水稻黄单胞菌水稻致病变种的超氧化物歧化酶活性及诱导

以水稻黄单胞菌水稻致病变种(Xanthomonas oryzae pv.oryzae)的毒性菌株PXO99Ａ和无毒菌株PXO99A(pBUavrXa10F1),检测液体培养中菌体超氧化物歧化酶(SOD)活性变化,以说明SOD与菌株致病性的关系。结果表明,菌体SOD活性高峰出现于延迟期末,之后下降;毒性菌株SOD活性高于无毒菌株。两个菌株的SOD活性的胞内定位均以胞质为主,占总活性的70%以上;酶体周质中SOD活性占总活性的20%～30%。以50～800μmol/L外源O-2处理细菌培养物1?h,可诱导菌体中SOD活性的增加。其 中以200?μmol/L O-2处理SOD活性最高;12?h菌龄培养物的诱导效果优于24?h培养物;对毒性菌株SOD的诱导作用更为明显。外源O-2处理后细菌存活率明显降低,2 4?h培养物的存活率下降大于12?h培养物;毒性菌株存活率下降大于无毒菌株。     

沙福芽孢杆菌GX-H6的分离鉴定及对水稻细菌性条斑病的防病效果

水稻细菌性条斑病（bacterial leaf streak, BLS）由稻黄单胞菌稻生致病变种（Xanthomonas oryzae pv. oryzicola, Xoc）侵染引起,已成为我国南方水稻种植区的一个重要病害。为了筛选防治BLS的生防菌,以Xoc野生型菌株GX01为指示菌,采用含菌平板稀释法和牛津杯法,从花生根际土壤中筛选到一株对Xoc具有拮抗作用的细菌,编号为GX-H6。根据形态学观察、生理生化特征以及16S rDNA和进化树分析,鉴定该菌株属于沙福芽孢杆菌（Bacillus safensis）。拮抗实验表明,B. safensis GX-H6菌株能够对多种黄单胞菌以及植物病原真菌具有较好的拮抗活性,尤其对引起水稻发生白叶枯病（bacterial blight, BB）的稻黄单胞菌稻致病变种（Xanthomonas oryzae pv. oryzae,Xoo）的拮抗效果最显著。温室和田间水稻植株试验表明,GX-H6菌株能较好地防治BLS和BB。对GX-H6菌株的基因组进行分析,发现该菌株的基因组中拥有与抗真菌、环境竞争相关的基因,同时也拥有地衣杆菌素（lichenysi...     

水稻白叶枯病菌广西分离株K74中主效TAL效应物的鉴定

水稻是世界上主要的粮食作物之一。水稻白叶枯病(Bacterial leaf blight)是危害水稻最严重的细菌性病害,可使水稻减产20%以上,严重时甚至绝产,给农业生产和经济造成巨大影响。水稻白叶枯病由水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)的侵染造成,而TAL效应物是Xoo的重要致病因子。近年来,该病在广西也有多发的趋势。2007年从防城港疫区分离得到Xoo K74菌株,经前期研究表明该菌株中有18个tal基因。本研究中,我们对K74菌株中的TAL效应物进行了功能研究。通过基因同源整合突变及互补等研究,我们鉴定到了K74菌株一个与致病力相关的主效TAL效应物。     

四种黄单胞菌的基因芯片检测方法的建立

结合双重PCR和基因芯片技术同时检测和鉴定我国检疫性细菌,包括水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)、水稻细菌性条斑病菌(X.oryzae pv.oryzicola,Xooc)、柑桔溃疡病菌(X.axonopodis pv.citri,Xac)以及严重危害十字花科作物的甘蓝黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)。以铁载体受体(Putative siderophore receptor)基因序列和RNA多聚酶西格玛因子(RNA polymerase sigma factor,rpoD)基因序列为靶标,设计引物和特异性探针能够同时检测这4种重要的病原菌。对17个细菌菌株进行芯片检测,仅4种靶标菌得到阳性结果,证明此方法具有很高的特异性。4种致病菌基因组DNA的检测灵敏度约为3 pg。检测结果表明,建立的基因芯片检测方法特异性强,能实现上述4种黄单胞菌的准确检测和鉴定,具有良好的应用前景。     

