ROLES OF XYLELLA FASTIDIOSA PROTEINS IN VIRULENCE Project Leader:

This work derives from a preliminary experiment by Civerolo and Bruening in which Chenopodium quinoa (Cq) was found to develop a localized chlorosis 24-48 hr after leaves were infiltrated with a suspension of live or heat-killed Xylella fastidiosa (Xf) cells. Excised electrophoresis gel regions were assayed for chlorosis-inducing activity, which was associated with a protein band with mobility corresponding to an estimated mass of 40K. Mass spectrometry of material the gel band revealed peptides corresponding to about 40% of the peptides predicted for the mopB gene. Xf mopB is an ompA protein. Members of the ompA group are located in the outer membrane of Gram-negative bacteria. The mopB translation product, signal peptide, and mature mopB protein were identified. In the present period, we showed that fluorescent anti-mopB IgG binds to intact Xf cells grown in liquid culture, confirming the location of mopB on the Xf cell surface. Previously we found that Xf cells extracted with SDS under specific conditions are depleted in most non-mopB proteins. This observation was extended by solubilizing mopB from the SDS-extracted cells at a slightly elevated pH, at 30°C in the presence of SDS and sodium perchlorate. Only traces of other proteins contaminated the mopB preparation. We postulate that mopB, as the major outer membrane protein of Xf, may participate in an interaction between Xf and the xylem sites at which colonization of the plant by Xf is initiated. Understanding a mopB-xylem interaction could direct strategies for interfering with Xf infection of grape and other Xf hosts. We demonstrated that thin balsa wood squares are able to absorb mopB from a mixture of mopB with other proteins, in the presence of non-ionic detergents known to be effective in solubilizing integral membrane proteins. Cellulose, in the form of washed filter paper, was similarly selective. These results support, but do not prove, our hypothesis about a role for mopB in Xf infection, possibly acting to bind Xf to the xylem interior. It may be possible to exploit mopB as a target for controlling Pierce’s disease. INTRODUCTION The bacterium, Xylella fastidiosa (Xf), is the causative agent of Pierce’s disease of grape. We reported previously the observation that untreated or heat-killed suspensions of 10 to 10 Xf cells/ml, pressure infiltrated into leaves of Chenopodium quinoa (Cq), induced a chlorosis in 24-48hr that conformed to the infiltrated area. The chlorosis reaction was the basis semi-quantitative assay for potency of Xf-derived preparations. The chlorosis-inducing activity survived treatment with the protein-denaturing detergent sodium dodecyl sulfate (SDS). Nevertheless, proteases inactivated the chlorosisinducing activity. Extraction of Xf suspensions with SDS under mild conditions (30 ̊C, pH approx. 8.5 buffer) removed many Xf proteins (analysis by gel electrophoresis, SDS-PAGE) from the insoluble fraction, but the chlorosis-inducing activity remained insoluble. Guided by stained bands in an SDS-PAGE gel, unstained gel segments were extracted, concentrated and tested by infiltration of Cq leaves. The bulk of the chlorosis-inducing activity was associated with material with mobility corresponding to molecular weight of about 40K. Analysis of tryptic digests by mass spectrometry revealed peptides derived from the Xf outer membrane protein mopB. The abundance of Xf mopB suggests that it is the major outer membrane protein of the bacterium. Although a minor contaminant of the mopB preparations could be responsible for the Cq chlorosis, most likely the chlorosis inducing agent is mopB itself. We identified the likely start of translation for the mopB gene, MKKKILT. (corresponding to a 40.7K translation product), recognized a candidate 22 amino acid residue signal peptide, and determined that the pyroglutaminyl-terminated peptide pyro-QEFDDR in tryptic digests mapped to the mopB gene sequence (Simpson, Reinach et al. 2000). Results from other experiments suggest that the pyroglutaminyl residue is the natural amino-terminal end of mature Xf mopB protein, predicted molecular weight 38.5, and is not created as an artifact of our analysis by cyclization of an amino-terminal glutamine residue. Xf mopB was further purified by solubilizing the insoluble fraction (from 30 ̊C SDS extraction of Xf cells) with hot SDS solution and chromatographing on 6% agarose beads. Pooled fractions were concentrated and used to raise polyclonal antibody. Attempts at cloning Xf mopB in E. coli, using constructions that encompassed the entire Xf mopB gene, including its putative promoter, were not successful. However, an inducible bacteriophage T7 RNA polymerase and T7 promoter system was adapted to create E. coli cultures that, when induced with IPTG, generated, at low levels, a protein with the mobility and immunological properties of mopB (work of Paul Feldstein). Xf mopB accumulation may sicken E. coli, accounting for the low level accumulation and requiring another approach for mopB production in E. coli.