The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active hrp type III secretion system that is flanked by virulence genes functionally unrelated to the hrp system

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrp/hrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.