Complete genome sequence of a Chinese Pseudomonas syringae pv. actinidiae strain Yunnan3.2.

Here, we report the complete genome sequence for Pseudomonas syringae pv. actinidiae strain Yunnan3.2, which was isolated from diseased kiwifruit grown in Yunnan province, China. The complete genome of Yunnan3.2 comprises a 6,564,315-bp chromosome with a GC content of 58.41% and a circular plasmid (74,466 bp).

miR2118 Negatively Regulates Bacterial Blight Resistance through Targeting Several Disease Resistance Genes in Rice.

Plant miRNAs are a class of noncoding RNA with a length of 21-24 nt that play an important role in plant responses to biotic and abiotic stresses. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases in rice. Our previous work showed that osa-miR2118b/n was induced by Xoo infection. However, the biological function of miR2118 has not yet been characterized in experiments. Herein, we constructed MIR2118b OE, as well as single and double mutants of MIR2118b/n using CRISPR/Cas9. Further results showed that osa-MIR2118b OE plants exhibited longer lesion lengths than the wild type after Xoo inoculation, while MIR2118 CRISPR plants exhibited shorter lesion lengths than the wild type after Xoo inoculation. Co-transformation experiments in rice protoplasts indicated that osa-miR2118 negatively regulated the transcripts of three nucleotide-binding sites and leucine-rich repeat (NLR) genes (LOC_Os08g42700.1, LOC_Os01g05600.1, and LOC_Os12g37290.1) which are predicted target genes of miR2118, but not the mutated NLR genes with a 3 bp insertion at the center of the binding sites. The transcriptional level of the three NLR genes was reversed relative to osa-miR2118 in the MIR2118b OE and MIR2118b CRISPR plants. The above results demonstrate that osa-miR2118b/n negatively regulates the resistance to bacterial blight through negatively regulating several NLR genes.

The Regulatory Functions of σ54 Factor in Phytopathogenic Bacteria.

σ54 factor (RpoN), a type of transcriptional regulatory factor, is widely found in pathogenic bacteria. It binds to core RNA polymerase (RNAP) and regulates the transcription of many functional genes in an enhancer-binding protein (EBP)-dependent manner. σ54 has two conserved functional domains: the activator-interacting domain located at the N-terminal and the DNA-binding domain located at the C-terminal. RpoN directly binds to the highly conserved sequence, GGN10GC, at the -24/-12 position relative to the transcription start site of target genes. In general, bacteria contain one or two RpoNs but multiple EBPs. A single RpoN can bind to different EBPs in order to regulate various biological functions. Thus, the overlapping and unique regulatory pathways of two RpoNs and multiple EBP-dependent regulatory pathways form a complex regulatory network in bacteria. However, the regulatory role of RpoN and EBPs is still poorly understood in phytopathogenic bacteria, which cause economically important crop diseases and pose a serious threat to world food security. In this review, we summarize the current knowledge on the regulatory function of RpoN, including swimming motility, flagella synthesis, bacterial growth, type IV pilus (T4Ps), twitching motility, type III secretion system (T3SS), and virulence-associated phenotypes in phytopathogenic bacteria. These findings and knowledge prove the key regulatory role of RpoN in bacterial growth and pathogenesis, as well as lay the groundwork for further elucidation of the complex regulatory network of RpoN in bacteria.

Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae.

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

Transcriptome Analysis Revealed Overlapping and Special Regulatory Roles of RpoN1 and RpoN2 in Motility, Virulence, and Growth of Xanthomonas oryzae pv. oryzae.

