Progress 12/15/05 to 12/14/09
Outputs
OUTPUTS: The main goals of this research were to better understand genetic mechanisms in bacterial pathogens associated with their interactions with plants. Objectives were achieved by studying Agrobacterium vitis, the cause of grape crown gall, and its interactions grape and with non-host plants like tobacco. A. vitis causes a grape-specific necrosis, a hypersensitive response, HR, on tobacco and strain F2.5 inhibits tumorigenic strains from causing crown gall on grape. F2.5 was shown to induce the expression of HR associated marker genes, HIN1, HSR201 and HSR203J in tobacco leaves whereas an HR minus mutant does not. Infiltration of tobacco leaves with F2.5 induced local and systemic expression of plant defense genes including PR1, PR2 and PR4 whereas the HR mutant only activated local expression. F2.5 also conferred local and systemic resistance to tobacco mosaic virus. A complex quorum sensing regulatory system was found to be involved in development of the HR and grape necrosis but not in biological control. A member of the LysR family, LhnR was essential for HR. Genes surrounding the regulatory genes were also in some cases involved in the HR. We also determined that quorum-sensing is involved in surfactant production and swarming motility of A. vitis. Other genes found to be associated with HR and grape necrosis include a large cluster that resembles clusters in marine bacteria related to type I polyketide synthase genes. Knockouts in any of the genes resulted in a reduced HR and necrosis but did not affect the biological control activity. Recently a related gene predicted to encode a phosphopantetheinyl transferases (PPTases) was identified that is predicted to modify type I polyketide synthases of prokaryotes. Knockouts of this gene resulted in total loss of HR, a reduced necrosis on grape and again did not affect biological control. Flagellin is the flagellum subunit in bacteria and is recognized as a PAMP (pathogen-associated molecular pattern) in plants and may induce HR on plants. Agrobacteriun and Rhizobium have highly divergent flagellin sequence and their flagellins have been reported to not stimulate the flagellin perception system in plants. The A. vitis genome, reveals seven flagellin genes and orthologs of one of them are not found in other sequenced Rhizobiales. Mutagenesis of this gene resulted in a HR-minus phenotype. Mutants of F2.5 that carry disruptions in a clpA gene totally lost HR ability, were reduced in grape necrosis and defective in biological control. ClpA typically functions as a chaperone that delivers proteins for degradation to the ClpP protease. The gene immediately upstream of clpA in A. vitis is a clpS that has no affect on biological control. ClpS is predicted to be a recruitment protein that delivers specific proteins to the ClpA-ClpP protease complex for modification or proteolysis. Two clpP homologs were also mutated and neither affected biological. clpP1 totally lost HR and necrosis activity. clpP2 had reduced HR and necrosis. These results suggest the ClpA chaperone as central to the biological control mechanism and possible divergent genetic pathways for HR, necrosis and biological control. PARTICIPANTS: Desen Zheng, Research Associate, Cornell, hired January 2007. Dr Zheng has assumed responsibilities of Dr. Hao. He is working on the regulatory systems of A. vitis as well as other genes associated with plant interactions. Luciana Cursino Parent, Postdoctoral Associate, Cornell, hired January 2006. Dr. Parent works half time on this project and is studying aspects related to biological control of A. vitis by a non pathogenic strain of the same bacterium. Dr. Parent is currently an Assistant Professor at Hobart and William Smith Colleges. Ji Huang, Postdoctoral Associate, Cornell, hired January 2009. Dr. Huang is investigating the expression of defense related genes in tobacco that are induced when leaves are infiltrated with A. vitis. TARGET AUDIENCES: The world scientific community with interests in plant pathology, bacteriology and plant microbe interactions. The wine and grape industries worldwide Other agricultural producers that grow crops affected by crown gall PROJECT MODIFICATIONS: The project was modified in that we did not attempt to identify a product produced by the polyketide gene cluster. This was decided because we determine this cluster reduced but did not abolish the HR. We therefore put more effort in discovery of other genes associated with the mechanisms of HR and necrosis. We also put more effort into understanding the mechanism of biological control.
