| Ralstonia solanacearum causes bacterial wilt disease of many economically important food crops. Understanding the virulence mechanisms of this pathogen at the molecular level has been limited by available technology and by the additive, redundant nature of R. solanacearum virulence factors. Some virulence genes with large pleiotropic phenotypes have been isolated, but they reflect only a small fraction of the pathogen's ∼5000 genes. Rather than seek new virulence factors through additional loss of function screens, I developed an in vivo expression technology (IVET) system for R. solanacearum to identify bacterial genes that are upregulated in tomato xylem tissue. This IVET screen isolated 153 genes that were specifically up-regulated in xylem. These in planta expressed (ipx) genes encode diverse metabolic and stress-responsive functions, as well as seven previously-identified virulence factors. A subset of these genes was variably expressed in tomato xylem five hours post inoculation and many genes were differentially regulated by seven known R. solanacearum virulence regulators. Further, two ipx genes putatively encoding L-ascorbate oxidase and a DNA repair enzyme, UvrA1, were only expressed in planta, where they were controlled by known virulence regulators. The initial development of IVET technology for R. solanacearum and the resulting findings have paved the way for ongoing IVET-based screens.; 9.8% of IVET-identified genes encode proteins involved in bacterial stress response pathways. Two genes from this class, acrA and dinF, encode putative multidrug efflux pumps. Mutations in these genes significantly reduced virulence, and transcription of acrA and dinF increased over 100-fold in planta relative to minimal medium. Expression of these genes was increased by known plant-derived toxins, which suggested they protect the bacterium from harmful plant defense toxins. Two other stress response genes isolated in the IVET screen are under investigation: uvrA1 and bcp. UvrA1 is predicted to repair DNA damage, while Bcp is a putative peroxidase that may neutralize reactive oxygen intermediates encountered by the bacterium as it infects the host. Lastly, four additional genes identified in my IVET screen are currently under study. |