σ54 factor (RpoN) plays a crucial role in bacterial motility, virulence, growth, and other biological functions. In our previous study, two homologous σ54 factors, RpoN1 and RpoN2, were identified in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice. However, their functional roles, i.e., whether they exert combined or independent effects, remain unknown. In the current study, rpoN1 or rpoN2 deletion in Xoo significantly disrupted bacterial swimming motility, flagellar assembly, and virulence. Transcriptome analysis led to the identification of 127 overlapping differentially expressed genes (DEGs) regulated by both RpoN1 and RpoN2. Furthermore, GO and KEGG classification demonstrated that these DEGs were highly enriched in flagellar assembly, chemotaxis, and c-di-GMP pathways. Interestingly, ropN1 deletion decreased ropN2 transcription, while rpoN2 deletion did not affect ropN1 transcription. No interaction between the rpoN2 promoter and RpoN1 was detected, suggesting that RpoN1 indirectly regulates rpoN2 transcription. In addition, RpoN1-regulated DEGs were specially enriched in ribosome, carbon, and nitrogen metabolism pathways. Besides, bacterial growth was remarkably repressed in ΔrpoN1 but not in ΔrpoN2. Taken together, this study demonstrates the overlapping and unique regulatory roles of RpoN1 and RpoN2 in motility, virulence, growth and provides new insights into the regulatory mechanism of σ54 factors in Xoo.

Bacterial Blight Induced Shifts in Endophytic Microbiome of Rice Leaves and the Enrichment of Specific Bacterial Strains With Pathogen Antagonism.

The endophytic microbiome plays an important role in plant health and pathogenesis. However, little is known about its relationship with bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo). The current study compared the community compositional structure of the endophytic microbiota in healthy and BB symptomatic leaves of rice through a metabarcoding approach, which revealed BB induced a decrease in the alpha-diversity of the fungal communities and an increase in the bacterial communities. BB-diseased rice leaves were enriched with saprophytic fungi that are capable of decomposing plant cell walls (e.g. Khuskia spp. and Leptosphaerulina spp.), while healthy rice leaves were found to be significantly more abundant with plant pathogens or mycotoxin-producing fungi (e.g. Fusarium, Magnaporthe, and Aspergillus). The endophytic bacterial communities of BB-diseased leaves were significantly enriched with Pantoea, Pseudomonas, and Curtobacterium, strains. Pantoea sp. isolates from BB leaves are identified as promising candidates for the biocontrol of BB for their ability to inhibit in vitro growth of Xoo, suppress the development of rice BB disease, and possess multiple PGP characteristics. Our study revealed BB-induced complexed changes in the endophytic fungal and bacterial communities of rice leaves and demonstrated that BB-associated enrichment of some endophytic bacterial taxa, e.g. Pantoea sp. isolates, may play important roles in suppressing the development of BB disease in rice.

Identification of the Regulatory Components Mediated by the Cyclic di-GMP Receptor Filp and Its Interactor PilZX3 and Functioning in Virulence of Xanthomonas oryzae pv. oryzae.

The degenerate GGDEF/EAL domain protein Filp was previously shown to function as a cyclic di-GMP (c-di-GMP) signal receptor through its specific interaction with an atypical PilZ domain protein PilZX3 (formerly PXO_02715) and that this interaction is involved in regulating virulence in Xanthomonas oryzae pv. oryzae. As a step toward understanding the regulatory role of Filp/PilZX3-mediated c-di-GMP signaling in the virulence of X. oryzae pv. oryzae, differentially expressed proteins (DEPs) downstream of Filp/PilZX3 were identified by isobaric tagging for relative and absolute quantitation (iTRAQ). A total of 2,346 proteins were identified, of which 157 displayed significant differential expression in different strains. Western blot and quantitative reverse transcription-PCR analyses showed that the expression of HrrP (histidine kinase-response regulator hybrid protein), PhrP (PhoPQ-regulated protein), ProP (prophage Lp2 protein 6) were increased in the ∆filp, ∆pilZX3, and ∆filp∆pilZX3 mutant strains, while expression of CheW1 (chemotaxis protein CheW1), EdpX2 (the second EAL domain protein identified in X. oryzae pv. oryzae), HGdpX2 (the second HD-GYP domain protein identified in X. oryzae pv. oryzae) was decreased in all mutant strains compared with that in the wild type, which was consistent with the iTRAQ data. Deletion of the hrrP and proP genes resulted in significant increases in virulence, whereas deletion of the cheW1, hGdpX2, or tdrX2 genes resulted in decreased virulence. Enzyme assays indicated that EdpX2 and HGdpX2 were active phosphodiesterases (PDEs). This study provides a proteomic description of putative regulatory pathway of Filp and PilZX3 and characterized novel factors that contributed to the virulence of X. oryzae pv. oryzae regulated by c-di-GMP signaling.

