Identification of traits of Xylella fastidiosa conferring virulence to grape and insect transmission by analysis of global gene expression using DNA microarrays
Xylella fastidiosa (Xf) regulates virulence factors important in both virulence to grape as well as colonization of sharpshooter vectors via its production of a fatty acid molecule (known as DSF) whose production is encoded by rpfF. The rpfF homologue of Xf strains that cause Pierce’s disease (PD), synthesizes a fatty acid cell-cell signal (DSF) that is apparently similar to that produced by Xanthomonas campestris pv. campestris. Xf rpfF mutants exhibit increased virulence to plants; however, they are unable to be spread from plant to plant by their insect vectors. While we have identified a key regulator of virulence and insect transmission in Xf we lack an understanding of the traits that are regulated by this pathogen in response to the DSF signal molecule. We thus are initiating studies to determine the rpf-regulation in Xf. The objectives of our study are: 1) determine those genes in Xf whose transcription is controlled by rpfF, the regulator of virulence and insect transmission, by assessing global gene expression using DNA microarrays, 2) determine the number and identity of genes in Xf that are expressed in grape plants but not in culture by assessing global gene expression using DNA microarrays, and 3) assess the contribution of individual genes of Xf whose transcription is dependent on rpfF to its virulence and insect transmissibility. We are exploiting a DNA microarray developed in another project that addresses host specificity genes in Xf to assess gene expression differences in isogenic rpfF+ and rpfF- strains of Xf strain Temecula. The microarray contains 2,555 gene-specific 70 bp oligodeoxynucleotides including negative and positive controls. We have isolated RNA from Xf strains grown both in culture as well as isolated from plants. After differential labeling with the fluorescent cyanine dyes Cy3 and Cy5, cDNAs made from these RNAs have been hybridized to the microarray. Preliminary results reveal that at least 150 genes are up-regulated in response to rpfF in Xf while at least 40 genes are repressed. Clearly this regulator has a large effect on the physiological function of Xf. Microarray-based gene expression results are being verified using quantitative Reverse Transcriptase-PCR. Work is also underway to determine the subset of Xf genes that might be plant-inducible and the identity of those whose expression is dependent on DSF production.