Synergistic Activation of the Pathogenicity-Related Proline Iminopeptidase Gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR Homolog

Xanthomonas campestris pv. campestris strain 8004 contains an orphan quorum-sensing (QS) locus, xccR-pip(Xcc), in which the proline iminopeptidase (pip(Xcc)) gene (where "Xcc" indicates that the pip gene is from X. campestris pv. campestris) is positively regulated by the LuxR homologue XccR by binding to the luxXc box of the pip(Xcc) promoter. The disruption of pip(Xcc) significantly attenuated the virulence of X. campestris pv. campestris. An imperfect plant-inducible promoter (PIP) box is located in the upstream region of the pip(Xcc) promoter, which is the putative binding site of the transcriptional activator HrpX. To explore whether the expression of the pip(Xcc) gene is regulated by HrpX, the expression level of a pip(Xcc) promoter-gusA fusion gene was assayed in an hrpX disruption mutant. The results showed that the lack of HrpX dramatically decreased the β-glucuronidase (GUS) activity. Further analyses using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR indicated that the imperfect PIP box in X. campestris pv. campestris is specifically bound to HrpX. These data demonstrated that the pip(Xcc) gene belongs to the hrp regulon and that the imperfect PIP box of the pip(Xcc) promoter could be a cis element for the HrpX protein. We further showed in a pulldown assay that XccR can bind HrpX, suggesting that these two regulatory proteins coactivate the virulence factor by binding to the different cis elements of the pip(Xcc) gene and adapt to the host environment during X. campestris pv. campestris infection.