The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the σ54 factor RpoN2 regulated bacterial virulence on rice in a flagellum-independent manner in Xoo. In this study, both yeast two-hybrid and pull-down assays revealed that RpoN2 directly and specifically interacted with PilRX, a homolog of the response regulator PilR of the two-component system PilS-PilR in the pilus gene cluster. Genomic sequence and reverse transcription PCR (RT-PCR) analysis showed 13 regulons containing 25 genes encoding T4P structural components and putative regulators. A consensus RpoN2-binding sequence GGN10 GC was identified in the promoter sequences of most T4P gene transcriptional units. Electrophoretic mobility shift assays confirmed the direct binding of RpoN2 to the promoter of the major pilin gene pilAX, the inner membrane platform protein gene pilCX, and pilRX. Promoter activity and quantitative RT-PCR assays demonstrated direct and indirect transcriptional regulation by RpoN2 of the T4P genes. In addition, individual deletions of pilAX, pilCX, and pilRX resulted in significantly reduced twitching and swimming motility, biofilm formation, and virulence in rice. Taken together, the findings from the current study suggest that the RpoN2-PilRX regulatory system controls bacterial motility and virulence by regulating T4P gene transcription in Xoo.

Identification of c-di-GMP Signaling Components in Xanthomonas oryzae and Their Orthologs in Xanthomonads Involved in Regulation of Bacterial Virulence Expression.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. Xoo produces a range of virulence-related factors to facilitate its pathogenesis in rice, however, the regulatory mechanisms of Xoo virulence expression have been not fully elucidated. Recent studies have revealed that virulence factor production is regulated via cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway that is well-conserved in Xoo and other Xanthomonas species. A set of GGDEF, EAL, HD-GYP, and PilZ domain proteins with diverse signal sensory domains for c-di-GMP synthesis, hydrolysis, and binding is encoded in the Xoo genome. Bioinformatic, genetic, and biochemical analysis has identified an array of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), as well as degenerate GGDEF/EAL, PilZ domain proteins along with a transcription regulator. These signaling components have been characterized to regulate various bacterial cellular processes, such as virulence, exopolysaccharide (EPS) production, biofilm formation, motility, and adaptation at the transcriptional, post-translational, and protein-protein interaction levels. This review summarized the recent progress in understanding the importance and complexity of c-di-GMP signaling in regulating bacterial virulence expression, highlighting the identified key signal elements and orthologs found in Xanthomonads, discussing the diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs, and effectors, and further exploration of the new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in regulation of bacterial pathogenesis.

Xanthomonas oryzae pv. oryzae Response Regulator TriP Regulates Virulence and Exopolysaccharide Production Via Interacting With c-di-GMP Phosphodiesterase PdeR.

PdeR, a response regulator of the two-component system (TCS) with the cognate histidine kinase PdeK, has been shown to be an active phosphodiesterase (PDE) for intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) turnover and positively regulates the virulence of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice. To further reveal the key components and pathways involved in the PdeR-mediated c-di-GMP regulation of virulence, 16 PdeR-interacting proteins were identified, using the yeast two-hybrid (Y2H) assay. Among them, PXO_04421 (named as TriP, a putative transcriptional regulator interacting with PdeR) was verified via Y2H and glutathione-S-transferase pull-down assays, and its regulatory functions in bacterial virulence and exopolysaccharide (EPS) production were assessed by biochemical and genetic analysis. The REC domain of TriP specifically interacted with the EAL domain of PdeR. TriP promoted the PDE activity of PdeR to degrade c-di-GMP in the presence of PdeK. In-frame deletion in triP abolished the polar localization of PdeR in the cell. Notably, the ∆triP mutant showed significantly reduced virulence on susceptible rice leaves and impaired EPS production compared with wild type, whereas the double mutant ∆triP∆pdeR, like ∆pdeR, caused shorter lesion lengths and produced less EPS than ∆triP. In addition, cross-complementation showed in trans expression of pdeR in ∆triP restored its EPS production to near wild-type levels but not vice versa. Taken together, our results suggest that TriP is a novel regulator that is epistatic to PdeR in positively regulating virulence expression in X. oryzae pv. oryzae.

