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Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB() and HpaP() mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.In general, plant-pathogenic bacteria possess hypersensitive response and pathogenicity (hrp) genes, which are clustered in their chromosomes. hrp genes encode a type III secretion system (TTSS) that delivers virulence and avirulence factors from the bacteria to plant cells and are required for pathogenesis in host plants and for triggering a hypersensitive response in nonhost plants (1,38). Transcriptional regulation of hrp genes depends on environmental conditions. The expression of hrp genes is generally suppressed in complex media and induced in planta and under certain in vitro conditions (6,24,35,39).In xanthomonads, the hrp cluster comprises six hrp loci, hrpA to hrpF, which are all required for full pathogenicity, and their expression is regulated by two genes, hrpX and hrpG, which are located outside the hrp gene cluster region (7, 36). The HrpG protein belongs to the OmpR family of two-component regulatory systems and activates the expression of hrpA and hrpX (37). HrpX, an AraC-type transcriptional activator, has been reported to control the expression of the operons hrpB to hrpF, which contain the hrp genes encoding a component of TTSS (36). It has also been suggested that HrpX controls some effector proteins (5). Several genes that are regulated in a HrpX-dependent manner possess the consensus nucleotide ...

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.