Progress 10/01/06 to 09/30/11
Outputs
OUTPUTS: OUTPUTS: During this project evolution and virulence mechanisms of the plant pathogenic bacterium Pseudomonas syringae were investigated using molecular and genomic approaches. Through collaborations some research was also extended to two additional plant pathogens: Xanthomonas ssp and Ralstonia solanacearum. The focus of this project was to understand the molecular mechanisms at the basis of host range and virulence evolution of P. syringae. The used approach consisted in identifying a group of closely related P. syringae strains followed by the characterization of these strains using molecular typing approaches and host range tests. As typing methods a technique called multilocus sequence typing (MLST) was widely used. MLST consist in sequencing fragments of 4 to 8 genes in all strains and to analyze the relationship between strains based on the DNA sequences of these gene fragments. To facilitate this approach a database with web interface was constructed. As second typing method whole genome sequencing was used. Dozens of P. syringae and R. solanacearum strains were sequenced. When differences between genomes were found, additional strains were tested for the presence or absence of these differences with a technique called: single nucleotide polymorphism (SNP) analysis. When differences between strains were found to be in potential virulence genes these differences were further investigated. The approach used for these investigations consisted in cloning these putative virulence genes and expressing them in strains that were missing them or that had different alleles of these genes. Finally, the phylogenetic position of strains, their host range, and the genetic differences identified between strains were correlated with each other to unravel the molecular mechanisms of host range and virulence evolution in P. syringae. MENTORING: Four graduate students and over one dozen undergraduate students were advised during this project. DISSEMINATION: Results were disseminated at the Annual Potomac Division Meetings of the American Phytopathological Society and at several international conferences, with the most important ones being: The International Congress on Plant - Microbe Interactions, Quebec City (Canada) in 2009, The International Congress on Plant Pathogenic Bacteria, Reunion Island (France) in 2010, the 8th International Conference on Pseudomonas syringae and Related Pathogen in Oxford (UK) in 2010. Besides distribution though peer-reviewed publications, results of this project have also been distributed through websites that we have set up: a molecular typing website for plant-associated bacteria ( www.PAMDB.org ) and a website with all the genomic data we have obtained so far ( genome.ppws.vt.edu ). PARTICIPANTS: Individuals No change, only PI Boris A Vinatzer Partner Organizations USDA ARS Salinas, California INRA, Avignon, France Sainsbury Laboratory, Norwich, UK CRA-Centro di Ricerca per l′Orticoltura, Battipaglia, Italy Federal University of Mato Grosso do Sul, Campo Grande, Brazil Biosciences, University of Exeter, Exeter, Devon, United Kingdom Universidad de los Andes, Bogota, Colombia Department of Plant Pathology, University of California, Davis, California Collaborators and contacts Within the PI's department: Chris Clarke (student) Rongman Cai (student) Shuangchun Yan (student) Anastasia Naumenko (student) Haijie Liu (technician) Withing the PI's institution: Joao Setubal (Virginia Bioinformatics Institue) Scotland Leman (Statistics Department) At other institutions: Carolee Bull (USDA ARS Salinas, California) Cindy Morris (INRA, Avignon, France) Kee Sohn (Sainsbury Laboratory, Norwich, UK) Massimo Zaccardelli (CRA-Centro di Ricerca per l′Orticoltura, Battipaglia, Italy) Nalvo Almeida (Federal University of Mato Grosso do Sul, Campo Grande, Brazil) David Studholme (Biosciences, University of Exeter, Exeter, Devon, United Kingdom) Adriana Bernal (Universidad de los Andes, Bogota, Colombia) Gitta Coaker (Department of Plant Pathology, University of California, Davis, California) Training or professional development The above listed graduate students were trained within the project and more than a dozen of undergraduates participated in the project also. TARGET AUDIENCES: The target audience of this project are mainly other researchers but also undergraduate students at Virginia Tech and at Concord University, a liberal arts college in West Virginia, and high school student from Roanoke Valley Governor School (RVGS) in Roanoke (VA). Undergraduate students were trained in formal classroom settings or received training by participating in the research in the PI's laboratory and/or by isolating bacteria from plants they collected. High school students were targeted during outreach activities. The PI visited RVGS several times and advised students in extracting bacteria from leaves and talked with students from RVGS during student visits to Virginia Tech. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