Phosphodiesterase EdpX1 Promotes Xanthomonas oryzae pv. oryzae Virulence, Exopolysaccharide Production, and Biofilm Formation.

In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, there are over 20 genes encoding GGDEF, EAL, and HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on the characterization of an EAL domain protein, EdpX1. Deletion of the edpX1 gene resulted in a 2-fold increase in the intracellular c-di-GMP levels, which were restored to the wild-type levels in the complemented ΔedpX1(pB-edpX1) strain, demonstrating that EdpX1 is an active phosphodiesterase (PDE) in X. oryzae pv. oryzae. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that the E153A mutation at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that the ΔedpX1 mutant was severely impaired in causing disease symptoms. In trans expression of wild-type edpX1, but not edpX1E153A, was able to complement the weakened virulence phenotype. These results indicated that an active EAL domain is required for EdpX1 to regulate the virulence of X. oryzae pv. oryzae. We then demonstrated that the ΔedpX1 mutant was defective in secreting exopolysaccharide (EPS) and forming biofilms. The expression of edpX1 in the ΔedpX1 mutant, but not edpX1E153A, restored the defective phenotypes to near-wild-type levels. In addition, we observed that EdpX1-green fluorescent protein (EdpX1-GFP) exhibited multiple subcellular localization foci, and this pattern was dependent on its transmembrane (TM) region, which did not seem to directly contribute to the regulatory function of EdpX1. Thus, we concluded that EdpX1 exhibits PDE activity to control c-di-GMP levels, and its EAL domain is necessary and sufficient for its regulation of virulence in X. oryzae pv. oryzae.IMPORTANCE Bacteria utilize c-di-GMP as a second messenger to regulate various biological functions. The synthesis and degradation of c-di-GMP are catalyzed by GGDEF domains and an EAL or HD-GYP domain, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome of X. oryzae pv. oryzae PXO99A, 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling in X. oryzae pv. oryzae, the enzymatic activities and regulatory functions of each GGDEF, EAL, and HD-GYP domain protein need to be elucidated. In this study, we showed that the EAL domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.

Overexpression of miR169o, an Overlapping MicroRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice.

Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs (miRNAs) in this phenomenon, 63 differentially expressed overlapping miRNAs in response to Xoo infection and N limitation stress in rice were identified through deep RNA sequencing and stem-loop quantitative real-time PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than the wild type (WT). Transcript level assays showed that under different N supply conditions, miR169o oppositely regulated NRT2, and this is reduced under normal N supply conditions but remarkably induced under N-limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with the WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to BB in rice. Consistently, transient expression of NF-YA genes in rice protoplasts promoted the transcripts of PR genes and NRT2 genes, while it reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the co ordinated regulatory mechanisms of cross-talk between Xoo infection and N deficiency responses in rice.

A ten gene-containing genomic island determines flagellin glycosylation: implication for its regulatory role in motility and virulence of Xanthomonas oryzae pv. oryzae.

