水稻白叶枯病菌Δrpfxoo基因缺失突变体DSF信号产生和毒性表达

【目的】旨在阐明3个DSF/Rpfxoo信号系统成员RpfFxoo、RpfCxoo和RpfGxoo在水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)毒性表达中的功能。【方法】用标记置换法缺失突变rpfFxoo、rpfCxoo和rpfGxoo基因,测定突变体及其互补菌株的DSF(diffusible signal factor)信号分子产生、胞外多糖(EPS)产生及其对水稻的致病性。【结果】从野生型菌株PXO99A基因组中克隆了推测与DSF信号生成和传导有关的基因rpfFxoo、rpfCxoo和rpfGxoo,获得了相应的单基因或双基因缺失突变体。与PXO99A产生DSF相比,ΔrpfFxoo、ΔrpfF+Cxoo和ΔrpfF+Gxoo均不产生DSF,ΔrpfCxoo过量产生,ΔrpfGxoo产量降低;rpfFxoo、rpfCxoo和rpfGxoo可以分别互补Xoo和Xcc的相应基因突变体,恢复DSF产生表型。除ΔrpfFxoo的EPS产生无明显变化外,其余突变体的均显著减少。所有突变体对水稻的致病性均显著下降。【结论】RpfFxoo、RpfCxoo和RpfGxoo调控了Xoo的DSF信号生成、EPS产生和致病性。     

水稻条斑病菌pilT基因在致病性中的功能分析

【目的】本实验室前期研究发现水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)RS105菌株中pil T基因Tn5转座子插入突变体在寄主水稻上致病性明显降低,在非寄主烟草上激发过敏反应(hypersensitive response,HR)的能力也明显减弱。为了揭示pilT基因在Xoc菌株中的功能,本文进行了深入研究。【方法】本实验通过无标记双交换敲除的方法获得Xoc RS105菌株pil T基因缺失突变体RΔpilT,并对该突变体的游动性以及生物膜等表型进行了检测。【结果】与野生型RS105菌株相比,敲除突变体RΔpilT不仅在感病水稻IR24上的致病性显著降低,在非寄主烟草上产生过敏反应的能力减弱,而且突变体游动性降低,生物膜含量增加,互补子能够恢复上述缺陷至野生型水平。qRT-PCR结果显示,在RΔpil T中,hrpG、hrpX、hrcC、clp、rpfG、pilA、pilC基因表达量明显降低。【结论】Xoc中pilT为重要的毒性相关基因,其致病性与游动性和生物膜含量变化相关,并且受clp、rpfG、pilA、pilC等基因调控。pil T基因编码的Pil T蛋白是构成IV型菌毛的亚基之一,为菌体运动提供能量。本文对pilT基因的功能研究,为进一步分析IV型菌毛在Xoc中的功能提供了线索。     

水稻细菌性条斑病菌中受DSF 调控的鞭毛基因flgD、flgE 的功能分析

[目的]为了阐明可扩散性信号分子(diffusible signal factor,DSF)调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)Rs105的致病性等方面的影响.[方法]采用PCR的方法克隆靶标基因flgDxoc和flgExoc,用同源重组法构建缺失突变体,测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型,用反转录PCR(RT-PCR)的方法验证Rs105和ArpfFxoc(rpfFxoc基因的缺失突变体,不产生DSF)中flgDxoc、、flgExoc表达量的差异.[结果]从Rs105基因组中克隆到flgDxoc、flgExoc基因,并证实这两个基因在基因组中均为单拷贝.PCR和Southern杂交结果显示,flgDxoc、flgExoc基因被成功敲除.与野生型相比,突变体的鞭毛产生能力丧失,游动性和趋化性能力减弱,接种水稻叶片显示其致病性部分减弱,基因互补可使其恢复.生长能力和对烟草叶片的致敏性无明显改变.RT-PCR结果显示,flgDxoc、flgExoc基因在△rpfFxoc中的转录水平明显降低.[结论]本试验表明:FlgD、FlgE是水稻细菌性条斑病菌鞭毛形成所必需的因子;进一步证明了DSF通过调控flgDxoc、flgExoc基因表达,从而影响条斑病菌的致病性等表型.为深入认识DSF对细菌性条斑病菌鞭毛基因簇的调控提供了科学依据.     

