病原细菌受体介导的c-di-GMP信号传导及其调控机制

细菌第二信使环二鸟苷酸(c-di-GMP)信号网络系统主要涉及信号代谢、识别、接受、传递、功能表达和调控。c-di-GMP胞内水平受到鸟苷酸环化酶(DGC)和磷酸二酯酶(PDE)的控制。c-di-GMP信号受体类型多样,包括转录调控因子、PilZ结构域蛋白、退化的GGDEF和EAL结构域蛋白、核糖体开关、多核苷酸磷酸化酶和新发现的蛋白激酶等。c-di-GMP受体接受信号后,可以在转录、翻译以及翻译后水平上对下游靶标进行调控,从而影响细菌的毒性、运动性、生物膜形成、细胞分裂等生理生化过程。本文结合本实验室对水稻白叶枯病菌的研究结果,综述了近年来国内外在c-di-GMP信号受体介导的调控机制等方面的研究进展。     

水稻白叶枯病菌毒性基因调控hrp基因表达的分析

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)侵染寄主水稻,引起水稻白叶枯病(bacterial leaf blight,BLB)。病原菌主要依赖hrp基因簇编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)将效应蛋白(T3SS effectors,T3SEs)注入水稻细胞中,激发水稻的抗(感)病性。同源性搜索结果显示,植物病原黄单胞菌中已鉴定的一些毒性基因在Xoo的代表菌株PXO99A中保守存在。为了明确这些毒性基因对hrp基因表达调控的影响,本研究利用pK18mob-W介导的定点突变方法,成功获得了14个毒性基因的突变体;在突变体中,利用hrp∶∶gusA融合表达体系,通过GUS活性定量测定检测了hrpG、hrpX和hrpB1的启动子活性;通过荧光定量PCR技术,检测了这3个基因的mRNA水平。结果显示,双组分调控系统ColR/ColS、RpfC/RpfG和转录调控子Clp负调控hrpG和hrpB1的表达;Trh和Xrv A通过HrpG-HrpX途径正调控hrpB1的表达;HpaR1和Fur不依赖于HrpG仅通过HrpX正调控hrpB1的表达。这些调控关系的鉴定为解析水稻黄单胞菌hrp调控网络提供了新的线索。     

水稻白叶枯病菌hrpG调控基因的鉴定

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)引起的白叶枯病(bacterial leaf blight,BLB)是水稻上最严重的细菌病害之一。Xoo与寄主水稻的互作依赖由hrp基因编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS),将效应蛋白(T3SS effectors,T3SEs)注射入水稻细胞,引起BLB症状的扩展。HrpG是hrp基因转录表达的主要调控因子。为了鉴定未知的hrpG调控子,本研究在以hrpG∷gusA为报道基因构建的转座子突变体库中,筛选获得4个候选的hrpG负调控子突变体G24-46、G48-22、G19-14和G57-41。GUS活性测定、荧光定量PCR以及烟草组织的GUS染色试验均显示,在这些突变体中hrpG的表达显著增加。Southern杂交结果显示,突变体中转座子均为单一位点的插入。插入位点分析结果显示,在G24-46、G48-22、G57-41和G19-14中转座子分别插入在minD、pilA、metB和wxoB基因中。毒性测定结果显示,这4个突变体在水稻上的毒性显著降低。这些hrpG调控子基因的鉴定为进一步解析稻黄单胞菌hrpG上游调控网络提供了新的科学线索。     

寄生蜂调控寄主分子机制的研究进展

在寄生蜂与寄主相互作用的研究中,揭示寄生蜂攻克寄主防御系统和调控寄主生长发育的分子机制,已成为生物防治领域的研究热点。有关研究表明:寄生蜂通过控制血细胞增殖与分化的转录/信号传导因子、调节血细胞延展/包囊的因子及诱导血细胞凋亡,以破坏寄主细胞免疫系统;干扰与血淋巴黑化相关基因及抗菌肽基因的表达以抑制寄主的体液免疫反应;调节与生长发育相关蛋白和酶类以扰乱寄主生长发育。作者从细胞免疫、体液免疫和对寄主生长发育三个方面综述了寄生蜂调控寄主分子机制的研究进展。     

