Concomitant Regulation by a LacI-Type Transcriptional Repressor XylR on Genes Involved in Xylan and Xylose Metabolism and the Type III Secretion System in Rice Pathogen Xanthomonas oryzae pv. oryzae.

The hypersensitive response and pathogenicity (hrp) genes of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, encode components of the type III secretion system and are essential for virulence. Expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX; HrpG regulates hrpX and hrpA, and HrpX regulates the other hrp genes on hrpB-hrpF operons. We previously reported the sugar-dependent quantitative regulation of HrpX; the regulator highly accumulates in the presence of xylose, followed by high hrp gene expression. Here, we found that, in a mutant lacking the LacI-type transcriptional regulator XylR, HrpX accumulation and hrp gene expression were high even in the medium without xylose, reaching the similar levels present in the wild type incubated in the xylose-containing medium. XylR also negatively regulated one of two xylose isomerase genes (xylA2 but not xylA1) by binding to the motif sequence in the upstream region of the gene. Xylose isomerase is an essential enzyme in xylose metabolism and interconverts between xylose and xylulose. Our results suggest that, in the presence of xylose, inactivation of XylR leads to greater xylan and xylose utilization and, simultaneously, to higher accumulation of HrpX, followed by higher hrp gene expression in the bacterium.

GamR, the LysR-Type Galactose Metabolism Regulator, Regulates hrp Gene Expression via Transcriptional Activation of Two Key hrp Regulators, HrpG and HrpX, in Xanthomonas oryzae pv. oryzae.

UNLABELLED: Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight of rice. For the virulence of the bacterium, the hrp genes, encoding components of the type III secretion system, are indispensable. The expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX: HrpG regulates hrpX, and HrpX regulates other hrp genes. Several other regulators have been shown to be involved in the regulation of hrp genes. Here, we found that a LysR-type transcriptional regulator that we named GamR, encoded by XOO_2767 of X. oryzae pv. oryzae strain MAFF311018, positively regulated the transcription of both hrpG and hrpX, which are adjacent to each other but have opposite orientations, with an intergenic upstream region in common. In a gel electrophoresis mobility shift assay, GamR bound directly to the middle of the upstream region common to hrpG and hrpX The loss of either GamR or its binding sites decreased hrpG and hrpX expression. Also, GamR bound to the upstream region of either a galactose metabolism-related gene (XOO_2768) or a galactose metabolism-related operon (XOO_2768 to XOO_2771) located next to gamR itself and positively regulated the genes. The deletion of the regulator gene resulted in less bacterial growth in a synthetic medium with galactose as a sole sugar source. Interestingly, induction of the galactose metabolism-related gene was dependent on galactose, while that of the hrp regulator genes was galactose independent. Our results indicate that the LysR-type transcriptional regulator that regulates the galactose metabolism-related gene(s) also acts in positive regulation of two key hrp regulators and the following hrp genes in X. oryzae pv. oryzae. IMPORTANCE: The expression of hrp genes encoding components of the type III secretion system is essential for the virulence of many plant-pathogenic bacteria, including Xanthomonas oryzae pv. oryzae. It is specifically induced during infection. Research has revealed that in this bacterium, hrp gene expression is controlled by two key hrp regulators, HrpG and HrpX, along with several other regulators in the complex regulatory network, but the details remain unclear. Here, we found that a novel LysR-type transcriptional activator, named GamR, functions as an hrp regulator by directly activating the transcription of both hrpG and hrpX Interestingly, GamR also regulates a galactose metabolism-related gene (or operon) in a galactose-dependent manner, while the regulation of hrpG and hrpX is independent of the sugar. Our finding of a novel hrp regulator that directly and simultaneously regulates two key hrp regulators provides new insights into an important and complex regulation system of X. oryzae pv. oryzae hrp genes.

The quantitative regulation of the hrp regulator HrpX is involved in sugar-source-dependent hrp gene expression in Xanthomonas oryzae pv. oryzae.

In Xanthomonas oryzae pv. oryzae, the pathogen of bacterial leaf blight of rice, hrp gene expression is regulated by the key hrp regulators HrpG and HrpX. HrpG regulates hrpX and hrpA, and HrpX regulates the other hrp genes on hrpB-hrpF operons. We previously examined the expression of the HrpX-regulated hrp gene hrcU and demonstrated that hrp gene expression is highly induced in a certain nutrient-poor medium containing xylose. In the present study, we found that the induction level of HrpX-regulated hrp genes was higher in medium with xylose than in media with any other sugar sources (glucose, sucrose and fructose), but that expression of hrpG, hrpX and hrpA was independent of the sugar sources. In western blot analysis, the accumulation of HrpX was reduced in media with a sugar other than xylose, probably as a result of proteolysis, but the addition of xylose canceled this reduced accumulation of the protein. The results suggest that proteolysis of HrpX is an important hrp regulatory mechanism and that xylose specifically suppresses this proteolysis, resulting in active hrp gene expression in X. oryzae pv. oryzae.

StoS, a hybrid histidine kinase sensor of Xanthomonas oryzae pv. oryzae, is activated by sensing low O2 concentration and is involved in stress tolerance and virulence.

Bacteria have two-component signal transduction systems (TCSTS), which are important devices for receiving various environmental signals. A TCSTS generally consists of a sensor histidine kinase (HK) and a response regulator (RR) that contains a receiver domain. There are also hybrid-type HK (HyHK) that comprise a HK with a receiver domain within one molecule. In this study, we show that the deletion mutant of a HyHK XOO_0635 (StoS) of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, had decreased stress tolerance to high osmolarity, sodium, and H2O2. Growth of the StoS mutant was delayed, and viability was lower than the wild type in medium and in rice leaves. We found that StoS regulates the expression of various genes including XOO_3715, XOO_0131, and stoS itself. A domain search revealed a PAS domain with a heme pocket in StoS, implying that the HyHK functions as an O2 sensor. When the bacteria were incubated in low oxygen, the StoS-dependent expression of XOO_0131 and XOO_3715 became higher. Therefore, StoS is activated by sensing a low O2 concentration in its environs and is involved in gene expression for adapting to various stressful conditions.

An H-NS-like protein involved in the negative regulation of hrp genes in Xanthomonas oryzae pv. oryzae.

hrp genes encode components of a type III secretion (T3S) system and play crucial roles in the pathogenicity of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). A histone-like nucleoid-structuring (H-NS) protein binds DNA and acts as a global transcriptional repressor. Here, we investigated the involvement of an h-ns-like gene, named xrvB, in the expression of hrp genes in Xoo. Under the hrp-inducing culture condition, the expression of a key hrp regulator HrpG increased in the XrvB mutant, followed by activation of the downstream gene expression. Also, in planta, the secretion of a T3S protein (XopR) was activated by the mutation in xrvB. Gel retardation assay indicated that XrvB has DNA-binding activity, but without a preference for the promoter region of hrpG. The results suggest that XrvB negatively regulates hrp gene expression and that an unknown factor(s) mediates the regulation of hrpG expression by XrvB.