Structural and biochemical essays of XAC3230, a putative virulence factor of NAD: arginin ADP-ribosyltransferase family of phytophatogen Xanthomonas axonopodis pv citri

Abstract

We have recently discovered that the Xanthomonas axonopodis pv citri (Xac) genome codes for a protein, XAC3230, which is a homolog of the HopU1, HopO1-1 and HopO1-2 virulence factors from Pseudomonas syringae pv. tomato. These proteins belong to the NAD:arginine ADP-ribosyltransferase family (ART) that covalently modifies arginine residues of target proteins with ADP moieties. Many bacterial toxins are members of the ART family, including instance cholera, pertussis and diphtheria toxin. HopU1, HopO1-1 e HopO1-2 are secreted by type III secretion system (T3SS) in P. syringae. It was recently shown that HopU-1 suppresses the host innate immune response by modifying RNA-binding proteins of the plant. Furthermore, the N-terminal portion (50 residues) of XAC3230 has a high identity with XAC0011 and XAC0286, putative virulence factors secreted by T3SS in Xanthomonas. This N-terminal sequence is probably a necessary condition to be secreted by T3SS. XAC3230 has not been characterized previously nor was it previously identified as a possible virulence factor in Xanthomonas. At present, no structure of a virulence factor of the ART family originating from phytopathogenic bacteria has been resolved. The aim of this work is to express, purify, crystallize and resolve the structure of XAC3230 protein. Enzymatic essays using the purified recombinant protein will also be executed. Finally, we aim to produce a xac3230 knockout strain for in vivo functional assays. In order to achieve these objectives, the following specific steps will be taken: a) Cloning of the xac3230 gene and expression of the XAC3230 protein; b) Purification of XAC323; c) NAD:arginine ADP ribosyltransferase enzymatic activity essays; d) Crystallization of the purified protein; e) Resolution of its three dimensional structure by X-ray diffraction; f) Production of a xac3230 knockout strain and subsequent complementation with the wild type gene and a mutant gene; g) In vivo pathogenicity assays in susceptible plants using wild type and mutant Xac strains. (AU)