水稻白叶枯病菌Zur和HrpG平行调控hrcC基因表达

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)的III型分泌系统(Type III secretion system, T3SS)由hrp基因簇编码,其决定在寄主水稻上的致病性。hrcC是hrp基因簇中hrpA转录单元仅有的一个基因,推测编码T3SS的核心组分蛋白。在Xoo中,hrcC在致病性中的功能以及受调控的机制仍未明确。本研究构建了hrcC的缺失突变体及其功能互补子,发现hrcC缺失使Xoo丧失了在寄主水稻上的致病性以及在非寄主烟草上激发过敏反应(Hypersensitive response, HR)的能力,功能互补子能够恢复这些表型至野生型水平。启动子GUS活性的定量测定和蛋白免疫杂交试验,证明hrcC的转录表达依赖于主要的hrp调控子HrpG,而不受HrpX调控;HrpG和铁转运家族类调控子(Ferric uptake regulator family)Zur以平行独立的方式正调控hrcC基因的转录表达。异源功能互补、启动子活性和蛋白表达试验发现Xoo和水稻条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)的hrcC基因在致病性上的功能具有互置性,以及受HrpG、HrpX和Zur的调控模式也具有相似性。这些研究为进一步解析黄单胞菌的hrp调控网络与全毒性调控网络之间的交叉提供了新的线索。     

水稻白叶枯病菌Zur和HrpG平行调控hrcC基因表达

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)的III型分泌系统(Type III secretion system, T3SS)由hrp基因簇编码,其决定在寄主水稻上的致病性。hrcC是hrp基因簇中hrpA转录单元仅有的一个基因,推测编码T3SS的核心组分蛋白。在Xoo中,hrcC在致病性中的功能以及受调控的机制仍未明确。本研究构建了hrcC的缺失突变体及其功能互补子,发现hrcC缺失使Xoo丧失了在寄主水稻上的致病性以及在非寄主烟草上激发过敏反应(Hypersensitive response, HR)的能力,功能互补子能够恢复这些表型至野生型水平。启动子GUS活性的定量测定和蛋白免疫杂交试验,证明hrcC的转录表达依赖于主要的hrp调控子HrpG,而不受HrpX调控;HrpG和铁转运家族类调控子(Ferric uptake regulator family)Zur以平行独立的方式正调控hrcC基因的转录表达。异源功能互补、启动子活性和蛋白表达试验发现Xoo和水稻条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)的hrcC基因在致病性上的功能具有互置性,以及受HrpG、HrpX和Zur的调控模式也具有相似性。这些研究为进一步解析黄单胞菌的hrp调控网络与全毒性调控网络之间的交叉提供了新的线索。     

植物寄生线虫效应蛋白调控寄主防卫反应分子机制研究进展

 在植物寄生线虫与寄主互作过程中,线虫分泌器官如食道腺细胞分泌的效应蛋白在寄主细胞壁修饰和调控寄主免疫反应以及取食位点形成和维护中发挥着关键作用。解析植物寄生线虫关键效应蛋白的功能及其与寄主互作机制将为探索植物寄生线虫防控新策略提供重要的理论基础。本文从效应蛋白降解寄主细胞壁、调控寄主基础免疫反应、诱导免疫反应机制和介导翻译后修饰调控寄主免疫反应以及植物激素代谢途径的调控机制等方面进行了概述。     

植物寄生线虫效应蛋白调控寄主防卫反应分子机制研究进展

 在植物寄生线虫与寄主互作过程中,线虫分泌器官如食道腺细胞分泌的效应蛋白在寄主细胞壁修饰和调控寄主免疫反应以及取食位点形成和维护中发挥着关键作用。解析植物寄生线虫关键效应蛋白的功能及其与寄主互作机制将为探索植物寄生线虫防控新策略提供重要的理论基础。本文从效应蛋白降解寄主细胞壁、调控寄主基础免疫反应、诱导免疫反应机制和介导翻译后修饰调控寄主免疫反应以及植物激素代谢途径的调控机制等方面进行了概述。     

植物病原黄单胞菌毒性相关基因对内源 过氧化氢的上游调控作用研究

 以植物病原细菌黄单胞菌的avrXa7基因和ahpC基因的2个突变体及其野生型菌株为研究对象,探讨植物病原细菌内源过氧化氢水平与菌株毒性的联系。AR/HRP法分析病原菌的过氧化氢清除力,组织化学法对细胞内过氧化氢进行定量和定位分析。结果表明,avrXa7基因的突变诱导病原菌毒性的显著降低,并引起病原菌内源过氧化氢积累水平显著下降;ahpC基因的突变诱导病原菌内源过氧化氢水平显著降低,但未能引起病原菌毒性产生显著的变化。研究结果说明,在植物病原细菌黄单胞病菌的致病机制中,病原菌内源过氧化氢的积累水平并不能直接决定菌株的毒性,而是处在avrXa7基因的下游,受到avrXa7或者更多毒性相关基因的调控,参与病原菌的致病过程。     

