Xoryp_08180 of Xanthomonas oryzae pv. oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv. oryzicola (Xoc) causes a destructive bacterial leaf streak disease in rice. Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice. A mutant, Mxoc1679, screened from our previous Tn5-tagged mutant library for Xoc strain RS105, showed reduced virulence in rice. In this mutant, a gene named as Xoryp_08180 was disrupted by Tn5 insertion. Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp. Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice, a delayed hypersensitive response (HR) in non-host tobacco, and a decrease in extracellular protease activity. The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans. In addition, the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition. These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production, HR induction and full virulence of Xoc.     

水稻条斑病菌致病力分化研究

在历年测定的基础上,选择了从国内各地收集的28个水稻条斑病菌株,用针刺法接种在一套有鉴别力的水稻品种上,结果表明,不同菌株间的致病力差异显著,条斑病菌株与水稻品种间的互作反应大多表现弱互作模式,部分菌株与品种间存在强互作关系,根据供试菌株在鉴别品种IRBB5、IRBB7、IRBB3、IRBB21和JG30上致病力的差异,可区分成5种反应模式,分别为RRRRS、RRRSS、RRSSS、RSSRS、RSSSS.     

水稻细菌性条斑病菌和白叶枯病菌的多重PCR检测体系开发

本研究设计水稻细菌性条斑病菌和水稻白叶枯病菌的专化性引物,建立了可同时检测这两种细菌的多重PCR检测体系。通过体系优化、特异性和灵敏度检查,结果表明：两对专化性引物X-b和X-t具有较高的特异性和灵敏度,对水稻细菌性条斑病菌液和水稻白叶枯病菌液的检测灵敏度分别为104 cfu/mL和105 cfu/mL;利用本研究建立的多重PCR体系,成功地从50种市售水稻种子中检测出1个水稻品种带有水稻细菌性条斑病菌,1个水稻品种水稻白叶枯病菌。本研究建立了多重PCR检测体系,为检疫部门控制这两种检疫性种传病害的入侵提供了有效检测技术和手段。     

水稻细菌性条斑病菌DNA多态性与致病性研究

利用６０个随机引物对来自我国不同稻区的水稻细菌性条斑病菌株基因组ＤＮＡ进行ＰＣＲ扩增,其中２０个引物均出现清晰和重复性的ＤＮＡ扩增片段,扩增片段大小在０．５ｋｂ至３．５ｋｂ;应用Ｓｔａｔｉｓｔｉｃｓ软件ＵＰＧＭＡ程序进行树状聚类分析,聚类结果表明,我国水稻细菌性条斑病菌具有明显的遗传分化,在遗传距离为０．３０时,供试菌株可划分为７个遗传相似组（谱系）。其中第１、第２和第５组为优势组群,分别由７、８、６个菌株组成。根据菌株在１２个已知基因品种上的抗感反应,将供试菌株分为１３个致病型,在遗传距离为０．５０水平上聚类归属于６个簇。菌株的致病类群与其ＤＮＡ的遗传变异具有弱相关性。     

The Twin-Arginine Translocation （Tat） Pathway Is Essential for Virulence,Flagellation, and Chemotaxis in Xanthomonas oryzae pv. oryzicola Strain RsGD42

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice （Oryza sativa）. Genetic determinants （tatABC genes） of the twin-arginine translocation （Tat） pathway from X. oryzae pv. oryzicola strain RsGD42 were cloned and characterized, meanwhile, a tatC disruption mutant was generated. The tatC mutant lacked detectable flagella and was highly impaired in motility and chemotaxis. Furthermore, it was observed that the tatC mutant exhibited a reduced production of extracellular polysaccharide （EPS） and a significant reduction of virulence on adult rice plants compared to wild type strain. However, the tatC mutation in X. oryzae pv. oryzieola strain RsGD42 did not affect the growth rate and the ability to induce hypersensitive response （HR） in nonhost tobacco （Nicotiana tabacum L. cv. Samsun）. In conclusion, the data indicated that the Tat pathway significantly contributed to the virulence of X. oryzae pv. oryzicola.     

西南地区水稻细菌性条斑病菌致病力的分化

以水稻品种IRBB4、IRBB5、IRBB14、IRBB18、IRBB21和IR24为鉴别品种,在孕穗期,采用针刺接种的方法,将水稻条斑病菌接种在水稻剑叶上,根据病菌与品种互作反应和亲和性的类型差异,将供试的75株条斑病菌菌株区分为13个小种群.大多数水稻条斑病菌株在鉴别品种上的互作反应表现为弱互作模式.云南、贵州、四川3省菌株小种组成的比较表明:以小种C9为目前3省的共同优势种群;云南省小种组成多样,所有的小种均有分布,贵州小种类型有6、9、11、13,四川小种类型有3、4、6、7、9、11、12、13,小种的分布受地理区域的影响.     

