【作者】 刘红霞；

【导师】 董汉松； 刘凤权；

【作者基本信息】 南京农业大学 ， 植物病理学， 2006， 博士

【摘要】 利用24个已知抗病基因的近等基因系材料对我国285个水稻白叶枯菌株的毒性进行了研究。除了IRBB51以外,含有多个抗病基因的水稻品种几乎对所有菌株都表现了高度的抗性。因此,我们选出对白叶枯菌株抗感反应有明显差异的13个含有单个抗病基因的水稻品种用于进一步的实验。IR24和IRBB10抗性很弱,仅对少数菌株具有抗性;而IRBB5、IRBB7以及IRBB21则具有较高的抗性,对大多数菌株表现出高度的抗性。其它单基因品种如IRBB2、IRBB3、IRBB4、IRBB7、IRBB13等在菌株间的抗感反应有着显著的差异。因此,根据白叶枯病菌与近等基因系水稻之间的互作,我们筛选出6个单基因品种（IRBB5,IRBB13,IRBB3,IRBB14,IRBB2,IR24）作为鉴别品种,从而将我国的白叶枯病菌菌株划分为9个小种,其互作模式依次为:RRRRRR、RRRRRS、RRRRSS、RRRSSS、RRSSSS、RSRRRS、RSSRRS、RSSSSS、SSSSSS,对应小种频率分别为10.18%、10.53%、4.91%、10.18%、24.21%、5.96%、11.23%、22.46%、0.35%。我国白叶枯菌株的毒性呈现多样性,不同时期、不同地区菌株毒性差异明显。与菲律宾菌株的毒性进行比较,也存在较大的差异。通过构建Xanthomonas oryzae pv.oryzae PXO99的hrpA、hrpF基因突变菌株PXO99/PMD-A（AOS）、PXO99/PMD-F（FOS）及其功能互补菌株AOS/pUFR034∷hrpA（cAOS）、FOS/pUFR034∷hrpF（cFOS）,并分别对之进行GFP荧光标记,产生PXO99/pHM1∷gfp（PXO99-GFP）、AOS/pHM1∷gfp（AOS-GFP）、FOS/pHM1∷gfp（FOS-GFP）、AOS/pHM1∷hrpA∷gfp（cAOS-GFP）、FOS/pHM1∷hrpF∷gfp（cFOS-GFP）,对水稻-白叶枯病菌互作进行研究。用PXO99、AOS、FOS、cAOS、cFOS、PXO99-GFP、AOS-GFP、FOS-GFP、cAOS-GFP、cFOS-GFP分别处理水稻及烟草。GFP标记菌株与非GFP标记菌株对水稻的致病性及在烟草上形成HR的能力没有明显差别。突变体FOS对水稻的致病性减弱,AOS几乎完全丧失了对水稻的致病性,形成褐斑。FOS仍然具有诱导烟草形成HR的能力,而AOS同时丧失了在烟草上形成HR的能力。功能互补菌株cAOS、cFOS对水稻的致病性比突变体有所恢复,但达不到野生型的水平。cAOS不能恢复AOS在烟草上形成HR的能力,cFOS仍然可诱导烟草叶片形成HR。病原菌喷雾处理水稻叶片的荧光观察显示,野生型菌株、功能互补菌株、突变体FOS-GFP通过伤口及自然孔口形成侵染,而突变体AOS不能形成侵染。电镜观察结果表明,野生型菌株多数以聚集的方式吸附在自然孔口周围,而突变菌株AOS则多数以分散的形式吸附于水稻叶片表面。以上结果显示,hrpA的突变可能对菌株的识别及聚集能力产生了影响,从而影响了菌株的侵染。已有研究试图确定harpins在植物细胞中的作用部位。但对不同的harpins如来自Erwinia amylovora的HrpN_Ea、Pseudomonas syringae pv.phaseolicola的HrpN_Psph等研究结果都不尽相同。因此,harpins的识别部位、结合受体等一直不清楚,在本研究中,作者利用荧光蛋白标记构建融合蛋白,以便今后进一步研究HrpA、HrpF在水稻上的作用部位。HrpA_Xoo、HrpF_Xoo和HrpA_Xooc、HrpF_Xooc分别是由水稻白叶枯病菌和水稻细菌性条斑病菌的hrpA、hrpF基因编码产生的蛋白质。从水稻白叶枯病菌和水稻细菌性条斑病菌中分别克隆hrpA和hrpF基因,与gfp（green fluorescent protein）构建融合基因,连接pET30a（+）载体,获得了重组质粒pET30a（+）∷hrpA_Xoo∷gfp、pET30a（+）∷hrpA_Xooc∷gfp、pET30a（+）∷hrpF_Xoo∷gfp和pET30a（+）∷hrpF_Xooc∷gfp,转化宿主菌BL21（DE3）产生表达菌株pAGGE、pARGE、pFGGK和pFRGK。表达菌株经IPTG诱导培养,收集菌体,超声波破碎后分别取上清和沉淀进行SDS-PAGE电泳,分别产生94.6 kD、94.8 kD、116.3 kD、116.3 kD大小的组氨酸标记的目的蛋白质与GFP的融合蛋白条带。水稻细菌性条斑病菌HpaG_Xooc蛋白质作为harpin效应分子可促进植物生长、诱导过敏性细胞死亡、诱导植物对病原菌的抗病性。其编码基因hpaG_Xooc包含两个富含甘氨酸的结构域（glycine-rich motif,GRM）和一个半胱氨酸（cysteine）。GRM是harpin类蛋白的共同特征,而一般harpin类蛋白结构中则不含有半胱氨酸。我们对hpaG_Xooc的GRM进行了缺失突变得到突变体hpaG_XoocMG（MG）,对hpaG_Xooc的半胱氨酸位点进行定点突变,将其半胱氨酸突变成苏氨酸得到突变体hpaG_XoocC47T（C47T）。hpaG_Xooc、MG、C47T经体外表达得到HpaG_Xooc、MG和C47T。用它们分别处理烟草,MG和C47T诱导烟草产生的死亡细胞数目分别是HpaG_Xooc的1.7和1.2倍;标记基因hin1、hsr203的表达结果与之一致。MG和C47T处理的植株的抗病性也明显增强。以HpaG_Xooc、MG、C47T处理的烟草再接种病斑TMV病毒,5天后调查,三种蛋白处理的植株叶片病斑分别减少了58%、92%、81%;以Pseudomonas syringae pv.tomato（DC3000）接种番茄,5天内对病原菌进行持续分离,统计其细胞数目。