MLST analysis and host range tests that were performed on a group of approximately 24 P. syringae strains isolated from different host plant species showed that MLST provides sufficient resolution to distinguish between strains that have different host ranges. Moreover, this study revealed that the exchange of DNA between ancestors of the analyzed strains was an important factor in their evolution. While it was not possible to infer host range of the most recent common ancestor of the analyzed strains, the study let to new hypotheses on plant pathogen evolution that will be tested in the future. Genome sequencing of five P. syringae strains from tomato and molecular analysis of over 100 additional strains from tomato isolated on four continents over 60 years revealed that there is one main genetic lineage of P. syrinhgae that causes the bacterial speck disease of tomato worldwide. Moreover, this lineage has evolved over the last 50 years apparently adapting to the tomato host by mutating the genes coding for some of the proteins that are recognized by the tomato immune system. These mutations appear to have allowed the pathogen to invade the tomato leaf more easily and to multiply inside the tomato leaf more efficiently. Based on these results, we can now make more informed choices on how to engineer tomatoes, and plants in general, for increased disease resistance. Moreover, the performed analysis revealed that P. syringae pv. tomato strains are frequently exchanged between Europe and North America, probably through the movement of tomato seed while pathogen populations in South America and Africa are more isolated and evolve independently. This is very important to know because it reveals the importance of limiting the import of pathogens even if strains of the same pathogen are already present in the USA. The above studies have also let us to investigate the importance of chemotaxis (i.e., the ability of organisms to move towards attractants typical of favorable environments and away from deterrents typical of unfavorable environments) during leaf invasion by P. syringae. A new approach of disease control based on chemotaxis is currently investigated. Over the duration of the project, the PAMDB database and website for typing plant-associated bacteria has been growing and is thus becoming an ever more powerful tool for diagnostics of the many plant diseases caused by P. syringae, Xanthomonas ssp., and R. solanacearum.
Publications
- Kumar A, Prameela TP, Suseela Bhai R, Siljon A, Biju CN, Anandaraj M, Vinatzer BA (2011, in press) Small Cardamom (Elettaria cardamomum Maton.) and Ginger (Zingiber officinale Roxb) bacterial wilt is caused by same strain of Ralstonia solanacearum: A revelation by multilocus sequence typing (MLST). Eur J Plant Pathol.
- Sohn KH, Saucet SB, Clarke CR, Vinatzer BA, O'Brien HE, Guttman DS, Jones JD (2011) HopAS1 recognition significantly contributes to Arabidopsis nonhost resistance to Pseudomonas syringae pathogens. New Phytologist doi: 10.1111/j.1469-8137.2011.03950.x.
- Cai R, Yan S, Haijie L, Leman S, Vinatzer BA (2011) Reconstructing Host Range Evolution of Bacterial Plant Pathogens using Pseudomonas syringae pv. tomato and Its Close Relatives as a Model. Infection, Genetics and Evolution 11(7):1738-51
- Cai R, Lewis J, Yan S, Liu H, Clarke CR, Campanile F, Almeida NF, Studholme DJ, Lindeberg M, Schneider DJ, Zaccardelli M, Setubal JC, Morales-Lizcano NP, Bernal A, Coaker G, Baker C, Bender CL, Leman S, Vinatzer BA (2011) The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity. PLoS Pathogens 7(8):e1002130
- Potnis N, Krasileva K, Chow V, Almeida NF, Patil PB, Ryan RP, Sharlach M, Behlau F, Dow JM, Momol M, White FF, Preston JF, Vinatzer BA, Koebnik R, Setubal JC, Norman DJ, Staskawicz BJ, Jones JB (2011) Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper. BMC Genomics 12:146
- Bull CT, Clarke CR, Cai R, Vinatzer BA, Jardini TM, Koike ST (2011) Multilocus Sequence Typing of Pseudomonas syringae Sensu Lato Confirms Previously Described Genomospecies and Permits Rapid Identification of P. syringae pv. coriandricola and P. syringae pv. apii Causing Bacterial Leaf Spot on Parsley. Phytopathology 101(7):847-58
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Last year's research was continued and extended and a new project area was developed based on last year's results. First of all, the recent evolutionary dynamics of the bacterial speck pathogen of tomato, Pseudomonas syringae pv. tomato, were further investigated by extending the previous analysis to more strains. This allowed performing statistical analyses to correlate the phylogeny of the analyzed strains with the geographic location of their isolation and the year of their isolation. Comparing the genomes of a subgroup of P. syringae pv. tomato strains also suggested that these strains may be under selection to become more efficient invaders of tomato leaves. Based on these results, the focus of the project shifted towards two aspects of invasion: 1. recognition of the bacterial flagellum by the plant immune system since detection of the flagellum by the plant immune system had been found to interfere with invasion, 2. Chemotactic ability of the pathogen to recognize tomato leaf exudates since an improved ability of the pathogen to find wounds or natural openings in the tomato leaf can be expected to lead to faster invasion. Moreover, we continued our effort in connecting our basic research with plant pathogen taxonomy and systematics for improved molecular diagnostics of bacterial plant diseases. In this regard, over forty different type strains of