Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Plant Pathology
Non Technical Summary
Ralstonia solanacearum (Rs) causes bacterial wilt diseases (BW). This pathogen indiscriminately stifles crop yields for subsistence and commercial farmers. Under the right conditions BW losses can be above 90%. The pathogen's wide host range, long environmental survival, and broad geographic distribution make it especially destructive. Rs attacks many economically relevant crops including potato, banana, tobacco, peanut, ginger, cloves, and tomato. BW is best managed by planting resistant cultivars. However, there is no known gene-for-gene wilt resistance in tomato, and nearly all BW-resistant tomatoes were derived from a single poorly-defined breeding line called Hawaii7996 (H7996). H7996 resistance is mediated by several quantitative trait loci (QTL), which makes it difficult to introgress this resistance into desirable commercial cultivars. Understanding the mechanistic basis of this resistance would improve wilt disease management. Further, the resistance in H7996 is overcome by Rs strain UW551, one of the R3B2 sub-group of Rs strains. We use a model system in tomato crop to investigate the mechanism of resistance to Ralstonia solanacearum (Rs) the causal agent of bacterial wilt disease. As a potential bioterrorism threat, R3B2 strains are highly regulated U.S. Select Agents. Thus, understanding how R3B2 overcomes our best form of resistance adds to the importance of this research.We are focused on better understanding this important source of wilt resistance through asystematic investigation of both the plant and pathogen side of resistant and susceptible interactions. This will help future plant breeders breed more resistant plants and prolong the durability of this valuable control method in an economically important crop. Effective crop resistance deployment benefits growers, consumers, and future agricultural sustainability.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
We know that certain compounds in xylem sap must fuel Rs growth, that H7996 resistance is QTL-mediated, and that resistance functions in stem tissue, but little is known about xylem chemistry in wilt-resistant plants1,7,41. I propose to address these knowledge gaps through bacterial mutagenesis, validated plant virulence assays, comparative and quantitative metabolomics, bacterial and plant gene expression analysis, all in the context of both resistant and susceptible tomato-Rs interactions.Objectives: Objective: 1Determine howRalstonia solanacearum (Rs) responds differently to resistant and susceptible hosts.Sub-objective 1.1:When growing in xylem sap from resistant Hawaii7996(H7996) tomato, RsUW551 will upregulate detoxification pathways.Sub-objective 1.2:The gene expression profiles of Rs strains in planta will differ depending on host resistance or susceptibility.Objective 2:Uncover the chemical composition of a resistant xylem environment.Sub-objective 2.1: H7996 sap contains Rs inhibitory compounds.Sub-objective 2.2: H7996 will have distinct transcriptomic responses to UW551 and GMI1000.
Project Methods
Specific Aims and Research Approach-We know that certain compounds in xylem sap must fuel Rs growth and that H7996 resistance is QTL-mediated and stem resistance plays a role, but little is known about xylem chemistry in wilt-resistant plants. I propose to address these knowledge gaps through bacterial mutagenesis, validated plant virulence assays, comparative and quantitative metabolomics, bacterial and plant gene expression analysis, all in the context of both resistant and susceptible tomato-Rs interactions.AIM 1: Determine howRs responds differently to resistant and susceptible hosts. UW551 breaks our best form of genetic resistance to Rs in tomato. Clarifying how UW551 overcomes plant defenses will provide vital information to manage this resistance. Approach: Compare Rs gene expression during bacterial growth in susceptible and resistant tomato stems and in ex-vivo xylem sap from these plants, following our lab's validated protocols for profiling Rs transcriptomes in culture and in plant. Experiments will include 4 independent biological replicates for each treatment. Transcriptomic results will be validated using qRT-PCR.Ho 1: When growing in xylem sap from resistant H7996 tomato, RsUW551 will upregulate detoxification pathways. My preliminary data show UW551 has no problem growing in sap from H7996 previously infected with GMI1000, even though this sap inhibited GMI1000. Interestingly, concentrating the H7996 sap 6-fold reduced GMI1000 growth even more but did not affect UW551. I expect RNAseq analysis will show that genes helping UW551 overcome this inhibition will be upregulated in H7996 sap relative to in bacteria growing in sap from susceptible BB plants. Approach: RNA for transcriptomic analysis will be extracted from strains GMI1000 and UW551 after 4 hours' growth in 6-fold concentrated xylem sap from H7996 plants previously infected with GMI1000. The control condition will be both strains growing in sap from BB plants.Ho 2: The gene expression profiles of Rs strains in planta will differ depending on host resistance or susceptibility. Although technically accessible, growth