经HpaG_Xooc、MG、C47T处理的番茄上的病原菌的数目分别约为对照的1/160、1/15860、1/1260。防卫相关基因Chia5、NPR1、PR-1a的表达结果与之一致。HpaG_Xooc、MG和C47T还可促进番茄生长,地上部分的增长幅度分别为15%、46%、125%、;地下部分的增长幅度分别为53%、106%、77%。细胞延展与生长相关基因EXP2的表达情况与之一致。这些结果说明HpaG_Xooc中富含甘氨酸的区域以及半胱氨酸的存在抑制了蛋白本身对植物的效应。HpaG_Xooc、HrpF_Xooc、HrpA_Xooc作为水稻细菌性条斑病菌的Ⅲ型蛋白,与病原菌的致病性密切相关。HpaG_Xooc作为效应分子,HrpF_Xooc参与病原菌Ⅲ型泌出系统通过寄主植物的转运结构的形成。HrpA_Xooc可能参与病原菌Ⅲ型泌出系统的泌出结构的形成。三者之间是否存在某种关系呢?通过酵母双杂交系统探讨Ⅲ型蛋白HrpAXooc、HrpFXooc与HpaGXooc之间的关系。分别构建AD载体pGADT7∷hrpA_Xooc、pGADT7∷hrpF_Xooc和DNA-BD载体pGBKT7∷hpaG_Xooc、pGBKT7∷MG、pGBKT7∷C47T。AD载体与DNA-BD载体共转化酵母菌株Y190,β-Galactosidase Assays显色表明,HrpA_Xooc、HrpF_Xooc与HpaG_Xooc、MG、C47T之间不存在直接的互作。本研究的创新点:（1）通过突变体阐释Ⅲ型蛋白HrpA和HrpF在水稻-黄单胞菌互作中的功能;（2）通过对HpaG_Xooc的结构的改变,明确Ⅲ型效应子HpaG_Xooc的关键结构特征对其植物效应的影响;（3）通过酵母双杂交系统探讨Ⅲ型蛋白HrpA_Xooc、HrpF_Xooc与HpaG_Xooc之间的关系。本研究的缺点:有些地方文字描述不够准确,个别地方缺乏统计数据。更多还原

【Abstract】 Xanthomonas oryzae pv.oryzae（Xoo） and pv.oryzicola（Xooc） cause leaf blight and leaf stripe,respectively,in rice.To provide a basis for studying mechanisms that underlie interactions between rice and the bacteria,virulence and race classification of Xoo isolates in China were determined.From more than 22 rice-growing provinces and districts in China,285 isolates of the pathogen were collected;these included 108 isolates collected during 1970-1992 and additional 177 in 2003-2004.To identify proper rice lines that can distinguish variations in virulence,24 near-isogenic rice lines,which contain 1-4 resistance genes,were determined for responses to inoculation with 91 of the 285 isolates.Most isolates were avirulent on the pyramided lines,except IRBB51,and hence,the pyramided lines can not be used as differentials for identifying Xoo races.The 13 rice lines with single gene were used further to test virulence of Xoo isolates.IR24 and IRBB 10 were susceptible to the isolates with several exceptions,whereas IRBB5,IRBB7,and IRBB21 were resistant. Based on these results,six single-gene rice cultivars（IRBB5,IRBB13,IRBB3,IRBB14, IRBB2,and IR24） were chosen as differentials,and the 285 tested isolates were classified into 9 races.The reaction patterns concerning responses of the rice lines to the 9 races sequentially were as follows:RRRRRR,RRRRRS,RRRRSS,RRRSSS,RRSSSS, RSRRRS,RSSRRS,RSSSSS,and SSSSSS,while "R" and "S" refer to resistant and susceptible,respectively.The race frequencies of the patterns were 10.18%,10.53%,4.91%, 10.18%,24.21%,5.96%,11.23%,22.46%,and 0.35%accordingly.So a system for identifying races of Xoo in China was established based on the results.The