胡萝卜软腐果胶杆菌鞭毛钩基因flgK 的功能

【目的】为了研究鞭毛钩基因flgK在胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovorum subsp.carotovorum,P.c.c)的功能。【方法】本研究采用两亲同源交换法构建了基因缺失突变体ΔflgKpcc并构建了互补菌株ΔflgKpcc-KH,测定突变体及其互补菌株的菌体形态、运动性、致病因子、致病性等表型。【结果】与野生菌株PccS1相比,ΔflgKpcc鞭毛缺失,菌体易沉降,在0.3%半固体培养基上运动能力明显降低,生长速率无明显变化,但是纤维素酶和蛋白酶的活性、生物膜形成能力明显下降,对感病寄主的致病力显著减弱。基因互补可以使上述突变表型恢复。【结论】实验表明,鞭毛基因flgK突变导致了菌体的运动性降低、病原菌毒性相关的酶活力下降,从而导致致病力下降。     

水稻白叶枯病菌△rpfxoo基因缺失突变体DSF信号产生和毒性表达

[目的]旨在阐明3个DSF/Rpfxoo信号系统成员RpfFxoo、RpfCxoo和RpfGxoo在水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)毒性表达中的功能.[方法]用标记置换法缺失突变rpfFxoo、rpfCxoo和rpfGxoo基因,测定突变体及其互补菌株的DSF(diffusible signal factor)信号分子产生、胞外多糖(EPS)产生及其对水稻的致病性.[结果]从野生型菌株PX099A 基因组中克隆了推测与DSF信号生成和传导有关的基因rpfFxoo、rpfCxoo和rpfGxoo,获得了相应的单基因或双基因缺失突变体.与PX099A 产生DSF相比,△rpfFxoo、△rpfY+Cxoo和△rpfr+Gxoo均不产生DSF,△rpfCxoo过量产生,△rpfGxoo产量降低;rpfFxoo、rpfCxoo和rpfGxoo可以分别互补Xoo和Xce的相应基因突变体,恢复DSF产生表型.除△rpfFxoo的EPS产生无明显变化外,其余突变体的均显著减少.所有突变体对水稻的致病性均显著下降.[结论]RpfFxoo、RpfCxoo和RpfGxoo调控了Xoo的DSF信号生成、EPS产生和致病性.     

天然氨基酸诱导野油菜黄单胞菌降解DSF-家族群体感应信号活性分析

【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris,Xcc)是十字花科植物黑腐病的致病菌。Xcc中DSF (Diffusible signal factor)信号依赖的群体感应系统和RpfB介导的群体感应退出机制均与其致病性密切相关。【目的】分别检测18种氨基酸对DSF-家族群体感应信号分子合成的影响,为研发新型生物防治方法提供思路。【方法】添加不同浓度的氨基酸到ΔrpfC菌株XYS培养体系中,接种后不同时间点取样提取DSF信号分子,利用高效液相色谱法(High performance liquid chromatography,HPLC)分析DSF和BDSF浓度。【结果】18种氨基酸中,甲硫氨酸、色氨酸和胱氨酸能有效降低ΔrpfC菌株培养体系中DSF和BDSF水平,抑制效果与氨基酸浓度密切相关;3种氨基酸对DSF信号分子的抑制作用存在叠加效应;甲硫氨酸、色氨酸或胱氨酸不影响ΔrpfCΔrpfB双突变体菌株中DSF和BDSF水平。【结论】首次发现了甲硫氨酸、色氨酸和胱氨酸通过RpfB诱导Xcc退出群体感应状态。     

新生隐球菌一个新的产孢相关蛋白Srp1的鉴定与功能分析

【目的】研究产孢相关蛋白Srp1在新生隐球菌有性产孢和致病性中的作用及机理。【方法】采用基因枪转化技术构建新生隐球菌SRP1基因缺失突变体及其互补菌株,并通过小鼠致病性实验和菌株交配实验检测Srp1在新生隐球菌有性产孢和致病性中的作用。【结果】与野生型菌株相比,srp1Δ突变体小鼠致病性无差异;srp1Δ突变体能够交配并形成双核菌丝,但丧失产生担孢子的能力;初步机理分析表明srp1Δ突变体交配后其减数分裂过程被阻断,从而导致srp1Δ突变体不能产生担孢子。【结论】产孢相关蛋白Srp1不影响新生隐球菌的致病性,但可通过调控减数分裂过程影响新生隐球菌的有性生殖。     

