Progress 10/01/10 to 09/30/14
Outputs
Target Audience: This project reached three major target audiences : plant pathologists and microbiologists studying the fundamental biology of plant-bacterial interactions, graduate and undergraduate students who were trained in these disciplines, and applied plant pathologists working to magage bacterial wilt disease and related agricultural problems. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student supported in part by this project earned a doctoral degree (Jonthan M. Jacobs, currently holding an NSF Postdoctoral Fellowship at IRD in Montpellier France). A second graduate student supported in part by this project (Beth L. Dalsing) is expected to defend her doctoral dissertation in summer 2015. How have the results been disseminated to communities of interest? By publications in the peer-reviewed literature (listed above and in previous reports) and by oral and poster presentations at national and international meetings (American Phytopathological Society, American Society for Microbiology), and the International Society for Plant-Microbe Interactions). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We compared the transcriptomes of two different Ralstonia solanacearum strains during infection of a common (and economically important) host, tomato. Following over a year of unsuccessful attempts to obtain latent infections that were consistent and predictable, the project was re-focused to instead use gene expression analysis to characyterize the in planta transcriptome of this major pathogen. We further examined differences in gene expression profiles between a tropical R. solanacearum strain, GMI1000, and a cool-temperate variant, Race 3 biovar 2 strain UW551 (Jacobs et al 2012). The resulting data generated many testable hypotheses about how this group of bacteria succeeds in the rhizospheres and xylem vessels of susceptible crop plants. We tested these hypotheses by creating targeted mutant strains of the pathogen lacking one or more candidate virulence genes. These mutants were then analyzed for their ability to grow in plants, compete with other R. solanacearum strains, and cause disease. We found that a sucrose-specific uptake system and degradation pathway contribute to bacterial wilt virulence, suggesting that this sugar is an important nutrient for the pathogen in planta (Jacobs et al 2013). We also found that R. solanacearum uses a broadly conserved effector protein, called PopS, to suppress salicylic acid-mediated defenses in tomato plants. Mutants lacking PopS were reduced in virulence on several -but not all- solanaceous plant hosts (Jacobs et al 2013). A bacterial mannose/fucose-binding lectin protein was differentially expressed at cool temperatures in strain UW551 and was required for full virulence at low temperatures (but not at tropical temperatures). Interestingly, mutant analyses showed that this lectin also contributed to the ability of the bacterium to attach to tyomato roots and to survive in potato tubers at the standard potato storage temperature of 4C (Meng et al, in review). Bacterial genes encoding metabolism of inorganic nitrogen compounds were among the most highly expressed in planta. We therefore explored the roles in virulence of nitrate, which is a common element of fertilizer and a relatively abundant ion in tomato xylem sap. We found that R. solanacearum requires its nitrate assimilatory pathway for full virulence and competitive fitness in tomato plants, and also, surprisingly, for normal regulation of a major virulence factor, extracellular polysaccharide (Dalsing et al, 2014). A comprehensive analysis of the R. solanacearum denitrifying pathway revealed that the pathogen uses this part of its metabolism for multiple purposes. Analysis of a set of five mutant strains each lacking one step in nitrate reduction or NO detoxification showed that nitrate serves as an alternate terminal electron acceptor that alows the bacterium to respire in the low-oxygen environment of plant xylem or in biofilms. In addition, R. solanacearum uses this pathway to detoxify reactive notrogen species like nitric oxide and nitrite, which are products of microbial respiration and may also derive from plant defense signaling (Dalsing et al, mBio, in review).
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Dalsing, B. L., A. N. Truchon, E. T. Gonzalez, A. S. Milling, and C. Allen. 201x. Inorganic nitrogen reduction and detoxification are necessary for full Ralstonia solanacearum virulence on tomato. In review at mBio
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2013
Citation:
Jacobs, J. M. 2013. BACTERIAL IN PLANTA TRANSCRIPTOMICS TO DEFINE BEHAVIOR OF PLANT PATHOGENIC RALSTONIA SPECIES. Ph.D. Dissertation, Department of Plant Pathology, University of Wisconsin-Madison.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Meng, F. L. Babujee, J. M. Jacobs, and C. Allen 201X. Comparative transcriptome analysis reveals cool virulence factors of Ralstonia solanacearum race 3 biovar 2. In review at PLoS ONE
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Target audiences reached were graduate students, research staff, and faculty who heard oral presentations at scientific meetings and peer institutions, and who read peer-reviewed publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project helped train three graduate students: Jennifer Colburn-Clifford (Ph.D. 2009, currently a postdoctoral researcher at Oregon State University); Fanhong Meng (Ph.D. 2011, currently a postdoctoral researcher at Texas A&M University); and Jonathan Jacobs (Ph.D. 2013, currently an NSF Postdoctoral Fellow at the Institute for Research in Development in Montpellier, France). How have the results been disseminated to communities of interest? By publication in the peer-reviewed scientific literature and oral and poster presentations at national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? We are continuing analysis of tomato responses to infection by R. solanacearum. The two primary goals for the coming year are: 1) to submit and publish a paper on virulence factors needed to cause bacterial wilt at cool temperate temperatures (research and most writing are complete), and 2) to complete the last experiments and analyses of tomato defense responses to R. solanacearum infection at temperate and tropical temperatures, and 3) to analyze the role of bacterial denitrification in R. solanacearum pathogemnesis.
Impacts
What was accomplished under these goals?
This project has supported studies of the biochemical mechanisms by which R. solanacearum succeeds in the low-nutrient, low-oxygen environment of plant xylem. Drawing on results of a screen for bacterial genes up-regulated during pathogenesis, we determined that R. solanacearum encounters an oxidative environment inside host plants, and that the stress response DNA-binding protein Dps is essential for bacterial success in planta and for survival of cold stress. We also identified a high-affinity cytochrome c oxidase that is essential for R. solanacearum growth under hypoxic conditions and for full virulence. In year 2, we initiated a comparative transciptomic analysis that identified genes differentially expressed during pathogenesis at two different temperatures by two different strains of R. solanacearum. This analysis yielded an excitingly comprehensive picture of bacterial physiology in planta, and also identified a lectin and a previously cryptic quorum sensing system that contribute to the epidemiologically important cold tolerance of R. solanacearum Race 3 strains. In addition, the transcriptomic data highlighted the unexpected importance of inorganic nitrogen metabolism during bacterial wilt pathogenesis. We found that the bacterium depends on nitrate assimilation to regulate expression of extracellular polysaccharide, a key virulence factor. Additional experiments on the denitrification pathway yielded preliminary data in support of a successful NSF proposal.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Dalsing, B. L., and C. Allen 2014. Nitrate assimilation contributes to Ralstonia solanacearum root attachment, stem colonization, and virulence. Journal of Bacteriology 196:949-960.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jacobs, J. M., A. Milling, R. M. Mitra, F. Ailloud, P. Prior, and C. Allen 2013. Ralstonia solanacearum requires PopS, an ancient AvrE-family effector, for virulence and to overcome salicylic acid-mediated defenses during tomato pathogenesis. mBio 4(6):e00875-13.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The objectives of this project were to analyze the transcriptome of the bacterial wilt pathogen Ralstonia solanacearum during infection of the tomato host plant, and, in parallel, to analyze the host plant's gene expression during this disease. To date, we have made substantial progress on both objectives. The first publication describing the pathogen transcriptome was published this year (see below). Using a modified hydroponic pathosystem, we sequenced the complete RNA set extracted from wilt-resistant and wilt-susceptible tomato plants infected with either a tropical or a temperate strain of R. solanacearum (or mock-inoculated with water). We harvested RNA at 24h post-inoculation, allowing us to capture a much earlier stage of the interaction than our previous analysis of gene expression after disease symptoms were already present. Interestingly, the tomato was already upregulating many known plant defense genes at this point, including a broad suite of genes encoding cell wall reinforcement and production of antimicrobial phenylpropanoid compounds. At the same moment in pathogenesis, the bacterial genes for degradation of these defenses were also highly expressed, suggesting a rapid interplay between plant and microbe early in the disease process. R. solanacearum mutants lacking the ability to degrade a subgroup of these, the hydroxycinnamic acids, were delayed in virulence, offering experimental confirmation of the biological importance of this interaction. PARTICIPANTS: Individuals working on this project at U. Wisconsin-Madison: Tiffany Lowe, PhD student; Jonathan Jacobs, PhD student; Tuan Minh Tran, PhD student; Melanie Mustful, B.S. student. Partner organizations and contacts: We are collaborating on aspects of this project with Professor Raka Mitra (Carleton College, Northfield, Minnesota); Dr. Philippe Prior (INRA and CIRAD, Reunion Island, France); and Professor Martha Hawes (University of Arizona). TARGET AUDIENCES: The target audience for this project is currently the basic biology research community working to understand the mechanisms of plant-microbe interactions and the basis of crop plant disease resistance. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We are collaborating on the plant transcriptome side of this project with Prof. Raka Mitra of Carleton College. This partnership with a faculty member from an small elite liberal arts college has expanded the impact of our work to additional undergraduate student researchers. In addition, Prof. Mitra's expertise in bioinformatics and plant biology makes her a valuable, complementary partner. This project is fostering a change in knowledge in the PhD and undergraduate students conducting these experiments and more broadly within our scientific community.
