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.

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.

Suppression of rice immunity by Xanthomonas oryzae type III effector Xoo2875.

Xanthomonas oryzae delivers effector proteins into host cells through a type III secretion system to inhibit host immune responses, but how these effectors suppress host immunity is largely unknown. Here we found that Xoo2875, one of the effectors of X. oryzae, strongly inhibited host resistance to X. oryzae. Transgenic rice plants expressing Xoo2875 exhibited semi-dwarfism and a reduction in Brassinolide-dependent laminar inclination, characteristics of brassinosteroid (BR)-insensitive mutants caused by mutations of the BR receptor. A yeast two-hybrid experiment indicated that Xoo2875 interacted with OsBAK1, an essential component of both microbe-associated molecular patterns (MAMPs) and BR receptors, suggesting that the virulent activity of Xoo2875 is mediated by inhibition of OsBAK1. Expression of Xoo2875 in Arabidopsis cells activated host immune responses, suggesting the presence of intracellular immune receptors that recognize Xoo2875. Because Xoo2875 homologs are highly conserved in Xanthomonas species, the development of Xoo2875-induced immunity is probably a useful strategy to avoid pathogen invasion.

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.

Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants.

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of "self-cloning," and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.

Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp.

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.

Identification of effector candidate genes of Rhizoctonia solani AG-1 IA expressed during infection in Brachypodium distachyon.

Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon. R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes (RsSEPGs) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1-3 and 29 RsSEPGs in the cluster 4-6 are expected to be involved in biotrophic and necrotrophic interactions, respectively.

Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae.

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.

Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A.

BACKGROUND: Xanthomonas oryzae pv. oryzae causes bacterial blight of rice (Oryza sativa L.), a major disease that constrains production of this staple crop in many parts of the world. We report here on the complete genome sequence of strain PXO99A and its comparison to two previously sequenced strains, KACC10331 and MAFF311018, which are highly similar to one another. RESULTS: The PXO99A genome is a single circular chromosome of 5,240,075 bp, considerably longer than the genomes of the other strains (4,941,439 bp and 4,940,217 bp, respectively), and it contains 5083 protein-coding genes, including 87 not found in KACC10331 or MAFF311018. PXO99A contains a greater number of virulence-associated transcription activator-like effector genes and has at least ten major chromosomal rearrangements relative to KACC10331 and MAFF311018. PXO99A contains numerous copies of diverse insertion sequence elements, members of which are associated with 7 out of 10 of the major rearrangements. A rapidly-evolving CRISPR (clustered regularly interspersed short palindromic repeats) region contains evidence of dozens of phage infections unique to the PXO99A lineage. PXO99A also contains a unique, near-perfect tandem repeat of 212 kilobases close to the replication terminus. CONCLUSION: Our results provide striking evidence of genome plasticity and rapid evolution within Xanthomonas oryzae pv. oryzae. The comparisons point to sources of genomic variation and candidates for strain-specific adaptations of this pathogen that help to explain the extraordinary diversity of Xanthomonas oryzae pv. oryzae genotypes and races that have been isolated from around the world.

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae.

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the 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.

The Colletotrichum lagenariu Ste12-like gene CST1 is essential for appressorium penetration.

Colletotrichum lagenarium is the causal agent of anthracnose of cucumber. This fungus produces a darkly melanized infection structure, appressoria, to penetrate the host leaves. The C. lagenarium CMK1 gene, a homologue of the Saccharomyces cerevisiae FUS3/KSS1 mitogen-activated protein (MAP) kinase genes, was shown to regulate conidial germination, appressorium formation, and invasive growth. In S. cerevisiae, Ste12p is known to be a transcriptional factor downstream of Fus3p/Kss1p MAP kinases. To evaluate the CMK1 MAP kinase pathway, we isolated the Ste12 homologue CST1 gene from C. lagenarium and characterized. The cst1delta strains were nonpathogenic on intact host leaves, but could form lesions when inoculated on wounded leaves. Conidia of the cst1delta strains could germinate and form melanized appressoria on both host leaf surface and artificial cellulose membrane, but could not produce infectious hyphae from appressoria, suggesting that CST1 is essential for appressorium penetration in C. lagenarium. In addition, matured appressoria of the cst1delta strains contained an extremely low level of lipid droplets compared with that of the wild-type strain. Lipid droplets were abundant in conidia of the cst1delta strains, but rapidly disappeared during appressorium formation. This misscheduled lipid degradation might be related to the failure of appressorium penetration in the cst1delta strain.