糖基转移酶基因rbfCxoo缺失突变导致水稻白叶枯病菌毒性表达增强

摘 要：【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)基因组中推导的脂多糖O抗原合成蛋白基因rbfCxoo (XOO2599) 的结构和生物学功能。【方法】通过基因克隆、序列分析、缺失突变和表型测定,对rbfCxoo的分子特征及其功能进行了鉴定。【结果】 用特异性引物进行PCR扩增,从野生型菌株PXO99A基因组DNA中成功地获得了与己测序菌株KACC10331序列完全一致的全长基因序列; RbfCxoo序列N端和C端分别具有一个糖基转移酶的保守结构域(Glycos_transf_2)。用标记置换法获得了基因缺失突变体△rbfCxoo。与PXO99A相比,△rbfCxoo 脂多糖O抗原合成能力并未发生变化,但鞭毛素糖基化能力有所降低。此外,△rbfCxoo鞭毛运动性、生物膜形成和胞外纤维素酶和木聚糖酶活性都无明显改变,但对水稻的致病性显著增强,毒性相关基因的表达也有所增加。【结论】RbfCxoo可能与细菌鞭毛素糖基化修饰以及毒性表达有关。     

膜泡运输蛋白VdSec22参与大丽轮枝菌胞外蛋白的分泌和致病性

摘要:【目的】验证大丽轮枝菌分泌途径中一个膜泡运输蛋白VdSec22的功能,为防治棉花黄萎病提供潜在的生物靶点。【方法】利用“正负双向筛选法”的方法,构建大丽轮枝菌VdSec22蛋白编码基因缺失的突变体菌株ΔQF。通过农杆菌介导的转化,将其编码基因VdSec22重新导入ΔQF构建了功能回补菌株CΔQF。以野生型菌株为对照,检测上述菌株分泌胞外蛋白(果胶酶、纤维素酶和毒素蛋白)的能力;采用蘸根接种的方法,检测上述菌株对棉花致病性的差异。同时通过定量PCR检测内质网分子伴侣表达量的方法,推断突变体菌株ΔQF中是否发生了内质网应激反应。【结果】成功构建了基因敲除突变体ΔQF和功能回补菌株CΔQF。突变体菌株ΔQF的果胶酶、纤维素酶、毒素蛋白的分泌能力和对棉花的致病性均较野生型减弱,并且产生了内质网应激反应。重新导入VdSec22基因可弥补突变体菌株ΔQF的上述缺陷。【结论】VdSec22是大丽轮枝菌的一个重要分泌途径蛋白,在大丽轮枝菌诸多胞外致病蛋白的分泌和棉花致病性中起重要作用。VdSec22可作为防治棉花黄萎病的潜在生物靶点。     

洛蒙德链霉菌S015中cutR/cutS双组分调控系统对洛蒙真菌素合成的调控

【背景】cutR/cutS双组分调控系统在链霉菌次级代谢过程中起重要作用。【目的】通过同源重组的方法在野生型S015菌株中分别敲除cutR和cutS,构建单基因缺失突变株,研究cutR/cutS双组分调控系统对洛蒙真菌素合成的调控。【方法】对突变株及野生型菌株的发酵产物进行高效液相色谱法(Highperformanceliquidchromatography,HPLC)分析,通过qPCR测定基因表达量的变化。【结果】HPLC产物分析发现,S015Δcut R和S015Δcut S中洛蒙真菌素产量分别达到了128.1±26.4 mg/L和61.8±4.5 mg/L,分别为野生型S015产量的11.5倍和5.5倍。qPCR检测发现,S015ΔcutR突变株中lomo14、lomo10、lphzB、lphzC、lphzE和lphzG的表达量分别达到野生型的1 151.7±88.8、110.5±5.8、129.3±7.7、380.2±34.6、348.2±42.1和299.8±38.2倍;S015ΔcutS突变株中lomo14、lomo10、lphzB、lphzC、lphzE和lphzG的表达量分别达到野生型的4.3±0.5、2.2±0.2、9.3±0.9、10.3±0.6、20.7±1.5和20.4±0.8倍。【结论】cutR/cutS双组分调控系统在洛蒙德链霉菌的洛蒙真菌素合成过程中对其合成途径核心基因和侧链修饰基因的表达有抑制作用,从而抑制其合成。     

