Understanding Bacterial Wilt Virulence from the Inside Out

NON-TECHNICAL SUMMARY: Ralstonia solanacearum Race 3, a quarantine plant pathogen and listed potential agroterrorism agent, forms latent (symptomless) infections of host plants that make detection and eradication very difficult. Latent infections also hinder control of the disease in the field. Our research will identify the specific genes that are expressed during latent infections of tomato plants , and determine how each gene functions to allow R. solanacearum to form these symptomless infections.

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APPROACH: Our working hypothesis is that different suites of R. solanacearum Race 3 genes are expressed during growth in the host plant in latent infection and full-blown wilt disease. We propose to test this hypothesis by comparing genes expressed during active pathogenesis on tomato plants, when the bacterium is causing disease symptoms, or during latent infection of the same host. These genes will be identified using the IVET (in vivo expression technology) system which we have already developed and tested with tomato. Briefly, IVET uses the host as a selective medium to screen for bacterial genes specifically induced during growth in the host. We have developed an IVET system for R. solanacearum and used it successfully to identify 156 unique genes induced in a Race 1 strain during growth in tomato plants. This bank of known plant-induced genes, together with the complete genome sequence of a Race 1 strain and the pending genome sequence of a Race 3 strain, gives us a comparative base that will allow rapid identification and analysis of the genes involved in latent infection. We have developed a rapid mutagenesis protocol and reporter gene-driven regulatory assays for use in this species. These will be used to measure the contribution of genes of interest, as well as their place in the complex R. solanacearum regulatory heirarchy.
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PROGRESS: 2003/10 TO 2007/09 <BR>
OUTPUTS: This project began with a focus on identifying virulence factors needed by the bacterial wilt pathogen, Ralstonia solanacearum, during its pathogenesis of host plants. Bacterial wilt is one of the world's most important plant diseases because it is widely distributed and also has an unusually broad host range that includes many economically and socially important crop plants. An earlier broad genetic screen for bacterial genes that are upregulated during growth in the model host tomato identified 153 unique in-planta-expressed (ipx) genes. These ipx genes encoded functions needed for bacterial taxis and motility, as well as functions needed to protect the pathogen from diverse stresses. We characterized the biological functions of several ipx genes by mutating the genes and studying the phenotypes of the resulting mutant strains. We found that the pathogen needs tactic motility (the ability to sense and move towards more favorable conditions) to invade and colonize host plants. Further, R. solanacearum depends on a specific type of motility, energy taxis or aerotaxis, to aggregate on plant roots (Yao 2007). A second group of plant-induced genes encoded multidrug efflux pumps. Experiments with mutant strains lacking these molecular pumps showed that they contribute to bacterial wilt virulence by allowing the pathogen to secrete and this avoid toxic plant antimicrobial compounds (Brown 2007). A general secretion system, called Tat (twin-arginine translocation) is also plant-induced and required for full pathogenic fitness. A bioinformatic analysis indicates the Tat system sectretes dozens of proteins, at least 4 of which play a direct role in virulence (Gonzalez 2007). The project's second portion has focused on identifying bacterial genes induced by plant root exudates. We developed an in vitro screen to tag such genes. Among the rex (root exudate-expressed) genes was a cluster encoding biosynthesis of a cyctochrome C oxidase complex.We hypothesize that this complex helps R. solanacearum survive and thrive in the low-oxygen microaerophilic environment of the rhizosphere and the plant vascular system. Experiments are underway to test this hypothesis. <BR> PARTICIPANTS: Darby Brown, PhD, MDTP (Now at UW-Richland Center) Jian Yao, PhD, Plant Pathology (Now at Michigan State University) Enid Gonzalez, PhD, MDTP and Biotechnology Training Program (now at UC-Davis) Jennifer Clifford, PhD candidate, Plant Pathology <BR> TARGET AUDIENCES: The target audience of our research are microbiologists, plant pathologists, and other scientists studying plant pathogens, plant rhizosphere microbiology, and ultimately more applied researchers working to develop novel and traditional strategies to control plant diseases. <BR> PROJECT MODIFICATIONS: We originally proposed comparing pathogen gene expression in actively and latently infected hosts. We spent about a year trying to identify experimental conditions that would reliably generate latently infected plants, but this proved to be impossible. The process appears to be driven by random factors that we did not identify. We therefore switch emphasis to identification of pathogen genes induced by plant root exudates, early in the host-pathogen interaction. This approach, although also technically challenging, has proven successful.
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IMPACT: 2003/10 TO 2007/09<BR>
This project has yielded basic biology knowledge about an important plant pathogen. Our in vivo expression screen identified an unusually large number of unique genes used by R. solanacearum during colonization of tomato xylem. This body of research has illuminated some of the molecular mechanisms that are critical for bacterial wilt pathogenesis and has begun to explore the mechanisms involved in the earliest stages of this pathogen's interactions with its host plants' root systems. Thus, it represents a significant advance in our understanding of this system, both in terms of specific mechanisms and in terms of the broad transcriptomic response of a pathogen to a host environment.