Involvement of Phosphoglucose Isomerase in Pathogenicity of Xanthomonas oryzae pv. oryzae.

ABSTRACT Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, was subjected to transposon mutagenesis to generate mutants defective in pathogenicity. A novel mutant 74M913 was attenuated in virulence but retained its ability to cause the hypersensitive response in leaf blight-resistant rice and tomato. Cloning and sequence analysis revealed that the transposon in 74M913 was inserted in a gene homologous to the phosphoglucose isomerase (pgi) gene of X. axonopodis pv. citri. Growth of the mutant in a synthetic medium containing fructose or xylose as a sole carbohydrate source was much reduced, indicating the transposon disrupted pgi function. The interaction between expression of pgi and hypersensitive response and pathogenicity (hrp) genes was investigated because we had demonstrated previously that expression of hrp genes of X. oryzae pv. oryzae is induced in a synthetic medium containing xylose. However, pgi and the hrp gene (hrcU) were expressed independently. This study suggests that PGI is involved in pathogenicity of X. oryzae pv. oryzae.

Bacterial effector modulation of host E3 ligase activity suppresses PAMP-triggered immunity in rice.

Pathogen effector proteins are delivered to host cells to suppress plant immunity. However, the mechanisms by which effector proteins function are largely unknown. Here we show that expression of XopP(Xoo), an effector of rice pathogen Xanthomonas oryzae pv. oryzae, in rice strongly suppresses peptidoglycan (PGN)- and chitin-triggered immunity and resistance to X. oryzae. XopP(Xoo) targets OsPUB44, a rice ubiquitin E3 ligase with a unique U-box domain. We find that XopP(Xoo) directly interacts with the OsPUB44 U-box domain and inhibits ligase activity. Two amino-acid residues specific for the OsPUB44 U-box domain are identified, which are responsible for the interaction with XopP(Xoo). Silencing of OsPUB44 suppresses PGN- and chitin-triggered immunity and X. oryzae resistance, indicating that OsPUB44 positively regulates immune responses. Thus, it is likely that XopP(Xoo) suppresses immune responses by directly interacting with and inhibiting a positive regulator of plant immunity.

A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity.

CERK1 is a lysine motif-containing plant pattern recognition receptor for chitin and peptidoglycan. Chitin recognition by OsCERK1 triggers rapid engagement of a rice MAP kinase cascade, which leads to defense response activation. How the MAP kinase cascades are engaged downstream of OsCERK1 remains obscure. Searching for host proteins that interact with Xoo1488, an effector of the rice pathogen Xanthomonas oryzae, we identified the rice receptor-like cytoplasmic kinase, OsRLCK185. Silencing OsRLCK185 suppressed peptidoglycan- and chitin-induced immune responses, including MAP kinase activation and defense-gene expression. In response to chitin, OsRLCK185 associates with, and is directly phosphorylated by, OsCERK1 at the plasma membrane. Xoo1488 inhibits peptidoglycan- and chitin-induced immunity and pathogen resistance. Additionally, OsCERK1-mediated phosphorylation of OsRLCK185 is suppressed by Xoo1488, resulting in the inhibition of chitin-induced MAP kinase activation. These data support a role for OsRLCK185 as an essential immediate downstream signaling partner of OsCERK1 in mediating chitin- and peptidoglycan-induced plant immunity.

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.

The Colletotrichum orbiculare SSD1 mutant enhances Nicotiana benthamiana basal resistance by activating a mitogen-activated protein kinase pathway.

Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance.

Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv. oryzae.

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.

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-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 beta-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.