Implication of hrpW in virulence of Pseudomonas cichorii

Pseudomonas cichorii strain SPC9018 harbours hrp genes that encode a type III secretion system. The hrp mutants have reduced virulence on lettuce and lose virulence on aubergine. Of the hrp genes, the B3 gene (B3) is located next to hrpW, encoding a harpin, and is predicted to encode a small (12 kDa) acidic (pI = 5·21) chaperone. In this study, involvement of the two genes in SPC9018 virulence was analysed. An hrpW-mutant (CWN1) and a B3-deleted mutant (ΔB3) of SPC9018 lacked virulence on aubergine and had reduced virulence on lettuce. Furthermore, both mutants also lost their ability to induce the hypersensitive response (HR) in tobacco leaves. The phenotypes were similar to those of the hrpL-deficient mutant (SPC9018-L). Transformation of CWN1 and ΔB3 with the plasmid containing hrpW and B3, respectively, originating from SPC9018 restored their virulence and ability to induce the HR in tobacco leaves. The population of CWN1 drastically decreased in aubergine following inoculation. In contrast, the population of ΔB3 was retained after inoculation, which was similar to that observed with SPC9018-L. RT-PCR analysis showed that hrpW and B3 are transcribed as a single transcript and its expression is dependent on HrpL. The two-hybrid analysis showed association of HrpW with the B3 protein. Therefore, HrpW can be implicated in the virulence of P. cichorii and its ability to induce the HR in tobacco, with some assistance from the B3 protein, probably acting as a chaperone.