野油菜黄单胞菌中3-羟基脂酰ACP脱水酶FabZ的生理功能

【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris, Xcc)引起十字花科植物黑腐病,在全球范围内造成经济损失,亟须深入研究其致病机理,开发新的黑腐病防控措施。细菌脂肪酸合成系统不仅为细胞膜合成提供原料,其中间代谢产物还是许多生物活性分子合成的底物,具有重要的生理功能,也是抗菌药物筛选的重要靶标。【目的】研究XccfabZ对扩散信号分子(diffusible signal factor, DSF)类信号产量、致病力、胞外酶、胞外多糖和运动性等方面的影响。【方法】利用报告菌株检测法分析了不同替换突变株的DSF类群体感应信号产量。利用同源重组原理,在DSF类信号高产菌株中获得替换突变株,利用高效液相色谱(highperformanceliquid chromatography, HPLC)法测定DSF类信号产量。利用剪叶法检测替换突变株对寄主植物甘蓝的致病力,并分析了不同菌株的胞外多糖、胞外酶和运动性差异。【结果】报告菌株检测法和HPLC法都证明大肠杆菌fabZ替换突变株(XccΔfabZ/pSRK-EcfabZ)中DSF类信号产量显著下降。...     

The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

水稻条斑病菌胞外多糖相关基因的鉴定

摘要:【目的】前期研究中从Tn5 转座子插入的水稻条斑病菌突变体库中获得了17 个胞外多糖改变的突变体。【方法】本文对这些突变体的Tn5 插入位点和基因类型进行了鉴定。【结果】结果显示,胞外多糖减少的11 个突变体中多数为Tn5 插入在已知的gum、xan 和wxoc 基因簇上,Xoryp_4217、Xoryp_2488 和Xoryp_0918为未知的与胞外多糖产生有关的基因,属首次报道;6 个胞外多糖增多的突变体中,fimO、pilY 和xopQ 与胞外多糖产生有关,但在水稻条斑病菌中未见报道;Xoryp2392、Xoryp_4221 和Xoryp_3511 为首次鉴定,其中Xoryp_3511 仅在水稻黄单胞菌中存在。毒性测定结果显示,胞外多糖减少的突变体在水稻上的毒性变弱,而胞外多糖增加的突变体在水稻上的毒性没有显著变化。【结论】这些结果为进一步分析水稻条斑病菌胞外多糖代谢途径以及与水稻的互作关系奠定了基础。     

Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence.     

Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.     

Construction of a Tn5-Tagged Mutant Library of Xanthomonas oryzae pv. oryzicola as An Invaluable Resource for Functional Genomics

To genome-widely mine pathogenesis-related genes of Xanthomonas oryzae pv. oryzicola (Xoc), which is the casual agent of bacterial leaf streak resulting in significant yield loss and poor quality in rice, a Tn5 transposon-mediated mutation library was generated. Twenty-five thousand transformants were produced by using Tn5 transposome, appropriately corresponding to 5 × ORF coverage of the genome, and inoculated into rice and tobacco, individually and respectively, for screening candidate virulence genes. Southern blot and thermal asymmetric interlaced polymerase chain reaction analysis of Tn5 insertion sites of randomly selected mutants suggested a random mode of transposition and a saturation library. Characterization of extracellular polysaccharides, extracellular protease activity, and pigment production of individual mutants in the growth media revealed that 11 mutants enhanced in growth, 12 reduced extracellular polysaccharide production, 12 lost extracellular protease activity completely or partially, and 21 were pigment deficient. In planta pathogenicity assays revealed 253 mutants reduced virulence in rice, but kept triggering hypersensitive response in tobacco; 49 lost the ability to elicit HR in tobacco and pathogenicity in rice; and 3 still induced hypersensitive response in tobacco, but lost pathogenicity in rice. The achieved mutant library of Xoc is of high-quality and nearly saturated and candidate virulence mutants provided a strong basis for functional genomics of Xoc.     

A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris (Xcc) is regulated by a cluster of genes called rpf (for regulation of pathogenicity factors). Two of the genes, rpfF and rpfB, have previously been implicated in the synthesis of a diffusible regulatory molecule, DSF. Here, we describe a screen of transposon insertion mutants of Xcc that identified two DSF-overproducing strains. In each mutant, the gene disrupted is rpfC, which encodes a hybrid two-component regulatory protein in which the sensor and regulator domains are fused and which contains an additional C-terminal phosphorelay (HPt) domain. We show that rpfC is in an operon with rpfH and rpfG. The predicted protein RpfG has a regulatory input domain attached to a specialized version of an HD domain, previously suggested to function in signal transduction. The predicted protein RpfH is structurally related to the sensory input domain of RpfC. We show that RpfC and RpfG act positively to regulate the synthesis of extracellular enzymes and EPS, but that RpfC acts negatively to regulate the synthesis of DSF. We propose that RpfGHC is a signal transduction system that couples the synthesis of pathogenicity factors to sensing of environmental signals that may include DSF itself.