EXPLOITING XYLELLA FASTIDIOSA PROTEINS FOR PIERCE’S DISEASE CONTROL Project Leaders:

The principal objective of this project is to construct and express in test plants, and then in grapevine rootstock, a protein or protein chimera capable of inactivating Xylella fastidiosa (Xf), the causative agent of Pierce’s disease (PD) of grapevine. Prior results from this project identified MopB as a, or possibly the, major outer membrane protein of Xf. We have shown that MopB is accessible on the Xf cell exterior and is a member of the OmpA family of outer membrane proteins of Gramnegative bacteria. The abundance of MopB in Xf cell extracts, known packing density of OmpA in a crystal, and Xf cell dimensions allowed us to estimate that MopB probably accounts for at least 10% of the Xf cell exterior. Thus, MopB is a highly suitable target for inactivation of Xf cells. Previous results suggested that some portion of the intact MopB gene from Xf is sickening to E. coli. Nevertheless, two E. coli strains were generated by substitution into the endogenous OmpA gene, one expressing mature MopB and the other a MopB-OmpA chimera with the amino-terminal half from MopB. Cells of both strains display MopB antigen on their surface, though accumulation is to a level much lower than MopB achieves in Xf. The strains are immune to bacteriophage K3, for which OmpA is the receptor. We modified and randomly mutated the OmpAbinding gp38 adhesin protein of bacteriophage K3 and will use Xf cells and the MopB-surface E. coli strains described above to select bacteriophage K3 variants that use MopB as the receptor. The selected gp38 gene will form the core of an anti-Xf protein. A readily transformed and regenerated tobacco line, SR-1, was identified as being susceptible to Xf and producing PD-like symptoms and cytology. SR-1 will be a used to test anti-Xf proteins and optimize constructions for grapevine transformation. High level expression of a fragment of MopB has been achieved and the same technology will be used to obtain sufficient quantities of MopB to complete its biological characterization. INTRODUCTION Resistant grapevine cultivars present the best approach to long term, effective, economical and sustainable control of Pierce’s disease (PD). This project has developed data showing that the OmpA class protein MopB of Xylella fastidiosa (Xf) is a major outer membrane protein of the bacterium. The demonstrated accessibility of parts of the MopB molecule on the cell surface and its abundance identify MopB as a high priority potential target for inactivation of the Xf cell or interference with the Xf infection cycle. As background, results from our prior research are summarized below (Bruening et al. 2005): a. Xf cells, fresh or heat-killed, when pressure-infiltrated into Chenopodium quinoa leaves, induce within two days chlorosis (chloroplast bleaching) that is limited to the infiltrated area of the leaf (CqC activity). b. The CqC activity is protease sensitive and was associated with a gel electrophoresis band that was found, by mass spectrometry, to contain predominantly the putative (OmpA class) Xf outer membrane protein MopB. c. The mature, 38.5K MopB protein was found to result from the release of a 22 amino acid leader peptide. The bulk of mature MopB molecules have a pyroglutaminyl amino end. d. MopB was partially purified in soluble form using sodium dodecyl sulfate (SDS) solutions but reducing, at the last step, SDS to very low levels. e. Application of anti-MopB antibody demonstrated that MopB is accessible on the Xf cell exterior and appears to be evenly distributed over the Xf cell surface. f. MopB is an abundant protein of Xf and may be the major outer membrane protein of the bacterium. g. E. coli did not tolerate plasmid constructions bearing the entire MopB gene from Xf. However, placing the MopB open reading frame under control of a bacteriophage-derived promoter allowed the production of low amounts of MopB in E. coli. h. Both purified MopB and MopB still embedded in Xf cells showed a strong propensity to associate tightly with porous materials of a variety of chemical types. This result is reminiscent of the observed association of a Pseudomonas fluorescens OmpA protein, OprF, with root surfaces (De Mot and Vanderleyden 1991, Deflaun et al. 1994), MopB may be involved in the association of Xf cells with the interior of xylem elements in the inoculated plants. Our principal objective is to construct and express in test plants, and then in grapevine rootstock, a MopB-binding protein (MBP) or protein chimera capable of inactivating Xf. We expect that a construction of suitable design will confer, on the