【作者】 邹丽芳；

【导师】 陈功友；

【作者基本信息】 南京农业大学 ， 植物病理学， 2007， 博士

【摘要】 水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)和水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)分别引起水稻白叶枯病(bacterial blight,BB)和水稻细菌性条斑病(baeterial leaf streak,BLS),在生产上造成水稻损失严重。这两种病原菌已成为水稻的两个模式病原菌。为精确和高通量地研究Xoo和Xooc的致病相关基因的功能、基因间相互作用以及调控,如hrp基因,即决定Xoo和Xooc在非寄主植物上激发过敏反应(hypersensitive response,HR)和在寄主植物上具有致病性(pathogenieity)的基因,本研究利用pK18mobGⅡ载体建立了一种基于单交换的基因定点插入突变体系和利用自杀性载体pKMS1建立一种基于负选择标记基因sacB的两次同源交换的无标记敲除技术。成功获得了水稻白叶枯病菌PXO99~A菌株的3个hrpF和4个hrcV插入突变体以及RS105的4个hrpF和4个hrcV插入突变体。RS105的hrcV,hrpF、hrpE、hpaB和hpa1的单基因敲除突变体,hpa1和hpa2、hpa1和hrpF、hrpE和hrpF以及hpaB和hrpF的双基因敲除突变体;hpaB、hpa1和hrpF及hpa2、hpa1和hrpF的三基因敲除突变体。多基因敲除技术的建立为研究水稻黄单胞菌致病相关基因的功能以及在致病过程中基因间相互作用奠定了遗传学基础。有证据表明水稻黄单胞菌都含有15个以上的avrBs3/PthA家族基因,它们具有几乎一样的5’端和3’端,不同的只是中间102 bp重复单元的重复数不同。本研究以avrBs3/pthA家族成员avrXa3的3’端含有核定位信号和酸性转录活化域的359bp的DNA片段构建探针,对中国菌系的白叶枯病菌和条斑病菌的基因组DNA进行Southern杂交分析,发现这两种病原菌的不同小种间所含有的avrBs3/pthA基因的拷贝数不同,有些条带在所有的小种中都存在,有些条带是某个小种所特有的。毒性弱的小种含有较少的avrBs3/pthA家族成员,毒性强的小种含有较多的avrBs3/PthA家族成员和较多的102 bp重复单元的重复数。以相同的359 bp的DNA片段为探针进行菌落原位杂交,从条斑病菌RS105菌株的基因文库中筛选到35个阳性克隆,经过酶切和Southern杂交后归类为28个avrBs3/PthA基因。34个阳性克隆分别导入白叶枯病菌PXO99~A菌株中,进行水稻近等基因系苗期注射接种和成株期剪叶接种,发现含有p6、p8、p9、p10和p14的克隆在含有抗病基因Xa10的水稻品种IRBB10上产生特异性的HR(Hypersensitive response)反应。序列测定结果显示它们均是avrBs3/pthA家族的成员,分别命名为avr/pth6、avr/pth8、avr/pth9、avr/pth10和avr/pth14。蛋白质序列比对发现,这5个蛋白质的C端均缺少水稻白叶枯病菌avrBs3/pthA家族特有的SGsVGGTI残基。基因旁侧序列比较发现,水稻条斑病菌的avrBs3/pthA基因在启动子区存在特有的GTTTCTG序列,avr/pth14在启动子区和终止子区存在一段顺式元件,具有转座子转移的特征.将avr/pth6和avr/pth8的BamHⅠ片段替换avrXa3基因相应的BamHⅠ片段,构建为新的融合基因avrBre6和avrBre8,这两个融合基因仍可使PXO99~A菌株在IRBB10上具有无毒性。从水稻条斑病菌中克隆avrBs3/pthA家族基因还未见报道。本研究的结果揭示,Xooc中存在avrBs3/pthA家族基因,可能因为Xooc中某种因子的存在,限制了avrBs3/PthA基因的无毒性功能,这也可能是水稻缺少抗条斑病的主效抗性基因的原因。hrcV基因在动植物病原细菌中高度保守,基因产物是Ⅲ型分泌系统的组成蛋白,推测形成一环状结构,位于细菌的內膜。本研究通过单交换同源重组分别获得了PX099~A的PXV268、PXV917、PXV106和PXV452插入突变体以及RS105的RSV268、RSV917、RSV106和RSV452突变体;通过双交换同源重组获得了RS105的菌株hrcV缺失突变体RS105△hrcV.PXV268和RSV268突变体在非寄主上仍能激发HR,在水稻仍具有致病性.其余的hrV突变体PXV917、PXV106、PXV452和RSV917、RSV106、RSV452丧失了在水稻上的致病性和在非寄主上激发HR反应的能力.HrcV的突变体与Ⅲ型组分蛋白HrcN、HrcJ、HrcR以及TTSS效应分子Avr/pth28、AvrXa3和Avr/pth13的酵母双杂交结果揭示:HrcJ和HrcR蛋白主要与HrcV蛋白N端互作;HrcN蛋白主要与HrcV蛋白C端互作;HrcV蛋白C缺失可能影响AvrBs3/pthA蛋白的分泌。黄单胞菌通过hrp基因编码的Ⅲ型分泌系统(typeⅢsecretion system,T3SS)经Hrppilus和HrpF形成的translocon将T3SS效应分子转运至寄主细胞中从而导致寄主产生抗(感)病性.hpa1编码的蛋白Hpa1是一类harpin蛋白,可在非寄主烟草上激发HR,hpa2基因在各类黄单胞菌中高度保守,编码类似溶菌酶的蛋白.