in ex vivo xylem sap is an imperfect proxy for pathogenesis of whole plants. I will expand on our previous transcriptomic study of GMI1000 and UW551 transcriptomes during infection of a susceptible host to compare the GMI1000 and UW551 response to infecting susceptible and resistant whole plants. Approach: Using our lab's standard assays, I will soil-soak inoculate BB and H7996 plants with UW551 or GMIl1000 and harvest bacterial RNA from midstems 5 days later for RNAseq analysis as previously described. Differentially expressed genes will be compared between the strains (UW551 vs. GMI1000) in each plant host, and also in the same strain in susceptible and resistant host (UW551 in BB vs. UW551 in H7996, GMI1000 in BB vs. GMI1000 in H7996). Depending on preliminary data, additional time-points may be included to define the arc of these interactions.Potential Pitfall: Because H7996 is resistant to RsGMI1000, only ~15% of soil-inoculated H7996 plants are sufficiently colonized by GMI1000 for sap harvest or RNA extraction. By using many plants, I can obtain enough xylem sap for my proposed experiments. However, if I cannot get enough high quality GMI1000 RNA from soil-inoculated plants for transcriptomic analysis, I will instead inoculate plants directly through cut leaf petioles as previously described.Expected results for Aim 1: Analyses of these transcriptomic datasets will generate a snapshot of the contrasting environments experienced by Rs inside xylem vessels of wilt-susceptible and resistant plants. Genes encoding degradation of specific plant defense compounds (e.g. terpenes, phytoalexins, trans-cinnamic acids) as well as general stress tolerance may be upregulated in strain UW551 successfully infecting H7996. Conversely, gene expression in RsGMI1000 will indicate failure to overcome resistance-linked toxicity (e.g. slowed growth, membrane, DNA and protein repair machinery) as previously suggested. I expect to observe more similar expression patterns in GMI1000 and UW551 during infection of BB, where both strains are successful.Aim 2. Uncover the chemical composition of a resistant xylem environment. Xylem sap from wilt-resistant H7996 tomato inhibits growth of RsGMI1000 and its chemistry may thus explain resistance. Approach: I will characterize both the metabolome of xylem sap from Rs-infected wilt-resistant and susceptible plants and the plants' transcriptional response to infection with strains that defeat (GMI1000) or overcome (UW551) host resistance.Ho 3: H7996 sap contains Rs inhibitory compounds. Characterizing the resistant host xylem environment will identify specific candidate inhibitors of Rs growth. Approach: To compare xylem metabolomic profiles, I will harvest xylem sap from H7996 and BB plants previously infected with GMI1000 or UW551. Then I will quantify Rs populations, filter sterilize xylem sap, and normalize for Rs population size. Experiments will include 5 biological replicates, each with pooled sap from 5-10 plants. Samples will be analyzed by West Coast Metabolomics (U. California-Davis) using GC/MS as described. In parallel, I will subject the ex vivo xylem sap to a series of selective tests including heat treatment, protease treatments, size exclusion filtration, and various crude chemical extractions to compile a list of inhibitor characteristics (e.g bigger or smaller than 100kD, heat-stable, non-polar, etc.). Fractions or treated sap samples will be assayed for activity using the validated Rs growth inhibition assay. If time permits, I will collaborate with colleagues in the UW-Madison School of Pharmacy to further purify and characterize candidate inhibitors.Ho 4: H7996 will have distinct transcriptomic responses to UW551 and GMI1000. H7996 launches defenses in response to both Rs strains, but it upregulates SA and ET signaling faster and more when infected by GMI1000. However, the specific downstream resistance mechanisms activated by these signals remain unknown. Approach: I will harvest plant RNA from BB and H7996 tomato midstems 3, 4, or 5 days after soil-soak inoculation with RsUW551 or RsGMIl1000. Harvest timing will be determined based on qPCR of validated Rs-responsive genes like PR1a and ACOC. Only RNA from plants containing comparable Rs populations will be analyzed. Experiment will include 3 biological replicates of 4 plants each per treatment. RNAseq and basic bioinformatic data analysis will be conducted by Novogene, Inc.Expected Results for Aim 2: Combined direct chemical analysis (xylem sap metabolomics), together with a list of differentially expressed plant pathways should yield specific testable hypotheses about why H7996 xylem sap inhibits most Rs strains and how RsUW551 overcomes that inhibition to break H7996 resistance. For example, a terpene degradation pathway upregulated in UW551 bacteria infecting H7996 may correlate with upregulated terpene biosynthesis and increased terpenes in sap from the resistant plants. Manipulative experiments using plants silenced for terpene synthesis and bacteria lacking or over-expressing terpene degradation can directly test the hypothesis that xylem sap terpenes are a mechanism of H7996 wilt resistance. These experiments are reasonable because of the malleable pathosystem.