浙江1株水稻细菌性条斑病菌株的分离鉴定

细菌性条斑病(简称细条病)是水稻的重要病害之一,随着气候的变暖,某些水稻品种有逐年发生加重的趋势,对产量影响较大。2013年浙江省金华市部分水稻种植区域发生细条病,发病严重的田块减产30%以上。采用组织分离法从发病水稻叶片分离获得6株细菌菌株,选择典型菌株JH01回接水稻幼苗,进行柯赫法则验证。接种后发病症状与自然发病症状一致,并重新分离得到此菌株,证明菌株JH01为水稻细条病的致病菌。通过形态学观察、常规生理生化指标测定、16S rDNA 序列测定和同源性分析,鉴定菌株JH01为稻黄单胞菌水稻致病变种（Xanthomonas oryzae pv. oryzicola,Xoc）。     

水稻白叶枯病菌udgH基因的功能研究

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,以下简称Xoo)是水稻病原菌的一种,其引起的水稻白叶枯病(bacterial leaf blight,BLB)可致使水稻大量减产。本研究发现一个假定的udg H基因的插入突变致使致病力急剧下降,并且胞外多糖、抗逆性、生物膜、游动性等几种生理表型与野生型相比发生了明显变化。互补菌株的致病力能恢复至80%以上,其他生理表型基本能恢复至野生型。本研究为进一步研究udg H基因的致病分子机理提供了研究线索。     

新型水稻黄单胞菌噬菌体资源的发掘与功能鉴定

由水稻黄单胞菌(Xanthomonas oryzae pv.oryzae,简称Xoo)引起的白叶枯病是水稻种植过程中毁灭性的细菌病害,对我国经济和食品安全造成巨大威胁.施用抗生素和化学生物防治手段的防控效果并不稳定,且易污染环境,还存在食品安全问题.为了应对该病害不可预知的爆发,利用噬菌体防控水稻白叶枯病可以作为一种备选方案,以减少化学杀菌剂和抗生素的使用.本研究利用中国不同水稻产区的9株水稻黄单胞菌和模式菌株PXO99A为靶标,从32份土壤样品中分离到了15个高效的Xoo噬菌体单株,说明黄单胞菌噬菌体广泛存在于中国各地的土壤中.选取其中Xoo_sp8和Xoo_sp9,电镜观察确定其具有二十面体的头部和细长的尾部,为典型的有尾噬菌体.宿主谱检测分析发现Xoo_sp8和Xoo_sp9都可以感染除PXO99A以外的9株不同生理小种的水稻黄单胞菌,且在培养基条件下能有效抑制其生长.测定其基因组序列后,根据末端酶大亚基(large terminase subunit,terL)相似性建立其与已报道的不同细菌噬菌体间的系统发育树,发现这两株噬菌体都与已报道的Xoo噬菌体亲缘关系很远,为新型的Xoo噬菌体.本研究分离发掘了15个对Xoo高效感染的噬菌体,为利用噬菌体防控水稻白叶枯病相关杀菌剂产品开发提供了宝贵的种质资源,同时也提出了土壤环境是分离Xoo噬菌体的重要来源的观点.     

水稻白叶枯病菌T3SS基因表达及其调控网络

植物病原细菌III型分泌系统(T3SS)在其毒性表达及与寄主互作中具有重要的功能。水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)hrp基因簇编码了T3SS装置,将毒性效应子蛋白分泌到水稻细胞内,抑制和破坏寄主免疫反应,或诱导感病基因表达,以达到成功侵染的目的。hrp基因表达受到严格调控,在模拟水稻内环境的贫瘠营养培养基中被诱导表达;hrp和效应子基因均受调控蛋白HrpG和Hrp X的调控。此外,hrp基因还受到其它毒性调控网络重要因子的调控,包括双组分调控因子、转录调控因子、DNA/RNA结合蛋白、糖代谢和c-di-GMP信号因子。结合本实验室的研究结果,综述了Xoo T3SS表达调控及其致病机理的研究进展,以期为水稻细菌病害发生机理的解析及其有效防控措施提供一些新见解、思路和途径。     

水稻条斑病菌gacA基因的克隆及功能初析

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacAXooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacAXooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。     

