Identification and structure-function analysis of protein-protein interactions in the plant pathogen Xanthomonas axonopodis pv citri

Abstract

In this project we propose to carry out genome-scale identification of protein-protein interactions in the recently sequenced bacterial plant pathogen Xanthomonas axonopodis pv citri. Interactions will be identified using Xanthomonas ORFS in a yeast two-hybrid system based on Gal4 DNA-binding and activation domains. The Xanthomonas ORFS are derived directly from the Xanthomonas pUC and M13 shotgun libraries produced during the sequencing project. Initially, we will focus on identifying interactions involving proteins coded by Xanthomonas pathogenicity-associated genes, type III secretory pathway genes and ORFS whose products have no known function. In this last category, we will initially target approximatelt 110 ORFS which are found in both Xanthomonas axonopodis pv citri and the recently sequenced citrus pathogen Xylella fastidiosa (Simpson et al. 2000), but have not as yet been found in any other organism. Results derived from protein interaction mapping in Xanthomonas will be integrated with data obtained from parallel projects involving transposon-mediated gene knockouts, gene expression microarrays and proteomics. The identification of protein-protein interactions of interest will open the door for future interaction and structure/function studies by classical biochemical and spectroscopic methods. In addition, in a second phase of the project, we hope to expand the interaction map to include all of the remaining ORFS enconding hypothetical proteins and proteins of unknown function. (AU)