Impacts
The research has greatly added to our understanding of mechanisms bacterial pathogens employ when interacting with plants to cause disease, induce disease defense or provide a biological control. It was demonstrated that A. vitis can upregulate defense genes in plants and with further research may be employed in development of disease control strategies. The research has been presented at professional academic meetings and to grower groups on a regular basis. The research has also helped us understand how crown gall infections occur on grapevines and provided important information on the mechanism of biological control. We took part in a project to sequence A. vitis strain S4 and recently we completed sequencing of F2.5. Having these two sequences now in hand is of great benefit to the research. Comparisons of the F2.5 sequence, a strain that provides biological control, to S4, a tumorigenic strain is becoming more valuable to understanding the mechanism of biological control. The discovery of how strain F2.5 prevents crown gall on grape will have major impact on the grape industry. The research has also contributed to the development of methods for indexing grapevines for A. vitis which survives systemically in grapevine cuttings. For example identifying specific genes of the pathogen has allowed the development of unique PCR amplicons that can be used to index plants for the presence of the pathogen. The ability to compare genomes of A. vitis with other Rhizobiaceae is also contributing to our understanding of how this important family of bacteria has evolved.
Publications
- S. Sule, L. Cursino, D. Zheng1, H.C. Hoch1 and T.J. Burr, 2009. Surface motility and associated surfactant production in Agrobacterium vitis. Letters in Applied Microbiology 49:596-601
- Carle, Sigrid A., et al. 2008. A gene cluster in A. vitis homologous to polyketide synthase operons is associated with grape necrosis and hypersensitive response induction on tobacco. FEMS Microbiol. Lett. 289:90-96.
- The Genomics of Agrobacterium: Insights into its Pathogenicity, Biocontrol, and Evolution 2009. Joao C. Setubal, Derek Wood, Thomas Burr, Stephen K. Farrand, Barry S. Goldman, Brad Goodner, Leon Otten, and Steven Slater IN Plant Pathogenic Bacteria: Genomics and Molecular Biology Caister Academic Press Robert W. Jackson School of Biological Sciences, University of Reading, Whiteknights, Reading, UK, editor
- Li, Y., Gronquist, M. R., Hao, G. Holden, M. R., Eberhardc, A., Scottd, R. A., Savka M. A., Szegedi,E., Sule, S., and Burr, T. J. 2006. Chromosome and plasmid-encoded N-acyl homoserine lactones produced by Agrobacterium vitis wildtype and mutants that differ in their interactions with grape and tobacco. Physiol. And Molec. Plant Pathol. 67:284-290. Zauner, S., Creasap, J. E., Burr, T. J., and Ullrich, C. I. 2006. Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus. Vitis 45:131-139.
- Hao, G. and Burr, T. J. 2006 Regulation of long-chain N-acyl-homoserine lactones in Agrobacterium vitis J. Bacteriology. 2006 188: 2173-2183.
- Hao, G., Zhang, H., Zheng, D. and Burr, T. J. 2005. A luxR homolog avhR in Agrobacterium vitis affects the development of a grape-specific necrosis and a tobacco hypersensitive response. J. Bacteriol. 187:185-192.
- Creasap, J. E., Reid, C. L., Goffinet, M. C., Aloni, R., Ulrich, C. and Burr, T. J. 2005. Effect of wound position, auxin and Agrobacterium vitis strain F2/5 on wound-healing and crown gall development in woody grapevine tissue. Phytopathology 95:362-367.
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Progress 12/15/07 to 12/14/08
Outputs
OUTPUTS: The main goals of this research are to understand genetic mechanisms in bacterial pathogens associated with how they interact with plants. The objectives are achieved by studying Agrobacterium vitis, the cause of grape crown gall, and its interactions with grape and non-host plants like tobacco. A. vitis causes a grape-specific necrosis, a hypersensitive response on tobacco and some strains such as F2.5 provide crown gall biological control. Four members of the LuxR family and one of the LysR family of transcriptional regulators were identified that affect HR and necrosis which was facilitated by having the genome sequence of strain S4 completed. In addition at least 5 genes downstream from the regulatory genes were identified and found to be necessary for the plant responses. In addition to being involved in regulation of HR and necrosis we determined that quorum-sensing regulation is also involved in surfactant production and swarming motility of A. vitis. The surfactant appears to be essential for swarming motility. Genes that possess characteristic promoter sequences that are typical of genes regulated by quorum-sensing were identified. Two of the genes have been knocked out and shown to be essential for expression of the HR. These include an autoinducer synthase gene and a gene that appears to be associated with sugar metabolism. A large gene cluster on chromosome two of A. vitis and necessary for expression of HR was also further characterized. Two large genes residing in the middle of the cluster were found to be co-expressed whereas the six other genes are expressed independently. The gene cluster is highly similar to operons found in marine bacteria that encode long chain polyunsaturated fatty acids. The primary strain of A. vitis that is used in the research F2.5 is able to prevent crown gall when applied to wounds on grape plants prior to application of the pathogenic strains. Mutants of F2.5 that carry disruptions in a clpA gene were defective in biological control. ClpA functions as a chaperone that delivers proteins for degradation to the ClpP protease. Interestingly the gene immediately upstream of clpA is a clpS homolog that. ClpS is predicted to be a recruitment protein that delivers