|SHI, XIANGYANG - University Of California|
Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Abstract Only
Publication Acceptance Date: 10/26/2012
Publication Date: 12/12/2012
Citation: Lin, H., Shi, X. 2012. Characterization of the virulence-related genes of Xylella fastidiosa. CDFA Pierce's Disease Progress Report, p.85.
Technical Abstract: To elucidate the role of virulence genes of Xylella fastidiosa (Xf), responsible for Pierce’s disease in grapes, a site-directed deletion method was employed to knock out virulence-related genes of Xf strain Temecula. Six virulence-related genes were selected, of which three genes were annotated to be transcriptional regulators; hfQ, xrvA and gcvR, and three genes were associated with two-component regulators (algR, colR1 and colR2). The mutant strains were obtained from selective media and the deleted loci were confirmed by PCR. To evaluate pathogenicity of the six mutants, greenhouse-grown Cabernet sauvignon grapevines were mechanically inoculated with mutant bacteria; wild type served as a control. Three months after inoculation, grapevines inoculated with algR, xrvA, and colR showed only mild symptoms when compared with grapes inoculated with wild type Xf. However, no symptoms were observed in grapes inoculated with mutant strains of gcvR, hfQ, and colR. Quantitative PCR analysis was conducted to estimate Xf titers in infected grapes. In vitro studies showed that while both mutants and wild type of Xf had similar growth curves all mutants appeared to have significantly reduced abilities to adhere to the well of culture tubes. Biofim production of gcvR, hfQ, algR, and colR was 5-fold less than that of wild type whereas xrvA and colR mutants produced 2.5-fold less biofilm than that of wild type. To further investigate metabolic pathways that involve regulation of virulence genes, chemical profiles of mutant and wild type were analyzed by gas chromatography-mass spectrometry. All six virulence mutants have been complemented via chromosome based recombination. These complemented strains are currently being subjected to inoculation experiments along with wild type to examine restoration of gene function. Characterization of virulence-related genes in Xf will provide insight into mechanisms of pathogenicity of Xf.