Specific Binding of the <i>Xanthomonas campestris</i> pv. vesicatoria AraC-Type Transcriptional Activator HrpX to Plant-Inducible Promoter Boxes

<jats:title>ABSTRACT</jats:title> <jats:p> The pathogenicity of the plant-pathogenic bacterium <jats:italic>Xanthomonas campestris</jats:italic> pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb <jats:italic>hrp</jats:italic> ( <jats:italic>h</jats:italic> ypersensitive <jats:italic>r</jats:italic> esponse and <jats:italic>p</jats:italic> athogenicity) gene cluster. Expression of the <jats:italic>hrp</jats:italic> operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved <jats:italic>cis</jats:italic> -regulatory element, the plant-inducible promoter (PIP) box (TTCGC-N <jats:sub>15</jats:sub> -TTCGC), present in the promoter regions of four <jats:italic>hrp</jats:italic> operons. No binding of HrpX was observed when DNA fragments lacking a PIP box were used. HrpX also bound to a DNA fragment containing an imperfect PIP box (TTCGC-N <jats:sub>8</jats:sub> -TTCGT). Dinucleotide replacements in each half-site of the PIP box strongly decreased binding of HrpX, while simultaneous dinucleotide replacements in both half-sites completely abolished binding. Based on the complete genome sequence of <jats:italic>Xanthomonas campestris</jats:italic> pv. vesicatoria, putative plant-inducible promoters consisting of a PIP box and a −10 promoter motif were identified in the promoter regions of almost all HrpX-activated genes. Bioinformatic analyses and reverse transcription-PCR experiments revealed novel HrpX-dependent genes, among them a NUDIX hydrolase gene and several genes with a predicted role in the degradation of the plant cell wall. We conclude that HrpX is the most downstream component of the <jats:italic>hrp</jats:italic> regulatory cascade, which is proposed to directly activate most genes of the <jats:italic>hrpX</jats:italic> regulon via binding to corresponding PIP boxes. </jats:p>