OxyR-regulated catalase CatB promotes the virulence in rice via detoxifying hydrogen peroxide in Xanthomonas oryzae pv. oryzae.

BACKGROUND: To facilitate infection, Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, needs to degrade hydrogen peroxide (H2O2) generated by the host defense response via a mechanism that is mediated by the transcriptional regulator OxyR. The catalase (CAT) gene catB has previously been shown to belong to the OxyR regulon in Xoo. However, its expression patterns and function in H2O2 detoxification and bacterial pathogenicity on rice remain to be elucidated. RESULTS: The catB gene encodes a putative catalase and is highly conserved in the sequenced strains of Xanthomonas spp. ß-galactosidase analysis and electrophoretic mobility shift assays (EMSA) showed that OxyR positively regulated the transcription of catB by directly binding to its promoter region. The quantitative real-time PCR (qRT-PCR) assays revealed that the expression levels of catB and oxyR were significantly induced by H2O2. Deletion of catB or oxyR drastically impaired bacterial viability in the presence of extracellular H2O2 and reduced CAT activity, demonstrating that CatB and OxyR contribute to H2O2 detoxification in Xoo. In addition, ΔcatB and ΔoxyR displayed shorter bacterial blight lesions and reduced bacterial growth in rice compared to the wild-type stain, indicating that CatB and OxyR play essential roles in the virulence of Xoo. CONCLUSIONS: Transcription of catB is enhanced by OxyR in response to exogenous H2O2. CatB functions as an active catalase that is required for the full virulence of Xoo in rice.

Alternative sigma factor RpoN2 is required for flagellar motility and full virulence of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, harbors a single polar flagellum for motility. How the flagellar system is regulated and how it is related to bacterial pathogenesis are not well understood. The genomic sequence of Xoo strain PXO99(A) revealed a flagellar regulon containing over 60 contiguous genes. A gene encoding alternative sigma factor 54 (σ(54)), named as rpoN2, is located in the central region of the regulon. RT-PCR analysis demonstrated rpoN2 was transcribed in an operon with flgRR, and fleQ. Single gene deletion mutants of the rpoN2 operon were generated. The rpoN2 and fleQ mutant lost swimming motility, whereas the flgRR mutant remained motile. Quantitative RT-PCR analysis further demonstrated that expression of regulatory genes fliA and flgM, and structural genes flgG, flhB, and fliC were significantly down-regulated in the rpoN2 and fleQ mutants. These results indicated that RpoN2 and FleQ synergistically controlled flagellar motility by regulating gene expression. Interestingly, the rpoN2 mutant, but not the fleQ mutant was impaired in its virulence on rice. In addition, we showed that the flagellin gene fliC mutant, which was non-motile, was not defective in virulence. Thus, we concluded that flagellar motility might not be essential for Xoo virulence in rice, and RpoN2 probably regulated bacterial virulence through a manner independent of its role in controlling flagellar gene expression.

The degenerate EAL-GGDEF domain protein Filp functions as a cyclic di-GMP receptor and specifically interacts with the PilZ-domain protein PXO_02715 to regulate virulence in Xanthomonas oryzae pv. oryzae.

Degenerate GGDEF and EAL domain proteins represent major types of cyclic diguanylic acid (c-di-GMP) receptors in pathogenic bacteria. Here, we characterized a FimX-like protein (Filp) which possesses both GGDEF and EAL domains in Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Both in silico analysis and enzyme assays indicated that the GGDEF and EAL domains of Filp were degenerate and enzymatically inactive. However, Filp bound to c-di-GMP efficiently within the EAL domain, where Q(477), E(653), and F(654) residues were crucial for the binding. Deletion of the filp gene in X. oryzae pv. oryzae resulted in attenuated virulence in rice and reduced type III secretion system (T3SS) gene expression. Complementation analysis with different truncated proteins indicated that REC, PAS, and EAL domains but not the GGDEF domain were required for the full activity of Filp in vivo. In addition, a PilZ-domain protein (PXO_02715) was identified as a Filp interactor by yeast two-hybrid and glutathione-S-transferase pull-down assays. Deletion of the PXO_02715 gene demonstrated changes in bacterial virulence and T3SS gene expression similar to Δfilp. Moreover, both mutants were impaired in their ability to induce hypersensitive response in nonhost plants. Thus, we concluded that Filp was a novel c-di-GMP receptor of X. oryzae pv. oryzae, and its function to regulate bacterial virulence expression might be via the interaction with PXO_02715.