P. syringae were analyzed using Multilocus Sequence Typing (MLST) and the results were deposited in our publicly accessible database PAMDB.org to aid plant disease diagnosticians in precise identification of diseases caused by P. syringae. MENTORING: Three graduate students and four undergraduate students were advised during this reporting year. DISSEMINATION: Results were disseminated at the Annual Potomac Division Meeting of the American Phytopathological Society in Ocean City (MD), the 12th International Congress on Plant Pathogenic Bacteria on Reunion Island (France), and at the 8th International Conference on Pseudomonas syringae and Related Pathogen in Oxford (UK). Also, besides the molecular typing website www.PAMDB.org a website with all the genomic data we have obtained so far has been added at genome.ppws.vt.edu PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The molecular analysis of over 100 P. syringae pv. tomato strains from four continents collected over 60 years revealed that there is one main genetic lineage of this pathogen that causes the bacterial speck disease of tomato worldwide. Moreover, this lineage has evolved over the last 50 years apparently adapting to the tomato host by mutating the genes coding for some of the proteins that are recognized by the tomato immune system. These mutations appear to have allowed the pathogen to invade the tomato leaf more easily and to multiply inside the tomato leaf more efficiently. Based on these results, we can now make more informed choices on how to engineer tomatoes, and plants in general, for increased disease resistance. Moreover, the performed analysis of a worldwide strain collection has revealed that P. syringae pv. tomato strains are frequently exchanged between Europe and North America, probably through the movement of tomato seed. Pathogen populations in South America and Africa instead are more isolated and evolve independently. This is very important to know because it reveals the importance of limiting the import of pathogens even if strains of the same pathogen are already present in the USA. Our studies on chemotaxis of P. syringae have only started but we have already determined that chemotaxis plays an important role in leaf invasion by P. syringae. We envision that the study of chemotaxis will lead to new approaches on how to control disease caused by P. syringae. The results from the MLST analysis have revealed that MLST can clearly distinguish between all described P. syringae pathovars making it a method of choice for molecular diagnostics of plant diseases caused by P. syringae. The PAMDB database and website will make it easy for diagnosticians to use the data we obtained.
Publications
- Bull CT, Manceau C, Lydon J, Kong H, Vinatzer BA, Fischer-Le Saux M (2010)Pseudomonas cannabina pv. cannabina pv. nov., and Pseudomonas cannabina pv.alisalensis (Cintas Koike and Bull, 2000) comb. nov., are members of the emended species Pseudomonas cannabina (ex Sutic & Dowson 1959) Gardan, Shafik, Belouin, Brosch, Grimont & Grimont 1999. Syst Appl Micobiol 33(3):105-15
- Moreira LM, Almeida NF Jr, Potnis N, Digiampietri LA, Adi SS, Bortolossi JC, da Silva AC, da Silva AM, de Moraes FE, de Oliveira JC, de Souza RF, Facincani AP, Ferraz AL, Ferro MI, Furlan LR, Gimenez DF, Jones JB, Kitajima EW, Laia ML, Leite RP Jr, Nishiyama MY, Rodrigues Neto J, Nociti LA, Norman DJ, Ostroski EH, Pereira HA Jr, Staskawicz BJ, Tezza RI, Ferro JA, Vinatzer BA, Setubal JC (2010) Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii. BMC Genomics 11:238.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: OUTPUTS: This year's project outcomes can be assigned to four focus areas: 1. reconstruction of the evolution of a group of closely related bacteria within the plant pathogen species Pseudomonas syringae. The research from the last reporting year was continued by modifying the choice of genes used for the phylogenetic reconstruction with the goal of obtaining a phylogeny with increased statistical support. The phylogenetic reconstruction was integrated with an ancestral state reconstruction to infer from the current host species of the analyzed bacteria the host species of ancestral bacteria. 2. Evolution and epidemiology of the Pseudomonas syringae pathogen that causes the "bacterial speck" disease of tomato. The genomes of five representative strains were compared with each other. Differences that were identified between the five genomes were then evaluated in an additional hundred strains isolated between 1940 and 2008 on four continents to reconstruct a very deep phylogeny of this pathogen. Phylogeny was correlated with time of isolation of strains, geographic location of isolation, and presence/absence of virulence genes and allelic variation in virulence genes. 3. Investigation of the plant resistance response of plant species to pathogens of other plant species, a response known as "nonhost resistance", a very strong and stable type of plant disease resistance that has the potential of contributing to the development of highly resistant crop plants in the future. In particular, the following question was investigated: Can a pathogen not cause disease on a nonhost plant species because it is missing certain virulence genes necessary to suppress the plant immune system of that plant species. To answer this question virulence genes were transferred from a pathogen of the model plant species Arabidopsis thaliana to a pathogen of tomato that cannot cause disease on A. thaliana. It was then determined if the tomato pathogen expressing these genes was able to cause disease on A. thaliana or at least grow o higher population density. 