水稻条斑病菌和白叶枯病菌致病相关基因的敲除

wxocA、wxocB、wxocD、wxocE和wzt是水稻条斑病菌（Xanthomonas oryzae pv．oryzicola,Xooc）的脂多糖（lipopolysaccharide,LPS）合成基因,avrXa3是水稻白叶枯病菌（Xanthomonas oryzae pv．oryzae,Xoo）的一个无毒基因。利用pBCSK（-）和pKNG101两个质粒的复制起点不被水稻黄单胞菌识别的特点,把wxocA、wxoeD、wxocE和wzt的中心区域克隆在pBCSK（-）上,获得突变转化单元pBCSK：：ΔwxocA、pBCSK：：hwxocD、pBCSK：：AwxocE和pBCSK：：Δwzt。把wxocB和avrXa3基因的旁侧序列和一个氯霉素抗性基因cm构建在pKNG101上,获得突变转化单元pKNG101：：ΔwxocB和pKNG101：：Δavr。以电转化方式把这些LPS合成基因突变转化单元DNA转入Xooc菌株RS105,把无毒基因突变转化单元转入Xoo菌株PXO99,通过同源重组分别获得了RS105中5个如相应基因突变体MwxocA、MwxocB、MwxocD、MwxocE、Mwzt和一个无毒基因的突变体PX099Δavr。SDS-PAGE分析发现,lps突变体的O抗原或核心寡糖的合成被破坏。致病性分析结果显示,基因wxocB、woxocE和wzt与致病性有关,前两基因的突变导致细菌完全失去毒性,而后一基因的突变导致细菌部分丧失毒性。与PX099相比无毒基因突变体PX099Δavr改变了原有的毒性表型,在IRBB50（Xa4／xa5）和Asominori（Xa17）上由较感转变为较抗表型。因此,本试验证明,以pBCSK（-）和pKNG101为载体敲除水稻条斑病菌和白叶枯病菌致病相关基因是可行的。     

水稻条斑病细菌dsp基因的克隆

用硫酸二乙酯(DES)化学诱变水稻条斑病细菌RS105菌株，获得一株dsp基因突变体AM11。该突变体的菌落形态、颜色、胞外多糖产生能力以及胞外酶(淀粉酶、蛋白酶、果胶酶和纤维素酶)的活性与野生型菌株RS105无明显差异。将水稻条斑病细菌RS105基因库1200个克隆逐个导入dsp基因突变体AM11中，致病性测定结果表明，dsp基因阳性克隆pC101可以恢复AM11在水稻上的致病性。酶切分析显示，克隆pC101携44．92kb的dsp基因片段。亚克隆和功能互补结果显示，克隆pE46和pE47均具有恢复dsp突变体AM11致病性的能力，这表明决定水稻条斑病细菌dsp表型的基因位点至少有2个。     

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp- mutants were produced from strain JXOIII of Xanthomonas oryzae pv.oryzae (Xoo) and strain RS105 of X.o.pv.oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che-mical. All the hrp- mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy-lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp- mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell-sonicated integrations of the wild type strains and the hrp- mutants demonstrated that there existed an HR eliciting substance which was heat-stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp- mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6-kb hrp fragment insert in pUHRX245 and a 39.3-kb insert in pUHRS138 revealed that a 3.3-kb SacⅠ fragment from pUHRX245 and a 4.5-kb BamHⅠ-KpnⅠ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp- mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3-kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.     

模块化质粒系统用于水稻条斑病菌基因表达的分析

水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)侵染水稻,引起细菌性条斑病(bacterial leaf streak,BLS)。近几年,在我国南方水稻产区,条斑病大面积发生,对水稻的安全生产构成严重威胁。为了准确地在Xoc中进行基因功能的分析,本研究设计出模块化的质粒系统,用于基因转录表达和蛋白表达的分析。为了验证该系统的可行性,选取hrpG、hrpX和hrpB1基因构建了相应的启动子载体和蛋白表达载体,将这些载体导入相应的hrp调控子基因trh、hrpG和hrpX的突变体中。GUS活性测定的结果显示,在XOM3培养基、寄主水稻组织和非寄主烟草组织中,启动子模块元件能够准确反映这3个hrp基因的调控表达模式。蛋白免疫杂交结果显示,HrpX蛋白表达水平反映的调控模式与hrpX启动子活性呈现的调控表达模式一致。水稻上致病性测定结果显示,有效表达的HrpG蛋白能够恢复hrpG突变体在寄主水稻上的致病性。这表明,这套模块化的载体系统能够有效应用于基因转录表达、蛋白表达和功能互补的分析。这将为Xoc-水稻互作研究提供有效的技术支持,加快Xoc毒性相关基因功能的研究。     

