual Meeting, Denver, CO. 5. 01/17/2004 'NSF Potato Genome Project and Center for Plant Genomics Training and Education'. Interagency Working Group (NSF-NIH-USDA-DOE-OSTP- AID-OMB) Outreach Workshop, San Diego CA. 6. 01/18/2004 'Structural and Functional Genomics of Disease Resistance in Solanum.' NSF Potato Genomics Project Annual Meeting, San Diego, CA. 7. 01/21/2004 'Keys to Successful Research and Professional Development. ' ARS National Scientific Leadership Conference, New Orleans, LA. 8. 04/22/2004 'Potato Functional Genomics: Application to Analysis of
Growth, Development, Metabolism and Responses to Biotic and Abiotic Stress.' University of Kentucky Genomics Symposium, Lexington, Kentucky. 9. 04/22/2004 'Molecular Genetic Studies of Plant Innate Immunity.' Plant Pathology Department, University of Kentucky, Lexington, Kentucky. 05/28/2004 'Genomic and Molecular Genetic Studies of Plant Innate Immunity.' Carnegie Institute Department of Plant Biology, Stanford, CA. 10. 06/01/2004 'Potato Functional Genomics: Application to Analysis of Growth, Development, Metabolism and Responses to Biotic and Abiotic Stress.' Plant Breeding Institute. Wageningen University, Wageningen, The Netherlands.
Impacts
(N/A)
Publications
- Erickson, F., Holzberg, S., Calderon-Urrea, A., Handley, V., Axtell, M., Corr, C., Baker, B.J. 1999. The helicase domain of the tmv replicase proteins induces the n-mediated defence response in tobacco. Plant Journal, 19(1) 67-75.
- Dinehsh-Kumar, S., Baker, B.J. 2000. Structure-function analysis of the tobacco mosaic virus resistance gene n. Proceedings of the National Academy of Sciences, 97(26) 14789-14794.
- Schornack, S., Ballvora, A., Gurlebeck, D., Peart, J., Ganal, M., Baker, B. J., Bonas, U., Lahaye, T. 2003. The tomato resistance protein bs4 is a predicted non-nuclear tir-nb-lrr protein that mediates defense responses to severely truncated derivatives of avrbs4 and overexpressed avrbs3. Plant Journal, 37(1) 46-60.
- Gianinna, B., Martin-Hermandez, A.M., Jin, H., Chen, J., Baulcombe, D.C., Baker, B.J., Jones, J.G. 2004. Virus-induced gene silencing in solanum species. Plant Journal, 39(2)264-271.
- Brigneti, G., Martin-Hernandezx, A.M., Jin, H., Chen, J., Baulcombe, D.C., Baker, B.J., Jones, J.G. 2004. Virus-induced gene silencing in solanum species. Plant Journal, 39(2): 264.
- BAKER, B.J., PARKER, J. BIOTIC INTERACTIONS - SIGNALS FROM THE ENVIRONMENT: THE GOOD, THE BAD AND THE UGLY! CURRENT OPINION IN PLANT BIOLOGY. 2003. 6(4):297-299.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Little is known about how resistance (R) gene products function. Our long- term goals are to understand how the N (necrosis) gene product functions to specifically recognize the viral pathogen, tobacco mosaic virus (TMV), and participate in signaling the induction of plant defense responses. Our specific objectives are to (1) identify sequences that encode functional domains of the N protein, (2) identify and isolate genes with structural and / or functional similarities to N for comparative studies and for isolation of new genes for crop protection, and (3) establish methods to identify components of the N signal transduction pathway in tomato. The results of these studies will lead to an understanding of the mechanism of pathogen recognition and signal transduction leading to pathogen resistance in plants. 2. How serious is the problem? Why does it matter? Infectious plant diseases cause
devastating crop losses resulting in human suffering and enormous economic losses. Protection of crops from disease can significantly improve agricultural production. This is a very serious problem. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Research on plant disease resistance will allow scientists to elucidate the molecular mechanisms of disease resistance for development of environmentally sound disease control strategies. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 year: -Why was research done? Genetic dissection of disease resistance has helped identify R gene pathway components, however much more information is required before an understanding of the mechanism of disease resistance is achieved. The TIR- NBS-LRR subfamily of plant R genes encodes proteins with a conserved Toll/ interleukin-1 receptor (TIR) domain, nucleotide
binding site (NBS) and a leucine rich repeat (LRR) region. N was the first member identified in this subfamily and the similarity of its N-terminal TIR domain to the cytoplasmic domain of the Drosophila Toll and human interleukin-1 receptors may indicate that N and other TIR-NBS-LRR resistance genes are evolutionarily related to receptors in the animal innate immune pathways. The structural similarity between the N protein and the Toll and IL-1 receptor proteins led us to postulate that the TMV resistance pathway may share signaling components with Toll and IL-1R defense pathways. B. Other Significant Accomplishment(s), if any. Due to their established regulatory role in activation of innate immunity in animals, MAPK cascades were of particular interest as candidate signaling components in plant disease resistance and innate immunity. MEKK1 encodes a MAPKKK that specifically phosphorylates and activates IkB kinase (IKK) in the animal innate immune pathway. The activated IKK complex
in turn phosphorylates the inhibitor of NFkB, IkB, targeting IkB for degradation and releasing transcription factor NF-kB for translocation into the nucleus. MEKK1 is also important in regulating cell survival and apoptosis; its role changes from a survival promoting kinase to an effector of cell death when cleaved by caspases. C. Significant Accomplishments/Activities that Support Target Populations: None to report. D. Progress Report: Many components of MAPK pathways, as well as some aspects of their regulation, are conserved throughout eukaryotic evolution. Using highly conserved signature motif sequences, analysis of the Arabidopsis genome has revealed 23 putative MAPKs, 10 putative MAPKKs and more than 25 MAPKKKs. It has been documented that plant MAPK cascades are activated by hormones, abiotic stresses, pathogens and pathogen-derived elicitors and are also activated at specific stages during the cell cycle. Considering the large number of plant MAPK cascade genes and their