4. Multilocus sequence analysis of strains of the plant pathogens Pseudomonas syringae, Xanthomonas, and Ralstonia solanacearum was used to compare the diversity that exists within each of these three pathogen groups in order to determine if a taxonomic revision within any of these pathogen groups is advisable. Moreover, the application of gene sequences for pathogen identification and plant disease diagnostics was investigated. MENTORING: Three graduate students and four undergraduate students were advised. An outreach project at Roanoke Valley Governor's School for Science and Technology was also performed to integrate research and education. DISSEMINATION: Results were disseminated at the Annual Potomac Division Meeting of the American Phytopathological Society in Gettysburg (PA) and at the International Congress on Plant Microbe Interactions in Quebec City (Canada). The website for typing plant pathogenic bacteria described in the last report was extended to additional species and a user-friendly interface was added ( www.pamdb.org ). PARTICIPANTS: The project has the same main participants as last year. However, one new collaborator has been added: Gitta Coaker (University of California, Davis) collaborates on the evolution and epidemiology of Pseudomonas syringae on tomato providing experience in proteomics and metabolomics and a wide collection of strains isolated from tomato in California. TARGET AUDIENCES: With the publication of the www.pamdb.org website for multilocus sequence analysis of bacterial plant pathogens the target audience of the project has been expanded to evolutionary biologists working with bacterial plant pathogens and to plant disease diagnosticians who want to use molecular tools for the precise identification of bacterial plant pathogens. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
PHYLOGENY AND HOST RANGE EVOLUTION OF CLOSELY RELATED STRAINS OF THE PLANT PATHOGEN PSEUDOMONAS SYRINGAE. It was possible to obtain a more robust phylogeny of a group of P. syringae strains compared to the phylogeny reported in last year's report by fine-tuning the choice of genes for phylogenetic reconstruction. However, correlating host specificity data with phylogeny, it has also become clear that only by including representative pathogen strains from wild plants in the analysis it will be possible to reconstruct the host specificity of the most recent common ancestor of the analyzed strains. Therefore, future sampling efforts will need to be directed to wild plants to obtain an in depth understanding of the evolution of crop pathogens necessary to develop approaches to avoid the evolution of more virulent pathogen variants. EVOLUTION AND EPIDEMIOLOGY OF THE PSEUDOMONAS SYRINGAE PATHOGEN THAT CAUSES THE "BACTERIAL SPECK" DISEASE OF TOMATO. Sequencing the genomes of five representative genomes of this pathogen and the analysis of mutations in selected genes in over hundred strains has given us unprecedented insight into the recent virulence evolution and epidemiology of a bacterial plant pathogen. It has been possible to determine the existence of pandemic strains exist in the bacterial speck pathogen population that have been largely replaced by new strains several times over the last 70 years. Moreover, strong selection from the plant immune system has caused loss of several genes present in the ancestral strain and allelic variation in virulence genes. The obtained results reveal that any agricultural practice anywhere in the world appears to influence the evolution of the worldwide population of P. syringae. International movement of plant material seems not to be restricted sufficiently to limit cross border movement of bacterial plant pathogens. More control of imported plant material is needed. INVESTIGATION OF THE PLANT RESISTANCE RESPONSE OF PLANT SPECIES TO PATHOGENS OF OTHER PLANT SPECIES. Comparing a pathogen of Arabidopsis thaliana with a pathogen that cannot cause disease on A. thaliana and by moving virulence genes between the two pathogens it has become clear that single genes cannot change host specificity. Surprisingly, when genes from the pathogen are expressed in the pathogen that cannot cause disease on the same plant reduces bacterial growth suggesting that virulence activity of pathogen genes strongly depend on other genes present in the same pathogen. The obtained results highlight a high degree of genetic complexity in the determination of pathogen host specificity. Understanding this complexity will be necessary to create crop plants with durable disease resistance. MULTILOCUS SEQUENCE ANALYSIS OF STRAINS OF THE PLANT PATHOGENS P. SYRINGAE, XANTHOMONAS, AND RALSTONIA SOLANACEARUM. Multilocus sequence analysis was found to be a powerful tool to compare pathogen diversity and to diagnose bacterial plant diseases. The developed website www.pamdb.org will greatly facilitate further evolutionary analysis of bacterial plant pathogens and the identification of the diseases they cause.
