EXPLOITING XYLELLA FASTIDIOSA PROTEINS FOR PIERCES DISEASE CONTROL

• Author(s): Bruening, George; Civerolo, Ed; Dandekar, Abhaya; Gupta, Goutam;
• Abstract: The Xylella fastidiosa (Xf) is the causal agent of Pierces disease of grape. In previous work, we discovered, partially purified, and investigated the processing of the Xf protein MopB, which previously had been known only from the nucleotide sequence of its gene. The amino acid sequence of MopB, the uniform staining of Xf cells with fluorescent anti-MopB antibody and the abundance of MopB in total protein extracts of Xf cells suggest that MopB is the major outer membrane protein of Xf. As such, MopB is expected to participate in Xf colonization of grape xylem elements. We previously demonstrated that partially purified MopB binds to (xylem-rich) balsa wood or cellulose (filter paper) disks under conditions in which other proteins do not adhere. Here we report improvements in our MopB purification procedure and observations on adherence of MopB in Xf cells to cellulose disks under conditions that eluted other Xf proteins. A high (0.25mM) concentration of the cellulose fragment cellotetraose did not interfere with the binding of MopB to cellulose, suggesting that the binding reaction of MopB is not specific for cellulose. We exposed Xf cells or MopB to each of three fibrous polymer disks and to cellulose disks and observed similar adherence of MopB from both sources to all four polymer disk types. Thus, MopB appears to associate with porous materials generally when it is exposed to such materials in purified form or as Xf cells. The abundance and exterior exposure of MopB makes MopB an ideal target for Pierces disease control strategies. We seek to develop soluble proteins with high affinity for MopB. We will apply, as an anti-Xf agent, a selected MopB-binding protein alone or as a chimera with a bacterial cell-inactivating peptide or protein. Our expectation is that expression of the anti-Xf protein, targeted to the xylem in grape rootstock, may result in the anti-Xf protein moving into and protecting the grafted scion. In this reporting period, experiments were initiated with the objective of creating a protein having high affinity for MopB. As a first step towards this objective, Project Scientist Paul Feldstein developed E. coli strains expressing surface elements of MopB protein, so that the experimentally compliant E. coli can be used to select proteins with high affinity for Xf MopB.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium