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-N 15 -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 ␤-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.Many gram-negative, plant-pathogenic bacteria possess clustered hypersensitive response and pathogenicity (hrp) genes. These genes encode proteins involved in the type III secretion system that delivers effector proteins from the bacteria to plant cells. These proteins are required for pathogenesis in host plants and for triggering a hypersensitive response (HR) in nonhost plants (reviewed in references 1, 6, and 41).The expression of hrp genes is highly regulated. These genes are induced only in plants or certain nutrient-poor synthetic media and are not induced in nutrient-rich complex media (3,16,20,30,31,33,40). There are two types of hrp regulatory systems in plant-pathogenic bacteria (1, 14). In group I systems, which are found in Pseudomonas syringae, Erwinia sp., and Pantoea stewartii, a member of the ECF family of alternative sigma factors, called HrpL, functions as the regulator for other hrp genes (21,42,43). On the other hand, in group II systems, which are found in Xanthomonas sp. and Ralstonia solanacearum, either the AraC-type transcriptional activator HrpX (Xanthomonas sp.) or HrpB (R. solanacearum) regulates expression of other hrp genes (13,29,39).In xanthomonads, the hrp cluster comprises six hrp loci, hrpA to hrpF, which encode components of a type III secretion system, and all of these genes are required for full pathogenicity (5). Two regulatory genes that control expression of the hrp genes have been identified in the genus Xanthomonas.

A novel regulatory gene, trh, which is involved in hrp gene expression, is identified in the plant pathogen Xanthomonas oryzae pv. oryzae. In the trh mutant, expression of HrpG, which is a key regulator for hrp gene expression, is reduced both under the in vitro hrp-inducing condition and in planta.