Disease Mechanisms and Management Strategies for Pierce's Disease

NON-TECHNICAL SUMMARY: We are conducting basic and applied research on Pierce's disease of grapevines whose goal is to better understand the disease process and identify potential methods to prevent or cure the disease.

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APPROACH: We have previously constructed a transposon library that contained over 1,000 Xf Tn5 mutants. These mutants were screened for altered pathogenicity in Vitis vinifera grapevines growing in the greenhouse. Nine of the mutants showed hypervirulent phenotypes (Guilhabert and Kirkpatrick, 2005). One of those mutants was in a hemagglutinin (HA) gene (HxfA), HAs are large membrane bound proteins that mediate cell-cell clumping of Xf in vitro. A second Xf HA, HxfB, was knocked out by site directed mutagenesis. We prepared antibodies to the Xf HAs and localized them to the outer membrane, a membrane vesicle fraction and as free secreted protein in culture media (Voegel and Kirkpatrick, submitted). Full-length and truncated HA genes containing putative cell-cell binding domains were cloned into Agrobacterium plant transformation vectors and HA-expressing transgenic tobacco and grapevines will be evaluated for their relative susceptibility to Xf. We hope that HAs expressed in transgenic grapevines will clump Xf cells in planta and slow their systemic movement in transgenic plants, thus offering a novel mechanism for Pierce's disease.
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Work by Purcell's lab at UC Berkeley showed that Xf infected grapevines can be cured by relatively severe cold temperatures. However the physiological basis for this cold-curing phenomena is not known. The goal of recent past and future research is to better understand the mechanism(s) mediating cold curing. We have determined optimum cold temperatures for curing Xf-infected grapevines without unacceptable plant mortality. We determined what physiological changes such as pH, osomolality, hydration etc occur in the plant host and then subject cultured Xf cells to these altered conditions and assessed what impact, if any, they have on Xf viability. Field plots, using potted healthy and Xf-infected grapevines, were established in several geographical regions with various cold temperature extremes. These vines were placed in these environments over the winter and xylem sap was expressed from the vines at various times during the winter using a pressure bomb. The xylem saps were analyzed for physiological and biochemical alterations. Significant changes in total phenolic content in cold exposed vines were correlated with increased PD curing. Future research will identify the chemical structures and potential Xf anti-microbial activity of these compounds. We hypothesize that i) some factor(s) induced in vines by cold stress are responsible for the cold curing phenomenon or ii) cold temperatures and accompanying desiccation are determined to the survival of Xf in cold exposed grapevines. If we can better understand the mechanism(s) responsible for the cold therapy phenomena it may be possible to induce these factors by means other than cold treatment.