hrp基因关键调控因子HrpG在水稻白叶枯病菌和条斑病菌中的交叉互补性研究

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)和条斑病菌(X. oryzae pv. oryzicola, Xoc)在非寄主烟草上产生过敏反应(HR)和在寄主水稻上具有致病性,是由hrp致病岛决定的,其中HrpG为关键调控因子。Xoo和Xoc侵染水稻途径和在水稻上产生病害症状的不同,是否与hrpG基因有关,还不清楚。本研究利用反向遗传学方法获得了Xoo和Xoc的hrpG突变体PΔhrpG和RΔhrpG,并用hrpG_Xoo基因和hrpG_Xoc基因分别互补上述2个突变体,获得了相应的互补菌株。致病性测定结果显示,PΔhrpG和RΔhrpG突变体丧失了在水稻上的致病性和在非寄主烟草上产生HR的能力,而hrpG_Xoo基因和hrpG_Xoc基因可分别互补上述突变体至野生型水平。利用GUS报告基因检测基因启动子活性发现,hrpG_Xoo和hrpG_Xoc的启动子活性没有显著差别;RT-PCR和Western杂交结果显示,hrpG基因交叉互补菌株中HrpG调控的下游基因hrpX、hpaR、hrcT、hpa2和hrpD6的表达没有差异,且III型分泌系统分泌蛋白Hpa2的分泌性没有受到影响。这些结果表明,稻黄单胞菌hrpG基因可在Xoo和Xoc中交叉互置,位于其上游和下游的调控途径可能相似,而决定Xoo和Xoc在水稻上的侵染途径以及所致病害症状差异可能与hrpG基因位点无关。     

The Xanthomonas oryzae pv. oryzae type IV pilus alignment subcomplex protein PilN contributes to regulation of bacterial surface-associated behaviours and T3SS system

The gram-negative plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) is able to infect the host rice and effectively colonize in vascular tissues. The type IV pilus (T4P) is one of the major virulence factors playing an important role in migration of Xoo through host vascular tissues. Here, we identified PilN, a T4P alignment subcomplex protein, which is involved in regulation of swimming motility, and analysed its contribution to bacterial surface-associated behaviours and virulence. We found that the pilN deletion mutant exhibited dramatically reduced twitching motility and scarcely detectable levels of T4P major pili PilA, as well as enhanced biofilm formation and exopolysaccharide (EPS) production. In addition, deletion of the pilN gene in Xoo resulted in impaired virulence in host rice and attenuated type III secretion system (T3SS) genes expression, which is independent of PilA assembly. Expression of the relevant pilN gene in trans was capable of restoring twitching motility and biofilm formation to the wild-type levels in the pilN mutant but partially recovering EPS production and virulence. Moreover, the expression of trh and xrvA genes, which encode the HrpG positive regulators, was decreased in the pilN mutant. Our results suggest that PilN executes versatile functions in bacterial virulence and cell surface-associated behaviours.     

十字花科黑腐病菌hrp基因致病功能分析

通过对Xcc 8004菌株的hrp基因进行逐一敲除,系统研究单个hrp基因对Xcc致病性的贡献。结果表明,9个hrc(hypersensitive response and conserved)基因单独突变后在满身红萝卜上的致病性和在辣椒ECW-10R上激发HR的能力完全丧失;9个hrp基因中,hrpW突变后在辣椒ECW-10R上仍能产生HR,满身红萝卜上致病性减弱,其余hrp基因突变后致病性和过敏反应均丧失;4个hpa(hrp associated protein)基因突变后,hpaA和hpaB突变体完全丧失在满身红萝卜上的毒性也不能在辣椒ECW-10R上引起HR,hpa1和hpaP突变后致病性和HR显著减弱。另外,通过RT-PCR对Xcc 8004 hrp基因簇的每个基因受hrpX和hrpG的调控情况进行分析,结果表明所有hrp基因在不同程度上都受到hrpG、hrpX的正向调控。     

Small molecules cause virulence attenuation of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. oryzae,the pathogen causing bacterial blight of rice

