Identification Of Novel Genes Involved In The Virulence Of Xanthomonas Oryzae Pv. Oryzae

It was previously shown that a cosmid clone pGXNGOOO containing Xanthomonas oryzae pv. oryzae (here after Xoo for short) 13751 DNA had at last four genes (rpfA rpfB rpfC rpfF), mutation in any of these genes resulted in significant reduction in the production of EPS and virulence of this bacteria. The plasmid pGXN3000 had been sequenced previously and sequence analysis revealved that there are at least four ORFs (pdeA,cheB,pcaD and pin) at the upstream of rpfA gene. The pdeA gene was 2262 bp, encoding a protein with 754 amino acids. PdeA has 96% similarity and 93% identity with c-di-GMP phosphodiesterase A of Xanthomonas axonopodis pv. citri str. 306. (here after Xac) .The cheB gene was 1077 bp, encoding a protein with 358 amino acids. CheB has 67% similarity and 67% identity with glutamate methylesterase of Xac. The pcaD gene was 813 bp,encoding a protein with 271 amino acids.PcaD has 92% similarity and 85% identity to beta-ketoadipate enol-lactonehydrolase of Xac. The pin gene was 651 bp. Amino acid 189-198 of P i n formed z i nc f i nger b i nd i ng doma i n (ZnF-NFX doma i n). There is one ORF dowmstream of rpfC, which encodes a protein having 99% similarity and 96% identity with Xcc RpfG, a regulator protein of two-component regulatory systems.In order to understand the function of the above mentioned five genes in Xoo 13751, nonpolar mutant of 13751 in pdeA(GXN1256), cheB (GXN1255), pcaD (GXN1261), pin (GXN1269) or rpfG (GXN1258) was constructed by homologous suicide plasmid integration.The lesion length of rice leaves caused by Xoo pin-(GXN1269) mutant was similar to that caused by Xoo wild type strain 13751, showing Xoo pin may not be necessary for the virulence of Xoo on rice. The virulence of Xoo rpfG- (GXN1258) , pdeA-(GXN1256), pcaD-(GXN1261) was significantly reduced compared to Xoo wild type strain 13751 both at high inoculation and low inoculation concentrations. The lesion length of rice leaves caused by Xoo rpfG-(GXN1258) , pdeA(GXN1256)and pcaD- (GXN1261) was 21. 37%, 64. 69% and 77. 44% of that caused by Xoo wi Id type 13751 at high inoculation concentration, respectively, and was 14.47%,77.99% and 76.10% of that caused by 13751 at low inoculation concentration, respectively, indicating that XoopdeA, rpfG and pcaD genes are required for the full virulence of Xoo on r i ce.The analysis of Xoo PdeA revealed that its ami no acid 317-488 formed DUF1 domain with c-di-GMP phosphodiesterase A activity and ami no acid 498-745 formed DUF2 domain (also named GGDEF motif) with diguanylate cyclase activity. Diguanylate cyclase is involved in the sythesis of c-di-GMP and phosphodiesterase A is involved in the turnover of c-di-GMP. Xoo pdek can restore the production of DSF of rpfG-mutant(GXN1258) and Xoo rpfG can restore the production of EPS and DSF of pdek mutant (GXN1256).The lesion length of rice caused by Xoo cheB-(GXN1255) with leaf-clipping inoculation was similar to that caused by Xoo wi Id type, while the disease index caused by Xoo cheB-(GXN1255) on rice with leaf-spraying inoculation was 48.91% of that of 13751. The chemotactic respone assay revealed the ability of chemotactic respone of Xoo cheB-(GXN1255) was significantly reduced, indicating that cheB is involved in chemotaxis of 13751. The role of cheB in the virulence of Xoo may through controlling the ability of bacteria to move towards hydrothodes. Chemotaxis may play an important role at the early stages in pathogenesis of phytopathogenic bacteria.