virulence of representative strains of 8 Philippine races on 6 differentials was determined and compared with the virulence of the Chinese races.The frequency distributions of Xoo races were described for different regions and different periods in China.HrpA_Xoo and HrpF_Xoo in Xoo,and HrpA_Xooc HrpF_Xooc in Xooc as well,are important type-Ⅲproteins.Mutagenesis in the hrpA and hrpF genes of the wild-type（WT） Xoo stain PXO99 was done by single recombinant,generating mutants PXO99/PMD-A（AOS） and PXO99/PMD-F（FOS）,which both diminished pathogenicity on rice.Complementing AOS and FOS with the pUFR034 vector containing WT hrpA and hrpF,respectively,produced the conjugates AOS/pUFRO34::hrpA（cAOS） and FOS/pUFRO34::hrpF（cFOS）,which partially restored pathogenicity of AOS and FOS on rice.The PXO99,AOS,FOS,cAOS, and cFOS were labeled by GFP,producing PXO99/pHM1::gfp（PXO99-GFP）, AOS/pHMI::gfp（AOS-GFP）,FOS/pHMI::gfp（FOS-GFP）,AOS/pHM1::hrpA::gfp （cAOS-GFP）,and FOS/pHM1::hrpF::gfp（cFOS-GFP）.Inoculation tests showed that the virulence of strains with GFP and without GFP were similar in pathogenicity on rice.AOS was avirulent to IR24 and did not induce HR in tomato.Virulence of FOS to IR24 markedly decreased compared to WT,but the induction of HR was not affected evidently.The virulence and inducing HR of mutants AOS and FOS can not been restored entirely by pUFR034 with hrpA or hrpF gene.Fluorescence microscopy（FM） and Electron microscopy（EM） revealed that PXO99-GFP,FOS-GFP,cAOS-GFP,and cFOS-GFP multiplied and congregated on aquaporin and adjacent areas of IR24 but AOS-GFP cells dispersed over epidermal surfaces.This result indicates that hrpA plays an important role in localization of the bacterial colonies on flee.The hrpA and hrpF genes from Xoo and Xooc,which encode HrpA and HrpF proteins in both pathovars,were cloned in pET30a（+） containing an IPTG-inducible promoter and fused with gfp in the vector,generating recombinant plasmids pET30::hrpA_Xoo::gfp（pOAG）, pET30::hrpA_Xooc::gfp（pRAG）,pEY30::hrpF_Xoo::gfp（pOFG）,and pET30::hrpF_Xooc::gfp （pRFG）.They subsequently were transferred into Escherichia coli cells.After the bacterial cells were cultured under inducting by IPTG,suspected proteins were produced based on SDS-PAGE analysis.The molecular masses of the 4 proteins are 94.6 kD,94.8 kD,116.3 kD and 116.3 kD,in consistence with the prediction by a pertinent software program.This result is an essential basis for further studies on functions of the gene and proteins in plants.HpaG_Xooc,produced by Xooc,is