Publications
- Almeida, N. F., Yan, S., Cai, R., Clarke, C. R., Morris, C. E., Schaad, N. W., Lacy, G. H., Jones, J. B., Castillo, J. A., Bull, C. T., Leman, S., Guttman, D. S., Setubal, J. C., Vinatzer, B. A. (2010) PAMDB, A Multilocus Sequence Typing & Analysis Database and Website for Plant-Associated and Plant-Pathogenic Microorganisms. Phytopathology (in press).
- Clarke, C. R., Cai, R., Studholme, D. J., Guttman, D. S., Vinatzer, B. A. (2010) Pseudomonas syringae Isolates Naturally Lacking the Canonical P. syringae hrp/hrc Locus are Common Leaf Colonizers Equipped with an Alternate Type III Secretion System. Mol. Plant-Microbe Interact (in press).
- Ferrante, P., Clarke, C. R., Cavanaugh, K. A., Michelmore, R. W., Buonaurio, R., Vinatzer, B. A. (2009) Contributions of the effector gene hopQ1-1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci. Mol. Plant Pathology (published online on August 20, DOI: 10.1111/j.1364-3703.2009.00577.x.).
- Almeida, N. F., Yan, S., Lindeberg, M., Studholme, D. J., Schneider, D. J., Condon, B., Liu, H.*, Viana, C. J., Warren, A., Evans, C., Kemen, E., MacLean, D., Angot, A., Martin, G. B., Jones, J. D., Collmer, A., Setubal, J. C., Vinatzer, B. A., 2009. A draft genome sequence of Pseudomonas syringae pv. tomato strain T1 reveals a repertoire of type III related genes significantly divergent from that of P. syringae pv tomato strain DC3000. Mol. Plant-Microbe Interact. 22:52-62
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: OUTPUTS. To accomplish the long-term goal of unraveling how plant pathogenic bacteria evolve to overcome plant disease resistance, closely related bacteria of the species Pseudomonas syringae are being compared in this project. The following analyses were performed this year: host range tests, phylogenetic analysis, and whole genome comparisons. Two dozen bacterial isolates were added to the analysis performed in previous years. Nine gene fragments were sequenced in all isolates and phylogenetic trees were constructed using three different methods: Bayesian inference, maximum likelihood, neighbor joining. Host range of the new isolates was determined on tomato, celery, cauliflower, and Arabidopsis thaliana. Six whole genomes were sequenced using high throughput genome sequencing. One genome sequenced in the previous year was annotated and genes coding for proteins secreted from the bacterium into plants were predicted. To understand how plants defend themselves against pathogens of other plants, A. thaliana plants and several mutant plants compromised in pathways of the plant immune system were infected with tomato isolates of P. syringae. The following data were compared between plants and between bacterial isolates: bacterial growth, disease symptoms, induction of the innate plant immune response. The tomato isolates were also mutated by either deleting genes or adding genes from other isolates that can cause disease on A. thaliana. The role of individual genes in either eliciting or interfering with the plant immune system can be determined in this way. To facilitate the transfer of genes between bacteria, gene libraries of two isolates were constructed. These will allow the simultaneous transfer of several genes at the time. Three graduate students and five undergraduate students were mentored during this project this year. Research was tightly integrated with teaching graduate students, undergraduate students and high school students. Students participated in collecting P. syringae isolates from weedy plants in Southwest Virginia and molecularly identified the isolated bacteria. Students were introduced to concepts of microbial ecology and evolution and molecular plant - microbe interactions. DISSEMINATION. Sequences of the analyzed nine gene fragments from the two dozen strains of this year and from previously analyzed strains have been added to a database with web interface: http://genome.ppws.vt.edu/pallele. This database contains the following data for each isolate: year of isolation, geographic region of isolation, plant species of isolation, and sequence data. This database is freely accessible to basic scientists and field pathologists to aid in the precise molecular identification of P. syringae isolates. Results were also disseminated through talks and posters at regional, national, and international meetings: the Annual Potomac Division Meeting of the American Phyotopathological Society in Morgantown (WV), the General Meeting of the American Society for Microbiology in Boston (MA), the International Conference on Arabidopsis Research in Montreal (Canada). PARTICIPANTS: One new individual has started to work on the project in 2008. Nalvo Almeida is a visiting professor from the Department of Computing and Statistics, Federal University of Mato Grosso do Sul, Brazil. He has worked the entire year in Joao Setubal's group at the Virginia Bioinformatics Institute. He contributed his bioinformatics expertise to the project. All other participants are the same as reported for 2007. TARGET AUDIENCES: High school students have been included as target audience in 2008. The PI Boris Vinatzer has advised a class of high school students at Roanoke Valley Governor School in isolating and molecularly characterizing bacteria from plants to introduce students to the microbial diversity in the environment and to teach concepts of microbial ecology and evolution. Other target audiences are the same as reported for 2007. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
