Effects on promoter activity of base substitutions in the cis-acting regulatory element of HrpXo regulons in Xanthomonas oryzae pv. oryzae

In Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, HrpXo is known to be a transcriptional regulator for the hypersensitive response and pathogenicity (hrp) genes. Several HrpXo regulons are preceded by a consensus sequence (TTCGC-N15-TTCGC), called the plant-inducible promoter (PIP) box, which is required for expression of the gene that follows. Thus, the PIP box can be an effective marker for screening HrpXo regulons from the genome database. It is not known, however, whether mutations in the PIP box cause a complete loss of promoter activity. In this study, we introduced base substitutions at each of the consensus nucleotides in the PIP box of the hrpC operon in X. oryzae pv. oryzae, and the promoter activity was examined by using a β-glucuronidase (GUS) reporter gene. Although the GUS activity was generally reduced by base substitutions, several mutated PIP boxes conferred considerable promoter activity. In several cases, even imperfect PIP boxes with two base substitutions retained 20% of the promoter activity found in the nonsubstituted PIP box. We screened HrpXo regulon candidates with an imperfect PIP box obtained from the genome database of X. oryzae pv. oryzae and found that at least two genes preceded by an imperfect PIP box with two base substitutions were actually expressed in an HrpXo-dependent manner. These results indicate that a base substitution in the PIP box is quite permissible for HrpXo-dependent expression and suggest that X. oryzae pv. oryzae may possess more HrpXo regulons than expected.