a member of harpin group of proteins that stimulate plant growth,hypersensitive cell death（HCD）,and pathogen defense.The protein contains two copies of the glyeine-rieh motif（GRM）,a characteristic of harpins,and a eysteine, which is absent in other harpins.Genetic modification generated the protein mutants HpaG_XoocMG（MG） by deleting GRMs and HpaG_XoocC47T（C47T） by replacing cysteine with threonine.When applied to tobacco plants,C47T and MG were 1.2-fold and 1.7-fold stronger,respectively,than HpaG_Xooc in inducing HCD,which occurred consistently with expression of the marker genes hinl and hsr203.The proteins markedly alleviated infection of tobacco by tobacco mosaic virus and tomato by Pseudomonas syringae.Treating tobacco plants with HpaG_Xooc,C47T,and MG decreased the viral infection by 58%,81%, and 92%,respectively.In tomato plants treated with HpaG_Xooc,C47T,or MG,P.syringae multiplication was inhibited;bacterial population multiplied in 5 d in these plants were ca. 160-fold,1,260-fold,or 15,860-fold smaller than that in control plants.So pathogen defense was induced in both plants.Defense-related genes Chia5,NPR1,and PR-1αwere expressed consistently with resistance.In response to HpaG_Xooc,C47T,and MG,aerial parts and roots of tomato plants increased growth by 15%and 53%,25%and 77%,and 46%and 106%,relative to controls.The expansin gene,EXP2,involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG_Xooc represses the effects of the protein in plants.To determine interactions between the bacterial type-Ⅲproteins,yeast two-hybrid screening was conducted.Multiple combinations were between pGADT7::hrpA_Xooc and pGADT7::hrpF_Xooc as AD vectors,and pGBKT7::hpaG_Xooc,pGBKT7::MG and pGBKT7::C47T as DNA-BD vectors.An AD vector and a BD vector were cotransformed into the yeast strain Y190.Theβ-galactosidase assay was used to observe the relationships of pairs HrpA_Xooc-HpaG_Xooc,HrpA_Xooc-MG,HrpA_Xooc-C,47T,HrpF_Xooc-HpaG_Xooc,HrpF_Xooc-MG, and HrpF_Xooc-C47T by.Results indicated that there was no interaction between HrpA_Xooc and HpaG_Xooc,and between HrpF_Xooc and HpaG_Xooc.In conclusion,this study has characterized specificity in the popular interaction between Xoo races and near-isogenic rice lines with importance in evaluating virulence variations,studied function of the critical hrpA and hrpF genes as well as the effector protein HpaG_Xooc in rice and nonhost plants.The production of several constructs will facilitate further studies on functions of the genes and proteins in plants.更多还原