[Binding of transcription regulator Clpxoo to promoter of endoglucanase gene engAxoo was inhibited by c-di-GMP in Xanthomonas oryzae pv. oryzae].

OBJECTIVE: To understand the regulatory mechanism by cyclic diguanylate (c-di-GMP) receptor and transcriptional regulator Clpxoo of expression of endoglucanase gene (engAxoo) in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice. METHODS: A plasmid to expressclpxoo gene was constructed and transformed into Escherichia coli for expression by isopropylthio-beta-D-galactoside (IPTG) induction. The recombinant protein was purified by Ni-NTA resin. The binding affinity between purified Clpxoo protein and the promoter of endoglucanase gene (engAxoo-p) was determined by electrophoretic mobility shift assay using fluoresce in (FAM)-labeled probes. The role of c-di-GMP on the binding was also examined. RESULTS: Under the optimized conditions, Clpxoo was expressed and purified successfully. Mobility shift of engAxoo-p in the presence of Clpxoo was observed, indicating that specific binding occurred between them. Moreover, addition of c-di-GMP molecules in the above reaction system abolished such binding. CONCLUSION: Once interacting with the signal molecule c-di-GMP, Clpxoo conformational structure may change substantially, which results in inhibition of binding to engAxoo-p; The optimized methods for Clpxoo protein purification and electrophoretic mobility shift assay (EMSA) can be used for subsequent identification of Clp regulon in a larger scale.

A novel two-component system PdeK/PdeR regulates c-di-GMP turnover and virulence of Xanthomonas oryzae pv. oryzae.

Two-component systems (TCS) consisting of histidine kinases (HK) and response regulators (RR) play essential roles in bacteria to sense environmental signals and regulate cell functions. One type of RR is involved in metabolism of cyclic diguanylate (c-di-GMP), a ubiquitous bacterial second messenger. Although genomic studies predicted a large number of them existing in different bacteria, only a few have been studied. In this work, we characterized a novel TCS consisting of PdeK(PXO_01018)/PdeR(PXO_ 01019) from Xanthomonas oryzae pv. oryzae, which causes the bacterial leaf blight of rice. PdeR (containing GGDEF, EAL, and REC domains) was shown to have phosphodiesterase (PDE) activity in vitro by colorimetric assays and high-performance liquid chromatography analysis. The PDE activity of full-length PdeR needs to be triggered by HK PdeK. Deletion of pdeK or pdeR in X. oryzae pv. oryzae PXO99(A) had attenuated its virulence on rice. ΔpdeK and ΔpdeR secreted less exopolysaccharide than the wild type but there were no changes in terms of motility or extracellular cellulase activity, suggesting the activity of PdeK/PdeR might be specific.

[Gene deletion and functional analysis of the EAL domain protein vieAxoo in Xanthomonas oryzae pv. oryzae].

OBJECTIVE: To better reveal the functions of key members involved in cyclic di-GMP signal metabolism pathways in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae (Xoo). METHODS: vieAxoo (PXO 04753), a gene putatively encoding the EAL domain proteins was investigated by gene deletion mutation using the marker exchange, complementation and phenotypic analysis. RESULT: The sequence of vieAxoo cloned from genomic DNA of the wild-type strain PXO99(A) was found to be highly conserved in plant-pathogenic Xanthomonas spp. VieAxoo was structurally featured with EAL and REC domains. No significant changes in virulence to rice, EPS production and flagellar motility were found in deltavieAxoo compared to PXO99(A), whereas remarkable changes in induction of hypersensitive responses (HR) in tobacco and biofilm formation were observed. CONCLUSION: VieAxoo might function as an important reponse regulator in cyclic di-GMP signaling and regulation of bacterial induction of HR and biofilm formation of Xoo.

[Diffusible signal factor production and virulence expression in deltarpfFxoo, deltarpfCxoo and deltarpfGxoo, the gene deletion mutants of DSF/Rpf signaling proteins of Xanthomonas oryzae pv. oryzae].

