Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Xylella fastidiosa is a Gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell-cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines. INTRODUCTIONXylella fastidiosa, a Gram-negative, xylem-inhabiting bacterium, causes economically important plant diseases including Pierce's disease (PD) of Vitis vinifera grapevines (Hopkins, 1989) and citrus variegated chlorosis in citrus trees (Rossetti et al., 1990). X. fastidiosa is transmitted by numerous xylem-feeding insects such as sharpshooters and spittlebugs (Hewitt et al., 1946). Once introduced by their vectors, the bacteria multiply in the xylem of infected plants and form microcolonies and three-dimensional biofilms that cause blockage of the xylem vessels. These blockages are thought to be the major cause of the development of symptoms that are similar, but not identical, to water stress and eventually result in the death of the grapevines (Hopkins, 1989; Thorne et al., 2006). X. fastidiosa was the first plant-pathogenic bacterium to have its genome sequenced (Simpson et al., 2000;Van Sluys et al., 2003). The comparatively small genome (2.5 Mb) may explain X. fastidiosa's limited niches, occupying only the insect foregut and the plant xylem. In both locations, the bacteria are exposed to high turbulence, an environment low in nutrients, and host defence responses (O'Toole et al., 1999). These conditions make the formation of a bacterial biofilm a key element in X. fastidiosa survival and replication (de Souza et al., 2003;Guilhabert & Kirkpatrick, 2005;Rodrigues et al., 2008). Biofilm formation in X. fastidiosa is thought to be a sequential process in which planktonic cells attach to host surfaces, self-aggregate with other cells and form a biofilm matrix which consists of exopolysaccharides, proteins and probably DNA (Guilhabert & Kirkpatrick, 2005;Lin, 2009;Roper et al., 2007). The first step in biofilm formation, attachment to host surfaces, is mediated by several different factors, including fimbrial and afimbrial adhesins on the bacterial surface (Li et al., 2007). Fimbrial adhesins can be divided into two classes: long (1-5.8 mm) type IV pili encoded by pil genes, and short (0.4-1.0 mm) type I pili encoded by the fim operon (Meng et al., ...

The spread of Pierce's disease (PD) from riparian hosts to grapevines in California's northcoastal grape-growing region is a function of the proportion of Graphocephala atropunctata (blue-green sharpshooters [BGSSs]) that acquire Xylella fastidiosa from infected plant tissue. Riparian hosts that do not maintain sufficient X. fastidiosa populations for acquisition may not be significant inoculum reservoirs. We examined X. fastidiosa populations in systemically infected riparian hosts (California blackberry, California grapevine, elderberry, Himalayan blackberry, periwinkle) at two coastal locations (Mendocino and Napa) with two methods of quantitation (culturing and real-time polymerase chain reaction) from 2003 to 2004. In summer and autumn, X. fastidiosa populations were above the threshold for BGSS acquisition in periwinkle, Himalayan blackberry, and California grapevine at both locations. The only X. fastidiosa-positive plants detected in spring at both locations were periwinkle and Himalayan blackberry, suggesting that these species may contribute to long-term survival of X. fastidiosa. California blackberry and elderberry may not be important reservoirs of X. fastidiosa, given that very few plants of either species maintained infections. Higher X. fastidiosa populations in California grapevine, Himalayan blackberry, and periwinkle in Napa, relative to plants in Mendocino, may partially explain the higher PD incidence in Napa vineyards.

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.