Flagellin glycosylation plays a crucial role in flagellar assembly, motility and virulence in several pathogenic bacteria. However, little is known about the genetic determinants and biological functions of flagellin glycosylation in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Here, the structure, regulation and functions of a ten-gene cluster gigX (glycosylation island genes of Xoo), which was embedded in a flagellar regulon, were characterized. gigX1 to gigX10 encoded putative enzymes or proteins involved in glycan biosynthesis and transfer, including a nucleotide sugar transaminase, an acyl-carrier protein (ACP), a 3-oxoacyl-ACP synthase, a 3-oxoacyl-ACP reductase, a dehydrogenase, an acetyltransferase, a ring hydroxylating dioxygenase, a hypothetical protein, a methyltransferanse and a glycosyltransferase, respectively. The gigX genes were co-transcribed in an operon and up-regulated by the upstream σ54 factor RpoN2 and transcriptional activator FleQ. In-frame deletion of each gigX gene affected flagellin glycosylation modification, meaning that the unglycosylated flagellin of the mutants was smaller than the glycosylated flagellin of the wild-type. No significant changes in flagellar filament and motility were observed in the ΔgigX mutants, among which only ΔgigX6 displayed increased swimming ability. Importantly, all mutants, except ΔgigX9, showed significantly increased virulence and bacterial growth in the susceptible rice cultivar IR24, and ΔgigX1 and ΔgigX10 showed enhanced type III secretion system (T3SS)-related gene expression. Moreover, the glycosylated flagellin of the wild-type induced higher H2 O2 levels in rice leaves than did the unglycosylated flagellins of ΔgigX1 or ΔgigX10. Taken together, this study reveals that the gigX cluster determines flagellin glycosylation, and implicates the regulatory role of post-translational modification with the glycosylation, acetylation and methylation of flagellin in the regulation of motility and virulence of Xoo.

RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence in Xanthomonas oryzae pv. oryzae.

BACKGROUND: Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important crop diseases in the world. More insights into the mechanistic regulation of bacterial pathogenesis will help us identify novel molecular targets for developing effective disease control strategies. A large flagellar gene cluster is regulated under a three-tiered hierarchy by σ54 factor RpoN2 and its activator FleQ, and σ28 factor FliA. A hypothetical protein gene fliTX is located upstream of rpoN2, however, how it is regulated and how it is related to bacterial behaviors remain to be elucidated. RESULTS: Sequence alignment analysis indicated that FliTX in Xoo is less well conserved compared with FliT proteins in Escherichia coli, Salmonella typhimurium, and Pseudomonas fluorescens. Co-transcription of fliTX with a cytosolic chaperone gene fliS and an atypical PilZ-domain gene flgZ in an operon was up-regulated by RpoN2/FleQ and FliA. Significantly shorter filament length and impaired swimming motility were observed in ∆fliTX compared with those in the wildtype strain. ∆fliTX also demonstrated reduced disease lesion length and in planta growth in rice, attenuated ability of induction of hypersensitive response (HR) in nonhost tobacco, and down-regulation of type III secretion system (T3SS)-related genes. In trans expression of fliTX gene in ∆fliTX restored these phenotypes to near wild-type levels. CONCLUSIONS: This study demonstrates that RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence and provides more insights into mechanistic regulation of T3SS expression in Xoo.

Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system.

The targeting of bacterial type III secretion systems (T3SSs), which are critical virulence factors in most Gram-negative pathogens, is regarded as an alternative strategy for the development of novel anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are two of the most important bacterial pathogens on rice, which cause leaf blight and leaf streak diseases, respectively. To identify potential anti-virulence drugs against these two pathogens, we screened a library of plant phenolic compounds and derivatives for their effects on the Xoo T3SS. Ten of 56 compounds significantly inhibited the promoter activity of a harpin gene, hpa1. These inhibitors were further tested for their impact on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results showed that pretreatment of Xoo with TS006 (o-coumaric acid, OCA), TS010, TS015 and TS018 resulted in significantly attenuated HR without affecting bacterial growth or survival. In addition, Cya translocation assays demonstrated that the translocation of two T3 effectors was suppressed by the four inhibitors. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced by treatment with the four inhibitors, suggesting that expression of the Xoo T3SS was suppressed. The expression of other virulence factors was not suppressed, which indicated possible T3SS-specific inhibition. Finally, we demonstrated that these inhibitors reduced the disease symptoms of Xoo and Xoc on the rice cultivar (Oryza sativa) IR24 to varying extents.

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.

Cross-talk between a regulatory small RNA, cyclic-di-GMP signalling and flagellar regulator FlhDC for virulence and bacterial behaviours.

Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression.

The Xanthomonas oryzae pv. oryzae PilZ Domain Proteins Function Differentially in Cyclic di-GMP Binding and Regulation of Virulence and Motility.

The PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R(141) and R(10) residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the full-length open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae.

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.

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.