central role in cellular regulation, relatively few mutants in MAPK pathways have been isolated. One explanation for this limited collection of loss-of-function mutants may be embryonic lethality of mutations in MAPK cascade genes. Virus induced gene silencing (VIGS) can generate loss of function mutations in essential genes in developed plants thus circumventing the otherwise lethal phenotype. Silencing of a host gene occurs after triggering the degradation of target RNA and, in N. benthamiana, potato virus X (PVX) and tobacco rattle virus (TRV) based silencing systems have been employed to ascertain plant gene function. The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic
acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. The Baker lab using PVX-induced gene silencing, demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. In collaboration, the Baker lab and the Zhang lab showed that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Together with previous reports that SIPK and WIPK are activated by TMV in a gene-for-gene- dependent manner, we conclude that the NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death. In collaboration with the Lahaye lab we isolated the Bs4 disease resistance gene from tomato. Bs4 encodes a TIR-NBS-LRR R protein and specifies recognition of Xanthomonas campestris pv. vesicatoria (Xcv)
strains that express the cognate AvrBs4 protein. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The tobacco resistance (R) gene N confers resistance to the plant pathogen tobacco mosaic virus (TMV). When my lab isolated the N gene in 1994 it was the first virus R gene and the first discovered to encode a predicted protein with similarity to innate immunity receptor proteins of insects and humans. Genetic resistance is an effective and environmentally benign means of controlling plant pathogens. Crop species often lack effective genetic resistance to some of their significant pathogens. Resistance to these pathogens can be found in other plant species, however barriers to interspecific crosses frequently prevent these resistance traits from being introduced by conventional breeding. It has been proposed that cloned plant R genes could be transferred from resistant wild species to susceptible plant species to control crop
plant diseases. We showed that tomato plants transformed with the N gene are resistant to TMV. This is an important finding because it paves the way for interspecies transfer of resistance traits. We found that the N gene mRNA is alternatively spliced to form 2 mRNAs, N-mRNA and N-mRNA truncated. We showed that expression of both N- mRNAs is required for complete TMV resistance suggesting that N encodes two proteins that work together to confer TMV resistance. Results from other studies in other plants including Arabidopsis suggest that other R genes may encode 2 proteins required for disease resistance. We have shown that many of the sequences of the TIR, NBS and LRR domains of N are required for TMV resistance. We have identified that N. repanda harbors a resistance gene similar in structure and function to N. We have identified at least five new genes required for N-mediated resistance using VIGs. 6. What do you expect to accomplish, year by year, over the next 3 years? Expected
accomplishments for the next three years: 1. Elucidation of several common and N-specific components of disease resistance signaling. 2. Identification of biochemical properties of the N protein. 7. 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? We have filed three patents concerning 1) the N gene, 2) a useful N cDNA- N/intron construct, 3) and an N-mediated pathogen free protective systemic resistance response. The first patent was issued. Together these technologies provide the means for design of environmentally friendly crop protection strategies. 1. With the cloned N gene in hand it is now possible to cross species barriers and protect crops from disease. 2. With our new construct technology, it will be even easier to produce disease resistant
plants; the shorter length cDNA-N/intron construct is easier to manipulate and less likely to pick up errors during the transformation process. 3. With the ability to induce the protective systemic acquired resistance response, crops can be protected against not only one specific pathogen, but against a broad spectrum of pathogens, including viruses, fungi, and bacteria. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). The NSF Potato Genome Project DBI 9975866, led by Principle Investigator Dr. Barbara Baker was renewed for five more years ($7.6 million). The project is being conducted as a collaboration between ARS, University and the TIRG genome scientists.
Impacts
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Publications
- JIN, H., LIU, Y., YANG, K., KIM, C., BAKER, B.J., ZHANG, S. FUNCTION OF A MITOGREN-ACTIVATED PROTEIN KINASE PATHWAY IN N GENE-MEDIATED RESISTANCE IN TOBACCO. PLANT JOURNAL. 2003. 33:719-731.
- RONNING, C.M., STEGALKINA, S.S., ASCENZI, R.A., BOUGRI, O., HART, A.L., UTTERBACH, T.R., VANAKEN, S.E., RIEDMULLER, S.B., WHITE, J.A., CHO, J., PERTEA, G.M., LEE, Y., KARAMYCHEVA, S., SULTANA, R., TSAI, J., QUACKENBUSH, J., GRIFFITHS, H.M., RESTREPO, S., SMART, C.D., FRY, W.E., VAN DER HOEVEN, R., TANKSLEY, S., ZHANG, P., JIN, H., YAMAMOTO, M.L., BAKER, B.J., BUELL, C. COMPARATIVE ANALYSES OF POTATO EXPRESSED SEQUENCE TAG LIBRARIES. PLANT PHYSIOLOGY. 2003. 131:419-429.
- BAKER, B.J. INTERACTION BETWEEN TWO MITOGEN-ACTIVATED PROTEIN KINASES DURING TOABCCOM DEFENSE SIGNALING. PLANT JOURNAL. 2003. 34:149-160.
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