OBJECTIVE: To better elucidate the functions of RpfFxoo, RpfCxoo and RpfGxoo, 3 proteins of diffusible signal factor (DSF)-dependent cell-cell signaling system in regulation of virulence expression of Xanthomonas oryzae pv. oryzae (Xoo). METHOD: Deltarpfxoo, the gene deletion mutants were generated from PXO99(A), the wild-type strain of Xoo via marker-exchanging and DSF biosynthesis and extracellular polysaccharide production and virulence to rice of the mutants were assayed. RESULT: rpfFxoo,rpfCxoo and rpfGxoo were cloned from the genomic DNA of PXO99(A) and the relative single or double mutants were constructed. Compared to PXO99(A), DSF production was deficient in deltarpfFxoo, deltarpfFCxoo and deltarpfFGxoo, while DSF was overproduced in deltarpfCxoo and reduced in deltarpfGxoo. DSF production of deltarpfFxcc, deltarpfCxcc and deltarpfGxcc, the mutants of X. campestris pv. campestris can be restored as XC1, the wild-type strain by in trans complementation of rpfFxoo, rpfCxoo and rpfGxoo. All the mutants except deltarpfFxoo were remarkably deficient in production All the mutants significantly exhibited the reduced bacterial virulence to rice. of extracellular polysaccharide. CONCLUSION: DSF signaling proteins RpfFxoo, RpfCxoo and RpfGxoo might function in regulation of DSF biogenesis and EPS production and bacterial virulence.

[Ferric iron absorption in deltar p f F xoo, a gene deletion mutant of Xanthomonas oryzae pv. oryzae, assayed using atomic absorption spectrophotometry].

The ferric iron absorption is one of the most important limiting factors of bacterial growth of Xanthomonas oryzae pv. oryzae. It has been previously speculated that r p f F xoo might be involved in the ferric iron metabolism of the pathogen. In the present study, deltar p f F xoo, a gene deletion mutant, was generated from the wild-type strain PXO99A of Xoo through the homologous recombination, and Fe content was assayed using flame atomic absorption in PXO99A and deltar p f F xoo. The results indicated that the recovery was 99.7% and the relative standard deviation was 1.89 under optimized AAS operating conditions. The increase in Fe absorption in PXO99A and deltar p f F xoo was observed with the increasing time. However, the ferric content of deltar p f F xoo was significantly lower than that of PXO99A (P < 0.05). It is suggested that r p f F xoo is involved in iron metabolism in Xanthomonas oryzae pv. oryzae.

[Identification and functional analysis of Tdrxoo, the member of TonB-dependent-receptor family proteins in Xanthomonas oryzae pv. oryzae].

OBJECTIVE: To demonstrate the novel regulatory pathways mediated in bacterial pathogenicity and motility in Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight in rice. METHODS: Molecular identification and functional characterization of Tdrxoo, which interacts with GacAxoo of the two-component regulatory system (GacSxoo/GacAxoo) in Xoo, were performed through gene cloning, sequencing and disrupt analysis. RESULTS: tdrxoo was successfully cloned from the genomic DNA of wild-type PXO99(A) by using polymerase chain reactions with the degenerated primers tdrxooF/R. The tdrxoo gene was found to be highly conserved in the plant-pathogenic Xanthomonas spp. Sequence analysis showed that Tdrxoo was homological to a protein with the TonB-Dependent-Receptor (TDR) domain. Tdrxoo is probably localized in the outer membrane of bacterial cells, recognizing the signals from extracellular environment, and inducing the intracellular signal transduction. delta tdrxoo, the disrupted mutant, was obtained after a single cross-over recombination event between tdrxoo and the plasmid pK-tdr with the tdrxoo segment. The mutant lost the ability of causing the disease, and was affected in growth in vitro compared to PXO99(A). In addition, the motility and the extracellular enzymes production of delta tdrxoo were reduced, which can be restored through complementation of the delta tdrxoo mutant by introduction of tdrxoo. tdrxoo deficiency didn't affect siderophore production. CONCLUSION: According to the existence of tdrxoo in Xoo genome and phenotype of delta tdrxoo, Tdrxoo, as the outer membrance protein, is proposed to be involved in regulation of pathogenicity, extracellular enzyme production, the growth and motility of Xoo.

[Roles of OxyRxoo, a transcriptional regulator of Xanthomonas oryzae pv. oryzae in regulation of detoxification of hydrogen peroxide].

