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

摘要

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.