Publications
- Jacobs, J. M., L. Babujee,, F. Meng, A. Milling, and C. Allen. 2012. The in planta transcriptome of Ralstonia solanacearum: Conserved physiological and virulence strategies during bacterial wilt of tomato. mBio 3(4):doi:10.1128/mBio.00114-12.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities: To identify the mechanisms involved in development and maintenance of latent (asymptomatic) tomato infections by the bacterial wilt pathogen Ralstonia solanacearum, we initiated experiments to compare the transcriptomes of both the bacterial pathogen and the host plant during latent and active (symptomatic) infection. Plant and pathogen RNA was extracted and purified from separated samples of the same wilt-resistant tomato plants. Developing the methods to reproducibly generate these RNAs was non-trivial and consumed considerable time in this first year of the project. This experiment was replicated 4 times, with each replicate containing pooled RNA from 6-8 individual plants. The resulting plant and bacterial cDNAs are currently being sequenced by RNA-seq at the University of Wisconsin-Madison Gene Expression Center. The next step is analysis of the transcriptomic data. In addition, we conducted a field experiment in commercial tomato production areas on the Eastern Shore of Virginia, where a bacterial wilt outbreak is in progress. We measured the frequency of latent infection in plants growing in naturally infested soil, and determined the population sizes of the pathogen in tissues from both symptomatic and asymptomatic plants. We further used these experiments as an opportunity to field-test some new detection methods for this pathogen. PARTICIPANTS: Individuals: Dr. Annett Milling, Assistant Scientist, conducted the plant transcriptomic research. Beth Dalsing, Tuan Tran, and Alejandra Huerta, all PhD. students, worked on the field research, the diagnostic method development, and the bacterial transcriptomic analysis. The work conducted here contributed to the training of these three students. Collaborators: Assoc. Prof. Steven Rideout (Virginia Tech University) collaborated with our field experiments. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Change in knowledge: The transcriptomic experiments generated a reliable protocol for extraction of high-quality plant and pathogen RNA from infected tomato plants. The Virginia field experiments revealed that latent infection is common in a naturally occurring tomato bacterial wilt outbreak in fields planted to a moderately wilt-resistant tomato cultivar. Population sizes of R. solanacearum in latently infected plants were extremely high, averaging 10e9 colony forming units (CFU) per gram tomato crown tissue. This result suggests that latently infected plants are a significant source of inoculum driving an ongoing epidemic. The field samples validated a new detection method for R. solanacearum, which has a sensitivity of at least 10e5 CFU in field samples. Change in actions: Nothing to report yet.
Publications
- No publications reported this period
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Progress 10/01/10 to 12/31/10
Outputs
OUTPUTS: This project began in October, 2010 so there are no significant outputs to report yet. The PhD student who is supported by this project is conducting preliminary experiments to optimize extraction of plant and pathogen RNA from tomato plants latently infected with the bacterial wilt pathogen Ralstonia solanacearum. She is also conducting experiments to measure the fitness cost to plants of hosting latent bacterial infections. PARTICIPANTS: The following people are working on this project: Professor Caitilyn Allen; Dr. Annett Milling; Beth Dalsing (PhD student) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project began in October, 2010, so there are no outcomes and/or impacts to report yet.
Publications
- This project began in October, 2010 so there are no publications to report yet.
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