PHYLOGENY OF CLOSELY RELATED PSEUDOMONAS SYRINGAE ISOLATES. The addition of two dozen isolates to the phylogenetic analysis of closely related isolates of P. syringae from tomato, cauliflower, celery, snapdragon, peach, cucumber, and from plants of the genus Berberis made it possible to get a deeper understanding of the evolution of this group of important plant pathogens. It is now clear that recombination between bacteria is a very important factor in the evolution of this group. However, recombination appears to have slowed down in the most recent evolution of P. syringae isolates from crops: over 50 isolates from tomato collected over the last 50 years are identical in all sequenced genes suggesting that they have evolved in isolation from other plant pathogens. HOST RANGE OF CLOSELY RELATED P. SYRINGAE ISOLATES. Analyzing the ability to grow and to cause disease of all analyzed isolates on tomato, celery, cauliflower and A. thaliana has confirmed earlier observations that some of the isolates are of wider host range than others. While some isolates cause disease on all plant species that were tested, other isolates cause disease only on one of the tested plant species or on neither of them. Correlating host range with the results of the phylogenetic analysis appears to confirm our previous hypothesis that some P. syringae bacteria, for example, the bacteria that cause the bacterial speck disease on tomato, have evolved from bacteria with relatively wide host range to become highly aggressive specialists on a single crop since the beginning of agriculture. This shows that agricultural practices may have a dramatic impact on the evolution of more aggressive plant pathogens. Therefore, it is fundamental to consider pathogen evolution when devising new agricultural practices to reduce the risk of the evolution of new pathogen variants as much as possible. THE GENOME OF P. SYRINGAE PV. TOMATO T1 AND OTHER CLOSELY RELATED BACTERIA. High throughput genome sequencing makes it possible today to sequence more than a dozen bacterial genomes for the price of sequencing one genome only five years ago. In this project, several closely related isolates that either cause disease on Arabidopsis and tomato or only on tomato have been sequenced. The first genome to be sequenced in this project was P. syringae pv. tomato T1. This isolate does not cause disease on Arabidopsis while the isolate DC3000, sequenced several years ago by other researchers, does. Genomes were compared thoroughly. In particular, it was found that isolate T1 contains 10 genes coding for proteins secreted into plants that are absent from DC3000. These proteins could be eliciting an immune response in A. thaliana blocking growth of T1. However, it was found that such an immune response is not sufficient to explain A. thaliana resistance to T1. Further characterization of which genes in isolate T1 and in A. thaliana determine the resistance of A. thaliana to isolate T1 will be instrumental in unraveling plant resistance in order to breed and engineer crops with durable (long lasting) disease resistance in the future.
Publications
- Mohr, T.J., Liu, H., Yan, S., Morris, C.E., Castillo, J.A., Jelenska, J., Vinatzer, B.A. (2008) Naturally Occurring Non-pathogenic Isolates of the Plant Pathogen Species Pseudomonas syringae Lack a Type III Secretion System and Effector Gene Orthologues. Journal of Bacteriology 190(8):2858-70
- Yan, S., Liu, H., Mohr, T.J., Jenrette, J., Chiodini, R., Zaccardelli, M., Setubal, J.C., Vinatzer, B.A. (2008) The Role of Recombination in the Evolution of The Model Plant Pathogen Pseudomonas syringae pv. tomato DC3000, a Very Atypical Tomato Strain. Applied and Environmental Microbiology 74(10):3171-81
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: To improve our basic understanding of virulence mechanisms, evolution, and epidemiology of plant pathogenic bacteria the following research activities have been performed this year: 1. Typing of plant pathogenic bacteria, 2. Host range tests of plant pathogenic bacteria, 3. Construction of mutant lines of plant pathogenic bacteria. 1. The technique multilocus sequence typing (MLST) was used to type bacteria belonging to the species complex Pseudomonas syringae. MLST is considered the most advanced typing technique for bacteria today. MLST profiles obtained for bacterial strains are based on DNA sequences. Profiles are thus qualitative. Once data are deposited in a database, any lab around the world can use them as reference allowing comparison of pathogens from all over the world to exactly identify them and to unravel their evolution and epidemiology. We have analyzed by MLST a group of closely related pathogens isolated from tomato, leafy greens, and celery to investigate
their evolution. We have analyzed a group of P. syringae pathogens from leafy greens that caused recent outbreak in California to exactly identify them, and we also analyzed P. syringae strains isolated from non-agricultural habitats (wild plants, rivers, snow, and rain) to investigate the worldwide epidemiology of P. syringae. 2. We determined the host range of the P. syringae strains, which we had analyzed by MLST, to correlate the host range of these strains with their phylogenetic relationship to unravel the evolution of host range. 3. We made mutations in known and putative virulence genes in P. syringae to investigate the contribution of these genes to virulence on tomato and leafy greens. Teaching and mentoring were integral parts of this year's activities. The PI mentored the graduate student Shuangchun Yan in the above described research activities and in learning the theoretical background to interpret the obtained data and to develop follow up experiments. Part of the
research activities were also integrated into a graduate course the PI teaches (Title: Plant Pathogenic Agents) to introduce students to modern typing methods and how these techniques can be applied to the identification of plant pathogenic bacteria. Graduate students learned in the lab section of this course how to isolate bacteria from plants and how to type them.