水稻抗白叶枯病基因及其应用研究进展

由黄单胞菌水稻变种Xanthomonas oryzae pv.Oryzae（Xoo）引起的白叶枯病是水稻重要病害之一。目前,已有37个水稻白叶枯抗性基因被鉴定并报道,其中28个被定位到染色体上,7个被克隆。本文简要综述了水稻白叶枯抗性基因的鉴定、定位和克隆的进展,并讨论了合理利用抗性基因防治白叶枯病的前景。     

hrpD6基因决定白叶枯病菌在烟草上的过敏反应和在水稻上的致病性

【目的】白叶枯病菌hrp基因簇由包括hrpD6在内的26个hpa-hrp-hrc基因组成,与植物互作后形成Ⅲ型分泌系统(T3S),将T3S效应分子注入寄主细胞中从而决定在非寄主上的过敏反应(HR)和在水稻上的致病性。但hrpD6基因是否参与了白叶枯病菌在非寄主上的过敏反应(HR)和在水稻上的致病性(pathogenicity)还不清楚。【方法】借助同源重组方法,本研究对白叶枯病菌hrpD6基因进行了突变。【结果】PCR和Southern杂交结果显示,hrpD6基因被成功敲除。烟草上测定结果显示,hrpD6突变体ΔPhrpD6丧失了HR激发能力。致病性测定发现,ΔPhrpD6在水稻苗期不能形成水渍症状,在成株期水稻上不具有致病性,并且细菌生长能力显著下降。功能互补结果显示,hrpD6基因可恢复ΔPhrpD6在烟草上激发HR和在水稻上的致病性以及在水稻组织中的生长能力。RT-PCR结果显示,hrpD6基因的转录表达不仅受水稻诱导,而且受hrpG和hrpX基因调控。不仅如此,hrpD6基因突变还影响T3S效应分子hpa1基因的转录表达和Hpa1蛋白的分泌,暗示hrpD6基因对hpa1基因转录表达具有调控作用。【结论】hrpD6基因的缺失导致白叶枯病菌不能激发烟草产生HR和和丧失在水稻上的致病性,主要是HrpD6对hpa1基因转录表达具有调控作用,并影响T3S效应分子Hpa1的分泌。这些结果为进一步分析hrpD6是否参与T3S分泌装置的形成和调控其它hrp基因的转录表达从而决定病菌在非寄主上的HR和在水稻上的致病性,提供了科学线索。     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Suppression of XopQ–XopX-induced immune responses of rice by the type III effector XopG

Effectors that suppress effector-triggered immunity (ETI) are an essential part of the arms race in the co-evolution of bacterial pathogens and their host plants. Xanthomonas oryzae pv. oryzae uses multiple type III secretion system (T3SS) secreted effectors such as XopU, XopV, XopP, XopG, and AvrBs2 to suppress rice immune responses that are induced by the interaction of two other effectors, XopQ and XopX. Here we show that each of these five suppressors can interact individually with both XopQ and XopX. One of the suppressors, XopG, is a predicted metallopeptidase that appears to have been introduced into X. oryzae pv. oryzae by horizontal gene transfer. XopQ and XopX interact with each other in the nucleus while interaction with XopG sequesters them in the cytoplasm. The XopG E76A and XopG E85A mutants are defective in interaction with XopQ and XopX, and are also defective in suppression of XopQ–XopX-mediated immune responses. Both mutations individually affect the virulence-promoting ability of XopG. These results indicate that XopG is important for X. oryzae pv. oryzae virulence and provide insights into the mechanisms by which this protein suppresses ETI in rice.     

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.     

水稻黄单胞菌黄色素合成相关基因的克隆与鉴定

用硫酸二乙酯(DES)诱变水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)和条斑病细菌(Xanthomonas oryzae pv. oryzicola, 简称Xooc),分别得到5株和13株黄色素缺失突变体,其中来自Xooc的M6和M12 还丧失了对水稻的致病性和在烟草上激发过敏反应的能力。以Xooc黄色素缺失突变体M51为受体菌交叉互补从Xoo JXOIII基因文库中筛选出一个黄色素合成相关的基因克隆pA341,以Xoo黄色素缺失突变体M1071为受体菌,从Xooc RS105基因文库中获得了一个黄色素合成相关的基因克隆pA270。功能互补显示,18株黄色素缺失突变体中的10株能分别被pA341和 pA270互补后正常产生黄色素,但这两个克隆不能同时互补同一株黄色素缺失突变体。能被pA341互补的黄色素缺失突变体M6没有恢复对水稻的致病性和在烟草上激发过敏反应,表明黄色素合成相关基因与hrp基因间不存在相关性。斑点杂交结果表明,pA270与pA341之间没有同源性。pA270亚克隆结果显示,与黄色素合成相关的基因约11.6kb大小,以基因簇的形式存在,不仅决定了黄色素的产生,还影响黄色素合成的数量和质量（吸收峰）。在紫外光条件下,黄色素能够提高菌体的存活率,提示黄色素对病原细菌有保护作用。     

Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.     

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.     

Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.