Chenyang He

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.

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.

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.

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

ABSTRACT 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 R141 and R10 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.

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.

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

BackgroundTo 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.ResultsThe catB gene encodes a putative catalase and is highly conserved in the sequenced strains of Xanthomonas spp. β-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.ConclusionsTranscription 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.

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.

The GGDEF-domain protein GdpX1 attenuates motility, exopolysaccharide production and virulence in Xanthomonas oryzae pv. oryzae.

Cyclic di‐GMP (c‐di‐GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF‐domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although 11 genes encoding GGDEF‐domain proteins have been shown in the genome of Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A, the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c‐di‐GMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF‐domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis.

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.

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

BackgroundBacterial 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.ResultsSequence 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.ConclusionsThis 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.