OBJECTIVE: OxyRxoo is a homologue of OxyR from Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of bacterial blight of rice. To elucidate the role of OxyRxoo in detoxification of hydrogen peroxide. METHODS: OxyRxoo was studied by gene cloning, sequencing, deletion,complement and phenotype analysis. RESULT: Compared to those of the wild-type strain PXO99(A), there was no difference in bacterial growth in vitro of delta oxyRxoo, but delta oxyRxoo is more sensitive to H2O2 with reduced catalase activity. In the presence of H2O2, the expression of catalase genes (ahpCxoo, catBxoo, katExoo and srpAxoo) was significantly down-regulated, while the expression of oxyRxoo was up-regulated in delta oxyRxoo. CONCLUSION: OxyRxoo functions as a transcription regulator in mediating and controlling H2O2 detoxification in Xoo.

[Deletion mutation of rbfCxoo, encoding a putative glycosyltransferase in Xanthomonas oryzae pv. oryzae leads to enhanced virulence expression].

OBJECTIVE: To better understand the structure and biological function of rbfCxoo, a gene with the putative function in lipopolysaccharide O-antigen synthesis in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. METHODS: The molecular identification and function analysis of rbfCxoo were performed through gene cloning, sequencing and deletion analysis. RESULTS: The sequence of rbfCxoo cloned from the genomic DNA of wild-type PXO99(A) was the same as that of the sequenced strain KACC10331. There were glycosyltransferase domains (Glycos_transf_2) at N and C terminal of RbfCxoo respectively. The deletion mutant delta rbfCxoo generated through a double crossover recombination and validated by PCR assay displayed the reduced flagellin glycosylation and no change in lipopolysaccharide O-antigen synthesis compared with PXO99(A). Moreover, no significant changes in flagellar mobility, biofilm formation and production of extracellular cellulase and xylanase in vitro were observed in delta rbfCxoo compared to PXO99(A). Most importantly, the deletion mutation of rbfCxoo resulted in enhanced virulence and gene expression. CONCLUSION: RbfCxoo might be related to flagellin glycosylation and virulence expression in Xoo.

The complete nucleotide sequence of the barley yellow dwarf GPV isolate from China shows that it is a new member of the genus Polerovirus.

The complete nucleotide sequence of the ssRNA genome of a Chinese GPV isolate of barley yellow dwarf virus (BYDV) was determined. It comprised 5673 nucleotides, and the deduced genome organization resembled that of members of the genus Polerovirus. It was most closely related to cereal yellow dwarf virus-RPV (77% nt identity over the entire genome; coat protein amino acid identity 79%). The GPV isolate also differs in vector specificity from other BYDV strains. Biological properties, phylogenetic analyses and detailed sequence comparisons suggest that GPV should be considered a member of a new species within the genus, and the name Wheat yellow dwarf virus-GPV is proposed.

[Molecular identification and functional analysis of Clpxoo, a homologue to the nucleotide receptor protein in Xanthomonas oryzae pv. oryzae].

OBJECTIVE: To better understand the mechanisms of cyclic di-GMP signaling in Xanthomonas oryzae pv. oryzae (Xoo),the casual pathogen of bacterial blight of rice, molecular identification of Clpxoo. METHODS: A putative signal receptor protein was performed through gene cloning, sequencing and deletion analysis. RESULTS: Sequence analysis showed Clpxoo was a homologue of Crp and Vfr, the cAMP receptor proteins in Escherichia coli and Pseudomonas aeruginosa respectively, which had the cNMP-binding domains (CAP_ED) at N terminal and the DNA-binding domains (HTH_CRP) at C terminal and is highly conserved in the plant-pathogenic Xanthomonas spp. We constructed delta clpxoo through a double crossover recombination and validated by PCR assay, delta clpxoo displayed the reduced motility and extracellular polysaccharide (EPS) production, and increased sensitive to H2O2 toxicity compared with PXO99(A). All these phenotype changes could be partly restored through complementation of mutants by introducing clpxoo. Moreover, we observed no significant changes in production of extracellular cellulase and xylanase in vitro, biofilm formation and induction of hypersensitive response (HR) on non-host tobacco in delta clpxoo compared to PXO99(A). CONCLUSION: Therefore, Clpxoo played a role as one of the global regulator in regulation of flagellar motility, EPS production and H2O2 resistance.