PARTICIPANTS: Individuals: The PI Boris Vinatzer (PPWS Department, Virginia Tech) developed and managed the project, advised the personnel, established collaborations, interpreted the obtained data, made choices on future directions, presented results at professional meetings, and wrote journal articles. The Laboratory and Research Practitioners Jenny Jenrette (until February 2007) and Haijie Liu (starting June 2007) performed a large part of the experiments for bacterial typing and host range tests. Collaborators and contacts: Joao Setubal, Associate Professor at the Computer Science Department (Virginia Tech) and the Virginia Bioinformatics Institute is the main collaborator in the project providing his expertise in bioinformatics and comparative genomics to the project. Dave Schneider, Research Leader at USDA ARS (Cornell University, Ithaca, NY) and Alan Collmer, Professor at the Plant Pathology Department at Cornell University) provide expertise in genomics and bacterial
pathogenesis respectively. Carolee Bull, research plant pathologist at USDA ARS (Salinas, CA) provides expertise in the taxonomy of plant pathogenic bacteria. Cindy Morris, research scientist at INRA (Avignon, France) conducts research into the importance of non-agricultural environments in the life cycle of the plant pathogen P. syringae and collaborates in regard to the evolution and epidemiology of P. syringae. Training or professional development: The graduate student Shuangchun Yan and the undergraduate students Eric Hall and Chandler Douglas are being trained in plant pathology, microbiology, and molecular biology as part of this project and contributed to various experiments.
TARGET AUDIENCES: Target audiences consisted mainly in other researchers that were reached through publications and presentations at scientific meetings. Also graduate students were reached by focusing the lab sections of the graduate level course with the title "Plant Pathogenic Agents" on the isolation and characterization of plant pathogenic bacteria and their host range giving students a hands-on experience in plant pathogen identification and molecular evolutionary analysis.
Impacts
In the discipline of taxonomy the diversity and relationships of bacteria are studied. Which criteria to use when describing bacterial plant pathogen species is not an obvious choice and is heatedly debated. Since 1980, all bacteria that cannot be distinguished from the species Pseudomonas syringae based on biochemical tests are part of the species P. syringae and are identified at the subspecies level as pathovars depending from which plant species they were isolated from, for example, isolates from tomato are identified as pv. tomato. The typing of closely related strains known as P. syringae using MLST and the determination of their host range has allowed us to gain understanding of the diversity within P. syringae. We were able to precisely resolve how closely related P. syringae strains are related to each other and how different the range of plants is that they attack. The results from this study can now be used to develop new rules on how to classify the diverse
group of bacteria known as P. syringae by dividing them into specific sub-groups and to finally decide if some of these sub-groups should be described as new species. This is very important for the discipline of phytopathology and for the control of plant diseases because a classification scheme that does not adequately describe the diversity of plant pathogens can lead to inadequate identification of plant pathogens in the field and consequently to inadequate and inefficient control measures. Correlating the phylogenetic relationship between P. syringae strains with their host range has also allowed us to develop a hypothesis on the evolution of plant pathogens since the beginning of agriculture. We inferred from the phylogentic relationship between P. syringae strains with different host ranges that the ancestor of the analyzed strains was probably a pathogen with a relatively wide host range able to infect various plant species. On the other hand, the most aggressive P. syringae
strains that are isolated from tomato today are of narrow host range and are able to infect only tomato. We hypothesize that pathogens of pre-agricultural times that lived in natural mixed-plant communities needed a relatively wide host range to be successful. Causing disease only on tomato would have limited their spread severely. However, with the advent of agriculture and the establishment of entire fields of the same crop species it probably became advantageous for pathogens to limit their host range to single crop species and to increase their aggressiveness on that single species. This hypothesis now provides us with a framework to study and compare P. syringae strains from wild plants and crop plants. Testing this hypothesis will provide us with important insights into short and long term evolution of plant pathogens. These insights will be fundamental for developing adequate agricultural practices and for breeding crop plants with durable disease resistance for a sustainable
and competitive Virginia and US agriculture.
