EXPLOITING XYLELLA FASTIDIOSA PROTEINS FOR PIERCES DISEASE CONTROL

• Author(s): Bruening, George; Civerolo, Ed; Dandekar, Abhaya; Feldstein, Paul; Francis, Marta; Gupta, Goutam;
• Abstract: 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 Pierces 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 Gram-negative 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 OmpA-binding 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.
• Publication Date: Dec 2005
• Journal: 2005 Pierce's Disease Research Symposium