中南4省水稻细菌性条斑病菌致病型的比较

以金刚30、窄叶青、XM5、XM6、M41等5个品种为鉴别品种,将来自4省的75个细条病菌菌株的毒力划分为0、Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ等7个致病型,0、Ⅰ、Ⅱ、Ⅲ、Ⅳ型毒力差异主要呈由弱到强的数量变化,占测试株的89．3Yo,其中较强毒力的Ⅲ、Ⅳ型菌占参试菌的54．7％;Ⅴ和Ⅵ型间呈交叉反应式的强互作关系,占参试菌的10．7％。4省菌株毒力的比较表明,福建省以Ⅲ、Ⅳ型菌为主;江西省以Ⅲ、Ⅵ型菌为主;湖北省以Ⅱ、Ⅲ型菌为主;湖南省没表现突出的优势群,但较强毒力的Ⅱ、Ⅲ、Ⅳ型菌所占比例为55．6％。     

水稻白叶枯病菌在非寄主植物上的过敏反应检测

为寻找一种有效检测水稻白叶枯病菌在非寄主植物上引起过敏反应的方法,将不同浓度的该菌菌液压渗入不同的供试植株叶片并观察比较分析。结果显示,Xoo 13751在接种浓度为5×108、109cfu/mL时,接种后48h在烟草叶片上形成的枯斑边缘模糊,不易观察;该菌以5×107~109cfu/mL浓度接种在蓖麻叶片上时,接种后48h均能观察到明显清晰的枯斑。表明蓖麻作为水稻白叶枯病菌引起过敏反应的检测植株,具有形成的枯斑清晰、灵敏度高的优点,是一种有效的检测方式。     

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv.oryzicola

Xanthomonas oryzae pv.oryzicola (Xoc),the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response (HR) in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.     

水稻白叶枯病菌毒性表达的负调控因子PXO_02944的分子鉴定

【目的】水稻黄单胞水稻致病变种（Xanthomonas oryzae pv. oryzae, 简称Xoo）侵染引起水稻白叶枯病,在侵染过程中Xoo可产生胞外多糖(EPS)、胞外酶、黏附因子、T3SS以及其产生的效应因子等毒性因子。细菌第二信使环鸟苷二磷酸（c-di-GMP）的水平在Xoo毒性调控中发挥了重要的作用,而PXO_02944是包含REC、GGDEF和EAL结构域的蛋白,是c-di-GMP信号蛋白家族的成员,研究旨在阐明水稻白叶枯病菌的环鸟苷二磷酸信号蛋白家族成员PXO_02944的基因结构和功能。【方法】根据PXO_02944基因序列信息,以PXO99A基因组DNA为模板,设计引物扩增PXO_02944基因全长,将其GGDEF结构域和EAL结构域分别与已经报道的保守的GGDEF结构域或EAL结构域蛋白进行氨基酸序列比对分析;分别扩增PXO_02944左右臂片段,将其与目的载体pK18mobsacB载体连接,得到质粒pK2944,将pK2944与pMD-GmR载体回收获得的GmR基因片段进行连接,获得重组质粒pK2944-GmR,用于构建基因突变体。将PXO_02944全长基因克隆到载体pHM1上,获得重组质粒pHM1-2944并转化到ΔPXO_02944中,即获得互补菌株ΔPXO_02944::C。对野生型菌株、突变体和互补菌株进行表型测定,分析PXO_02944 基因缺失突变对Xoo致病性和EPS产生、生物膜形成、胞外酶活性和鞭毛运动性的影响,并通过qRT-PCR方法测定EPS合成基因和毒性相关基因的表达。【结果】用特异性引物进行PCR扩增,成功地从野生型菌株PXO99A中克隆了PXO_02944;生物信息学分析发现,PXO_02944蛋白具有磷酸化信号接收的REC输入结构域和GGDEF、EAL输出结构域,但GGDEF和EAL结构域保守序列发生了突变。与野生型菌株PXO99A相比,虽然PXO_02944突变体胞外纤维素酶和木聚糖酶活性、鞭毛运动性都无明显变化, 但其对水稻的致病性、EPS产生和生物膜形成能力显著增强,T3SS调控基因hrpG和EPS合成基因gumG的转录水平也明显升高。【结论】应答调控因子PXO_02944负调控了水稻白叶枯病菌致病性、EPS产生和生物膜形成这些毒性因子的表达。     

受条斑病菌侵染的水稻cDNA文库构建

提取经条斑病菌(Xanthomonas oryzae pv.oryzicola)侵染诱导后的水稻mRNA,利用SMARTTM技术于载体pGADT7-SfiAB上构建了水稻cDNA文库.检测发现,文库中含有水稻病程相关蛋白基因OsPR-1a、OsPR-1b和PAL.随机提取cDNA文库克隆的质粒,酶切分析发现,该cDNA文库的插入片段分布在500～2 000 bp之间,平均大小约为1 kb,重组率达95%.上述结果表明,水稻受条斑病菌侵染的cDNA文库质量较好,这为研究条斑病菌致病性因子与水稻互作机制奠定了工作基础.     