specific proteins to the ClpA-ClpP protease complex for modification or proteolysis. Whereas clpS was found to be essential for HR expression on tobacco it is not required for biological control. This provides a possible branch in mechanistic pathways between HR and biological control. Two clpP homologs in F2.5 have been knocked out and one clpP1 was found to be essential for HR. The role of the clp genes in biological control is being investigated. PARTICIPANTS: Desen Zheng, Research Associate, Cornell, hired January 2007. Dr Zheng has assumed responsibilities of Dr. Hao. He is working on the regulatory systems of A. vitis as well as other genes associated with plant interactions. Luciana Cursino Parent, Postdoctoral Associate, Cornell, hired January 2006. Dr. Parent works half time on this project and is studying aspects related to biological control of A. vitis by a non pathogenic strain of the same bacterium. Ji Huang, Postdoctoral Associate, Cornell, hired January 2009. Dr. Huang is investigating the expression of defense related genes in tobacco that are induced when leaves are infiltrated with A. vitis. TARGET AUDIENCES: The world scientific community with interests in plant microbe interactions. The US wine and grape industries PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This research is improving our understanding of mechanisms bacterial pathogens employ when interacting with plants. In this case the bacterium may cause HR or necrosis or function as a biological control depending on the plant species and tissue it is applied to. The research has advanced our knowledge of how disease is induced as well as the induction of defense mechanisms that are employed by plants against disease. The research has been presented at professional academic meetings and to grower groups on a regular basis. The information is important for helping understand how crown gall infections occur and how the bacterium interacts with its grapevine host. It has also aided in the development of methods for indexing grapevines for the pathogen and also for implementing disease management strategies. For example identifying specific genes of the pathogen has allowed the development of specific probes that can be used to index plants for the presence of the pathogen. Having access to the A. vitis genome sequence has greatly assisted our efforts in gene discovery and understanding their functions. Because one strain of the bacterium we are working on is able to prevent crown gall disease, the research is being used to improve biological control under commercial conditions. The discovery of how strain F2.5 prevents crown gall on grape will have major impact on the grape industry. The ability to compare genomes of A. vitis with other Rhizobiaceae is contributing to our understanding of how this important family of bacteria has evolved.
Publications
- Carle, S. A., Hao, G., Zheng, D., Sanni-Sanoussi, T., Talarico, M. J., Hilton, J. S. and Burr, T. J. 2008. A gene cluster in Agrobacterium vitis homologous to polyketide synthase operons is associated with grape necrosis and hypersensitive response induction on tobacco. FEMS Microbiol. Lett. 289:90-96.
- Otten, L., Burr, T. J. and Szegedi, E. 2008. Agrobacterium: a disease-causing bacterium. IN Agrobacterium: From Biology to Biotechnology, Tzvi, T and Citovsky, V. (Eds.) ISBN: 978-0-387-72289-4
- Bini, F., Kuczmog, A., Putnoky, P., Otten, L., Bazzi, C., Burr T. J., and Szegedi, E. 2008. Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens. Vitis 43 (3).
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Progress 12/15/06 to 12/14/07
Outputs
OUTPUTS: The main goal of this research is to understand genetic mechanisms in bacterial pathogens associated with their interactions with plants. The objectives are achieved by studying Agrobacterium vitis, the cause of grape crown gall, and its interactions with grape and non-host plants. A. vitis causes a grape-specific necrosis, a hypersensitive response on tobacco and some strains such as F2.5 provide crown gall biological control. Employing the genome sequence of A. vitis strain S4 members of the LuxR family and LysR family of transcriptional regulators that affect bacterial plant interactions were identified. Currently four LuxR and one LysR regulator have been found to be associated with HR regulation. In addition at least 5 genes downstream from the regulators were found by targeted knockout to be necessary for the plant responses. In addition to the HR and necrosis responses we have now determined that quorum-sensing regulation is also involved in swarming motility of A.
vitis and also in production of surfactant. The surfactant is essential for swarming motility. Further research identified genes with characteristic promoter sequences that are typical of those regulated by quorum-sensing. Two of these genes have been knocked out and shown to be essential for expression of the HR. These include an autoinducer synthase gene and a gene that appears to be associated with sugar metabolism. A large gene cluster on chromosome two of A. vitis and necessary for expression of HR was also further characterized. Two large genes residing in the middle of the cluster were found to co-expressed whereas six other genes in the cluster appear to be expressed independently. The two gene operon is highly similar to genes found in marine bacteria that encode long chain polyunsaturated fatty acids. Finally, one strain of A. vitis that we utilize F2.5 is able to prevent crown gall when applied to wounds on grape plants prior to application of the pathogenic strains. A
mutation in a protease gene clpA resulted in loss of HR and biological control. A mutation in a downstream gene clpS was important for HR but not for biological control. Further analyses will help to determine mechanisms of both phenotypes.