Publications
- Vinatzer, B.A., Yan, S. (2007) Mining the genomes of plant pathogenic bacteria: how not to drown in gigabases of sequence. Molecular Plant Pathology (published as OnlineEarly)
- Morris C,E., Sands, D.C., Vinatzer, B.A., Glaux, C., Guilbaud, C., Buffiere, A., Yan, S., Dominguez, H., Thompson, B.M. (2008) The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. The ISME Journal: in press
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Progress 10/01/05 to 09/30/06
Outputs
The goal of this Hatch proposal is to investigate the molecular basis of host switching in the plant pathogenic bacterium Pseudomonas syringae. We want to understand for example how a pathogen on tomato can become a pathogen on cauliflower. Towards this goal we have set up in our lab a molecular approach that allows us to clone and sequence large genomic regions of P. syringae that distinguish isolates from different host plants. We use a technique called Long and Accurate Polymerase Chain Reaction (LA-PCR). Since the beginning of this proposal we have implemented this approach to clone and sequence differences between the pathogenic P. syringae strain PsyB728a isolated from bean and a nonpathogenic P. syringae strain Psy508 isolated from a healthy apple leaf. We found differences in three different regions of the genomes between these strains. Each of these regions contains in the pathogenic PsyB728a strain genes for effector proteins that are secreted from P.
syringae into plants during infection. We wanted to ask the question if these genes are present in the non-pathogenic Psy508 strain. We found that neither of these regions contains any effector gene or any other genes involved in virulence in the nonpathogenic strain. We then asked if the nonpathogenic strain had retained its ability to secrete effector proteins into plants using a Type 3 Secretion System (T3SS). We used a technique called Plasmid Rescue to clone and sequence the genomic region in Psy508 that corresponds to the hrp/hrc region that codes for the T3SS in the pathogenic PsyB728 strain. Surprisingly, we found that the entire region in Psy508 was replaced by a bacteriophage genome. We conclude that Psy508 evolved from a pathogenic strain into a nonpathogenic strain by losing its ability to secrete effector proteins into plants due to loss of its T3SS. Once it lost its ability to secrete effectors, it probably lost the genes coding for effectors since there was no selection
pressure to retain them. These results show that our approach is efficient and informative to find and sequence genomic differences between P. syringae strains to understand the evolution of P. syringae strains. We also wanted to improve our conditions for LA-PCR. The longer the products that we can obtain by LA-PCR, the more differences between P. syringae strains can be cloned using this approach. We therefore tested different parameters in LA-PCR. In the preliminary data section of the Hatch proposal we reported that we could obtain 22,000 bp long products. We have since then increased the length of LA-PCR products to 30,000bp. This increase will allow us to clone and sequence the majority of differences between P. syringae strains. We have also extended our collection of very closely related P. syringae strains isolated from different hosts that we can now analyze by LA-PCR to understand how they adapted to different hosts. This will ultimately help us to unravel the molecular
basis of host switching.
Impacts
Infectious diseases of humans, animals, and plants have a constant humanitarian and economical impact on our country and the world. It is a prerogative to understand how infectious diseases evolve to learn how to control them. This Hatch proposal has the long-term goal to understand basic molecular mechanisms involved in the evolution of infectious diseases and has thus a potentially very deep impact on human health, animal health, and agricultural productivity. In the medium term the results of this Hatch proposal will help understanding the molecular basis of nonhost resistance, i.e. understanding why most plant are resistant against most pathogens. This will positively impact agricultural productivity since it will provide the knowledge on how to engineer crop plants for resistance to various diseases. In the short term finding any differences between bacterial plant pathogens will aid in distinguishing very similar isolates which in turn will help understanding
how bacterial plant pathogens spread. This information can then be used in measures to avoid contamination of seed and other propagation material with bacterial pathogens. This will lead to a reduction in disease incidence and consequently an increase in agricultural production, in particular on the Eastern Shore in Virginia, where bacterial plant pathogens seriously reduce quality and yield of tomato production year after year.
Publications
- Vinatzer BA, Teitzel GM, Lee MW, et al. The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non-host plants. Molecular Microbiology 62 (1): 26-44 OCT 2006
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