IDENTIFICATION OF TRAITS OF XYLELLA FASTIDIOSA CONFERRING VIRULENCE TO GRAPE AND INSECT TRANSMISSION BY ANALYSIS OF GLOBAL GENE EXPRESSION USING DNA MICROARRAYS Project Leader:

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 (Xcc). 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. 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 2555 genespecific 70 bp oligodeoxynucleotides including negative and positive controls. Microarray analysis was performed to identify genes that are controlled by DSF and/or RpfC. DSF bioassay with reporter strain Xcc 8523 (pKLN55) indicated that DSF production is most abundant 10 days after inoculation when rpfF expression is most active. Preliminary results reveal that at least 124 genes are controlled in response to rpfF in Xf, including those encoding gum production, type IV pili and hemagglutinin. Clearly this regulator has a large effect on the physiological function of Xf. Microarray analysis revealed that more than 300 genes are also controlled by RpfC, including some of the same genes regulated by rpfF as well as genes such as tonB. Microarray-based gene expression results are being verified using quantitative Reverse Transcriptase-PCR. Comparison of the RpfF and RpfC regulons reveals that a complex pattern of expression of potential virulence genes contribute to the virulence of Xf and explains the hypervirulence of rpfF mutants and the reduced virulence of rpfC mutants. Work is also underway to determine the subset of Xf genes that is plant-inducible and the identity of those whose plantinducible genes whose expression is also dependent on DSF production. INTRODUCTION PD of grape, a chronic problem in the grape industry in California now promises to be a far more devastating disease due to the introduction of the glassy-winged sharpshooter, which is a far more effective vector of the pathogen Xf (Purcell 1997). Xf apparently causes disease by multiplying within, and thus blocking, xylem vessels (Hill and Purcell 1995, Hopkins 1989). The colonization by Xf in grape and sharpshooters shows great similarities to microbial biofilms that form in other aquatic systems. Biofilms of bacteria develop on solid surfaces that are exposed to a continuous flow of nutrients to form thick layers. These structures consist primarily of an EPS matrix in which the bacteria are embedded. Cells in biofilms are inherently more resistant to many stresses such as antimicrobial compounds, viruses, and predators. The EPS matrix aids in the nutrition on the cells by accumulating various types of nutrients in a way analogous to an ion exchange column (Wolfaardt et al. 1994). Thus, cells in such aggregates are much more able to grow and survive than planktonic cells, which might be thought of as “scouts” for other colonization sites. Small molecules such as N-acyl homoserine lactones (AHLs), small peptides, butyrolactone derivatives or a fatty acid (known as DSF), play key roles as signals (Bassler 2002,Whitehead et al. 2001) in biofilm formation in numerous species of bacteria. The signals, which increase in concentration with population density, typically coordinate the expression of genes involved in exploitation of a host organism. The virulence of many pathogens is usually greatly reduced when the ability to produce signaling compounds is disrupted by mutation. Much evidence now indicates that Xf regulates virulence factors via its production of a fatty acid molecule (known as DSF) whose production is encoded by rpfF. Xf rpfF mutants exhibit dramatically increased virulence to plants, however, the rpfC mutant showed decreased virulence to plants (Newman et al. 2004). Numerous genes with various functions were identified to be controlled by rpfF in the plant pathogens Xanthomonas campestris pv campestris (Xcc), and Xanthomonas oryzae pv oryzae (Xoo) (Chatterjee and Sonti 2002). The RpfF homologue of Xf strains that cause PD, synthesizes a fatty acid cell-cell signal (DSF) similar to that of Xcc (Newman et al. 2004, Scarpari et al. 2003). Once DSF concentration reaches a threshold level, Xf senses DSF by RpfC, a two-component regulator containing both sensor kinase and response regulator domains, which then controls downstream genes. While DSF levels clearly are involved in regulation of virulence and behavior of Xf, we still do not understand what virulence factors are, nor how they are regulated in this pathogen. We thus are investigating the regulon dependent on DSF (rpfF regulon) as well as those genes dependent on rpfC both in culture and in plants to determine those factors that influence the interactions of the pathogen with plants and insects.