PARTICIPANTS: Desen Zheng, Research Associate, Cornell, hired January 2007. Dr Zheng has assumed responsibilities of Dr. Hao. He is working on the regulatory systems of A. vitis as well as other genes associated with plant interactions. Luciana Cursino Parent, Postdoctoral Associate, Cornell, hired January 2006. Dr. Parent works half time on this project and is studying aspects related to biological control of A. vitis by a non pathogenic strain of the same bacterium.
TARGET AUDIENCES: The world scientific community with interests in plant microbe interactions. The US wine and grape industries
Impacts
This research is providing an improved understanding of mechanisms used by bacterial pathogens as they interact with plants. It has improved our knowledge of how disease is induced as well as defense mechanisms that are employed by plants against disease. The research has been presented to grower groups on a regular basis. It is helping growers understand how crown gall infections occur and how the bacterium interacts with its grapevine host. It has also aided in the development of methods for indexing grapevines for the pathogen and also for implementing disease management strategies. Having access to the A. vitis genome sequence has greatly assisted our efforts in gene discovery and understanding their functions. Because one strain of the bacterium we are working on is able to prevent crown gall disease, the research is being used to improve biological control under commercial conditions. Also being able to compare genomes of A. vitis with other Rhizobiaceae is
contributing to our understanding of how this important family of bacteria has evolved.
Publications
- No publications reported this period
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Progress 12/15/05 to 12/15/06
Outputs
The main goal of this research is to understand genetic mechanisms in bacterial pathogens associated with their interactions with plants. The objectives are achieved by studying Agrobacterium vitis, the cause of grape crown gall, and its interactions with grape and non-host plants. A. vitis causes a grape-specific necrosis, a hypersensitive response on tobacco and some strains such as F2.5 provide crown gall biological control of. We determined that quorum sensing regulation is involved with the HR and necrosis. At least ten genes belonging to the luxR family and three to the luxI family are present in the A. vitis genome. Four of the luxR group including aviR, avhR, asvR, and ahyR and one in luxI group, avsI, are essential for the HR and necrosis. The gene pair of avsR and avsI is also essential for HR and necrosis and is involved in production of long- chain autoinducer signal molecules. Eight ORFs have been identified so far that have characteristic motifs in their
promoter regions suggestive of regulation by quorum sensing. A lysR homolog, lhnR was also found to be essential for the HR and necrosis. lhnR is part of a gene cluster that includes at least four genes that are also essential. lhnR and essential luxR homologs appear to regulate HR and necrosis independently and lhnR negatively regulates downstream genes. An apparent operon in A. vitis composed of at least eight genes is also essential for the HR and necrosis. Knockouts of the individual genes all affect the HR and necrosis. Non-tumorigenic A. vitis strain F2.5 is able to prevent crown gall when it is applied prior to application of pathogen. A mutation in a gene homologous to clpA resulted in loss of HR and biological control. Three genes flanking clpA gene were assessed for their functions and one, a clpS homolog, affected HR development on tobacco.
Impacts
This research will provide an improved understanding of mechanisms used by bacterial pathogens as they interact with plants. It will improve our knowledge of how disease is induced as well as defense mechanisms that are employed by plants against disease. The work will facilitate the development of new disease controls. Sequence comparisons between A. vitis strains and other Rhizobiaceae will contribute to our understanding of how this important family of bacteria has evolved.
Publications
- Zauner, S., Creasap, J.E., Burr, T.J. and Ullrich, C.I. 2006. Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus. Vitis 45:131-139.
- Hao, G. and Burr, T.J. 2006. Regulation of long-chain N-acyl-homoserine lactones in Agrobacterium vitis J. Bacteriol. 188: 2173-2183.
- Li, Y., Gronquist, M.R., Hao, G., Holden, M.R., Eberhard, A., Scott, R.A., Savka, M.A., Szegedi, E., Sule, S. and Burr, T.J. 2006. Chromosome and plasmid-encoded N-acyl homoserine lactones produced by Agrobacterium vitis wildtype and mutants that differ in their interactions with grape and tobacco. Physiol. And Molec. Plant Pathol. 67:284-290.
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