湖北省水稻细菌性条斑病菌与噬菌体互作关系的研究

通过对细菌夺冠滤器过滤，氯仿处理以及用稻细条病病叶组织浸提液直接分离噬菌体等方法的比较，结果表明：三者中相比之下，细菌过滤器过滤法是最为简便可靠的噬菌体分离方法，通过对湖北省10个水稻细菌性条斑病菌与20个噬菌体互作反应的研究表明：不同来源的细条病菌与不同来源的噬菌体之间，存在着亲和性的分化，根据它们相互的亲和关系，可把10个细条病菌菌株划分为6个组，把20个噬菌体划分为7个溶菌型；其中RP、RS、RT、RX、RY等5个细条病菌菌株可与所有参试的20个噬菌体发生亲和反应，据此在用噬菌体技术进行湖北省水稻细菌性条斑病的测报工作中，友谊赛些广亲和性的菌株可逡为指示菌。     

水稻白叶枯病菌hrp调节基因hrpXoo的克隆与序列分析

 用化学方法诱变水稻白叶枯病菌PXO99A 菌株 ,获得 6株hrp-突变体 ,此突变体除丧失在非寄主烟草上激发过敏反应和在感病寄主水稻上的致病能力外 ,有些缺乏激发烟草产生HR的信号物质 ,有些在胞内存在此信号物质 ,而不能泌至胞外。来自Xanthomonasoryzaepv .oryzaeJXOIII粘粒基因文库的hrp基因克隆pUHRX2 4 5 ,所携hrp基因片段大小为 36 .8kb。系列亚克隆 36 .8kbhrp基因片段及各亚克隆对hrp-突变体功能互补作用的结果显示 ,3.3kbSacI片段为最小酶切功能片段。序列测定和分析结果表明 ,3.3kbhrp片段含hrpX oo基因和含与热激蛋白 90家族有关的 2个开放阅读框hspORF1和hspORF2。HspORF1在蛋白质数据库中未发现序列蛋白。HspORF2与Hsp90 Xo的同源性达 99%。hrpXoo与黄单胞菌中已报道的hrpX基因有很高的同源性(90 %以上 )。在黄单胞菌中高度保守的编码α 螺旋 转 α 螺旋结构的 6 0 bp核苷酸序列 ,在交叉功能互补时是必需的。不含此结构的hrpXoo (1.1kb)片段 ,可使JXOIII的hrp-突变体在水稻上具致病性和在非寄主烟草上激发产生HR ,但不能使来自PXO99A 和RS10 5的hrp-突变体恢复在烟草上激发产生HR的功能。黄单胞菌中已知HrpX的同列比较显示 ,X .oryzae和X .campestris种间在 88、196和 2 4 7位点的氨基酸上有     

Virulence of Xanthomonas oryzae pv.oryzae on Rice Near-lsogenic Lines with Single Resistance Gene and Pyramiding Lines in China

Ninety one isolates of Xanthomonas oryzae pv. Oryzae were collected from different rice growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes:IRBB1 (Xa1),IRBB2 (Xa2),IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11),IRBB13 (xa13),IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21),IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21),IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10,less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR,RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48％ respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the evelopment of rice resistance breeding to bacterial blight in China.     

水稻细菌性条斑病菌(Xanthomonas campestris pv.Oryzicola Dye)的致病力变异和菌系鉴别

1988-1991年,从福建省内外收集和分离了161个菌株,用针刺接种法,先后在25个水稻品种上进行了病菌致病力的测定.根据从中选择的17个水稻品种对选择的16个菌株所做的试验结果表明,稻条斑病菌菌株间存在明显的致病力差异,而且这种差异是数量性的.1991年,根据125个菌株对选择的4个鉴别品种的侵染反应,将这些菌株划分为致病力强弱不同的4个菌群.0群菌致病力退化;Ⅰ群菌致病力弱;Ⅱ群菌致病力中等;Ⅲ群菌致病力最强.其中Ⅱ和Ⅲ群菌占优势,在福建省34个县市已有分布.