Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv. oryzae.

We have cloned a hrp gene cluster from Xanthomonas oryzae pv. oryzae. Bacteria with mutations in the hrp region have reduced growth in rice leaves and lose the ability to elicit a hypersensitive response (HR) on the appropriate resistant cultivars of rice and the nonhost plant tomato. A 12,165-bp portion of nucleotide sequence from the presumed left end and extending through the hrpB operon was determined. The region was most similar to hrp genes from Xanthomonas campestris pv. vesicatoria and Ralstonia solanacearum. Two new hrp-associated loci, named hpa1 and hpa2, were located beyond the hrpA operon. The hpa1 gene encoded a 13-kDa glycine-rich protein with a composition similar to those of harpins and PopA. The product of hpa2 was similar to lysozyme-like proteins. Perfect PIP boxes were present in the hrpB and hpa1 operons, while a variant PIP box was located upstream of hpa2. A strain with a deletion encompassing hpa1 and hpa2 had reduced pathogenicity and elicited a weak HR on nonhost and resistant host plants. Experiments using single mutations in hpa1 and hpa2 indicated that the loss of hpa1 was the principal cause of the reduced pathogenicity of the deletion strain. A 1,519-bp insertion element was located immediately downstream of hpa2. Hybridization with hpa2 indicated that the gene was present in all of the strains of Xanthomonas examined. Hybridization experiments with hpa1 and IS1114 indicated that these sequences were detectable in all strains of X. oryzae pv. oryzae and some other Xanthomonas species.