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight in rice, has developed resistant strains due to rampant usage of chemical pesticides and disease resistant rice cultivars. Virulence attenuating agents could be an alternative strategy to combat this pathogen; therefore, the main objective of the present study was to screen phenolic compounds of plant origin as well as a thiadiazole derivative, acibenzolar S methyl for their effect on various virulence determinants of Xanthomonas oryzae pv. oryzae such as the type three secretion system, motility, extracellular polysaccharide production and secretion of exoenzymes through the type two secretion system. From the compounds screened, cinnamic acid and its derivatives like ferulic acid, caffiec acid and p-coumaric acid showed significant reduction in promoter activity of xopQ, an effector secreted through type three secretion system. None of the compounds used in this study showed substantial inhibitory effect on other virulence determinants indicating that their effect was specific on the type three secretion system except acibenzolar S methyl which, along with inhibitory effects on promoter activity of xopQ, also showed reduction in motility of bacteria. Wheat-bran-derived natural medium was effective for induction of type three and type two secretion systems at detectable levels. We also performed virulence assay that showed significant reduction in disease symptoms confirming cinnamic acid, ferulic acid, caffeic acid, p-coumaric acid as effective virulence attenuating agents of Xanthomonas oryzae pv. oryzae BXO43. These studies demonstrate the potential of selected small molecules for development of new alternative strategies to control the bacterial blight of rice.     

Type-II secretion pathway structural gene xpsE, xylanase- and cellulase secretion and virulence in Xanthomonas oryzae pv. oryzae

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases in rice-growing areas worldwide. Four virulence-deficient mutants were identified from a transposon mutagenesis library of X. oryzae pv. oryzae . Sequence-analysis revealed that the transposon of the four mutants inserted at different sites in the same ORF, which is homologous to the xpsE gene encoding a component of the type-II secretion system in many bacterial pathogens. Extracellular enzymes, such as xylanase and cellulase, were not secreted to the extracellular space in the mutants. Analysis of the protein profile of the extracellular, periplasmic and intracellular fractions indicated that at least two secreted proteins accumulated in the periplasmic space in the mutants. After genetic complementation of these mutants with a functional xpsE gene, the xpsE gene could express normally and the pathogenicity of the mutants and their secretion of extracellular enzymes were restored. Western blot analysis with an anticellulase antiserum also showed that cellulase was secreted normally in the complemented strains. The results show that the type-II secretion pathway structural gene xpsE is required for xylanase and cellulase secretion and full virulence in X. oryzae pv. oryzae .     

水稻条斑病菌异柠檬酸脱氢酶在致病性中的功能分析

 异柠檬酸脱氢酶(isocitrate dehydrogenase,IcdH)催化异柠檬酸转化成α-酮戊二酸,参与碳代谢途径末端的三羧酸(tricarboxylic acid,TCA)循环。然而,编码异柠檬酸脱氢酶基因是否参与水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的致病性,我们并不清楚。为了阐明IcdH的作用,通过同源重组技术获得了Xoc的icdH基因缺失突变体(RΔicdH),并对该突变体进行了相关功能研究。研究表明:该突变体不能利用苹果酸、丙酮酸和柠檬酸,在寄主水稻上的生长能力和致病力相对于野生型均显著降低,其游动性也显著减弱; 功能互补子恢复RΔicdH的上述表型至野生型水平; Real-time PCR结果显示,六碳单糖、蔗糖、苹果酸、丙酮酸与柠檬酸能显著诱导icdH基因的转录表达;与水稻细胞互作时icdH基因受诱导表达,并受HrpX和HrpG负调控。这些结果说明:icdH基因是Xoc获取碳源和在寄主水稻上具有致病性所需的。     

The filamentous phage ϕRSS1 enhances virulence of phytopathogenic Ralstonia solanacearum on tomato

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ?RSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ?RSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ?RSS1, resulting in early wilting. Tomato plants inoculated with ?RSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ?RSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ?RSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ?RSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.     

<Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactone-mediated quorum sensing blockage,a novel strategy for attenuating pathogenicity of Gram-negative bacterial plant pathogens

Quorum sensing is a bacterial communication mechanism by which bacteria sense their own population size and couple specific gene expression to cell density. In Gram-negative bacteria, the most commonly used quorum sensing signals are N-acyl homoserine lactones (AHLs). It is now apparent that many pathogenic bacteria employ quorum sensing to control premature expression of virulence factors. This control is thought to decrease the likelihood that the plant host would detect the pathogens presence and activate its defense system. Novel strategies that target bacterial quorum sensing systems in order to control plant bacterial diseases are discussed.