The characterization and role of Xylella fastidiosa plant cell wall degrading enzymes and exopolysaccharide in Pierce's disease of grapevine

Xylella fastidiosa (Xf), a gram negative fastidious bacterium, is the causal agent of Pierce's disease (PD) of grapevine (Vitis vinifera) and several other economically important diseases. Xylem vessel blockage is the accepted cause of the disease and symptoms in grapevine include leaf scorch, raisining of berries, general stunting, and vine death. PD has devastated some viticulture areas in California and understanding how Xf causes disease is the first step to successfully controlling PD. In silico analysis of the Xf genome has provided insight into the possible mechanisms of pathogenicity used by this bacterium. The goals of this study were to investigate plant cell wall degrading enzymes (CWDEs) and exopolysaccharide (EPS) as possible virulence determinants of Xf in grapevine.; There are several open reading frames; (ORFs) in the Xf genome encoding for putative plant CWDEs and it is hypothesized that the bacterium utilizes these enzymes to facilitate its systemic movement throughout the xylem network of grapevines. In this study we confirmed that two ORFs, PD 1485 and PD 1851, encode a functional polygalacturonase (PG) and a beta-1,4 endoglucanase, respectively. Moreover, mutants deficient in PG were unable to move efficiently from the point of inoculation and unable to cause disease in grapevine, thereby indicating the PG as the first major pathogenicity factor described for Xf.; The Xf genome contains an operon very similar to the gum operon found in Xanthomonas campestris that is responsible for the production of the EPS, xanthan gum. Other phytopathogenic bacteria require EPS for full virulence and it is hypothesized that Xf EPS may also contribute to virulence. In this study we confirmed that Xf is capable of producing an EPS that is similar in structure and composition to what was predicted by in silico analysis. This is the first biochemical evidence that Xf produces EPS. Using immunolocalization, we demonstrated that EPS is a major component of Xf biofilms formed in vitro and in planta and showed that Xf EPS contributes to xylem vessel occlusion in grapevine. However, the role of EPS in virulence is still unclear.