Ralstonia (Pseudomonas) solanacearum causes a lethal wilting disease of over 200 different plants worldwide. Secreted plant cell-wall-degrading exoenzymes and the EPS I exopolysaccharide, both contribute to its successful colonization of a plant's vascular system after root invasion. Because EPS I blocks water flow in the xylem, it is also a primary cause of wilting and killing. Biosynthesis and export of EPS I, a long unbranched polymer of 3 amino sugars, are encoded by the 16-kb eps operon. Transcription of the eps operon, as well as genes encoding some exoenzymes and other virulence genes, is controlled by a large, interactive regulatory network of over 12 proteins that is responsive to multiple environmental signals. This network is comprised of two distinct two-component regulatory systems (VsrAD and VsrBC), the unique signal integrator protein XpsR and the unusual Phc signal transduction system. Preliminary data suggest that the Phc module employs an atypical phosphorelay cascade that responds to 3-hydroxypalmitic acid methyl ester (3-OH PAME), a new type of volatile, quorum-indicating molecule. Phc and 3-OH PAME in turn regulate phcA, encoding a global, LysR-type transcriptional regulator that controls reversible switching between two very different physiological states, one adapted for virulence in plants, the other for saprophytic survival. A major goal of this project is to understand at the molecular level how the Phc module responds to 3-OH PAME, and how the Phc components subsequently control phcA function. Using two different approaches, a genetic one (transposon mutagenesis) or a biochemical one (DNA-affinity chromatography of R. solanacearum extracts on columns with the eps regulatory region covalently bound), missing genes that mediate regulation of eps by XpsR and VsrC), will be searched for, cloned, and characterized. These studies of the R. solanacearum virulence regulatory network are important because one of the most critical factors for successful pathogenesis is the ability to coordinate production of virulence and pathogenicity factors in response to environmental signals, and because the ability of the R. solanacearum network to process and integrate multiple signal inputs is largely unparalleled. These studies should give insight into the ways multiple environmental cues are processed by pathogens to adjust virulence gene expression. Finally, genes required by R. solanacearum for rapid, efficient colonization of plants by cloning and analysis of genes encoding several vsrD-regulated exoproteins will be searched for. In parallel, in vivo expression technology will be adapted and applied to R. solanacearum. This technology identifies and isolates genes that are highly expressed. by a pathogen only during colonization of a host. Either or both of these approaches will provide a way to access new, important genes, and provide insight into a pathogen's strategies for successful colonization of plants.