在本研究发现,ArpF和hpal基因单突变以及hpa1和hap2基因双突变后Xooc(RS105)仍可在非寄主烟草上产生HR以及在水稻上可形成水渍(water soaking)症状,但是症状不能有效扩展.hrpF和hpa1基因双突变体以及hrpF、hpa1和hpa2的三突变体則丧失了激发烟草产生HR反应和在水稻上形成水渍症状的能力。喷雾接种发现,hrpF突变体不能侵染水稻形成条斑症状,hpa1突变体以及hpa1和hpa2的双突变体能够形成短小的条斑。电镜扫描显示,hpa1和hrpF单突变体均可通过气孔侵染水稻,而hrpF和hpa1双突变体以及hrpF,hpa1和hpa2的三突变体則丧失了在水稻气孔细胞处聚集的能力。扫描电镜显示,hrpF和hpa1单突变以及hpa1和hpa2双突变体均能与水稻细胞互作,但hrpF和hpa1双突变体以及hrpF、hpa1和hpa2的三突变体則丧失了与水稻细胞互作的能力。这提示,hrpF和hpa1基因协同作用是水稻条斑病菌水渍症状形成的主要因素,或者HrpF和Hpa1是两个主要的转运蛋白;hrpF基因是水渍症状扩展的主要因子;hrpF和hpa1基因突变不影响病菌通过气孔侵染水稻,但因突变后影响其它效应分子与水稻细胞的互作从而影响了病害症状的扩展或者与水稻建立营养寄生关系的能力。hrpF和hpa1基因功能的分析将加快Xooc与水稻的互作研究,为从分子水平解析条斑病水渍症状的形成机制奠定了基础。hpaB和hrpE基因XooC在水稻上致病性和烟草上HR反应所必需的。hrpB和hrpE基因突变,則Xooc丧失了在烟草上激发HR的能力和在水稻上的致病性。hpaB和hrpF双突变体仍能使XooC保持在烟草上的HR反应,但丧失了在水稻上的致病性.hrpF、hpaB和hpa1三突变体丧失了激发烟草HR应的能力和在水稻上的致病性。突变体与水稻愈伤细胞的互作结果显示:hpaB突变体以及hrpF和hpaB的双突变体都能够与水稻愈伤细胞互作,hrpF、hpaB和APa1的三突变体以及ArpE突变体丧失了与水稻愈伤细胞互作的能力。扫描电镜的结果显示:hrpE基因突变,Hrp plius装置不能够形成是致病性丧失的主要因素。HpaB可能特异性控制能够引起水渍症状的效应分子的分泌;不依赖HpaB分泌但经址HrpF转运进入植物细胞的某些(个)效应分子能够抑制烟草的HR反应;Hpa1是不经过HrpF进入植物细胞的一类Harpin蛋白。本研究获得了水稻白叶枯病菌PXO99~A菌株的三个hrpF突变体PXF322、PXF410和PXF744。三个突变体通过注射接菌在苗期水稻叶片中仍能够引起水渍状病斑和具有激发烟草产生HR反应的能力。通过剪叶接种成株期水稻结果发现,三个突变体丧失了在水稻叶片上引起白叶枯症状的能力,功能互补子能够恢复突变体在水稻上形成与野生型相似的病斑长度。以上结果证明,Xoo的hrpF是影响水稻白叶枯病菌致病性的关键基因,但是hrpF的突变并不能完全阻止T3SS效应分子与寄主和非寄主植物的互作。更多还原

【Abstract】 Xanthomonas oryzae pv.oryzae(Xoo) and Xanthomonas oryzae pv.oryzicola(Xooc) cause bacterial blight(BB) and bacterial leaf streak(BLS) in rice(Oryza sativa), respectively.BB is one of the most serious diseases in rice,and BLS is emerging in importance in rice.Xoo and Xooc are considered as the model pathogens of rice,which is taken as the model cereal crop.A high throughput and reliable strategy is required to study the function,interregulation,and interplays among repertoire genes in pathogenicity,such as the hrp genes which determine the ability of the bacterium to trigger hypersensitive response(HR) in non-host plants and pathogenicity in host rice.Here we reported a novel target inserted mutagenesis in Xoo and Xooc through single exchange by a vector pK18mobGⅡand knock-out mutagenesis for single and multiple genes in Xooc through two homologous crossover events mediated by a suicide vector pKMS1 containing a sacB gene.Successfully obtain three hrpF and four hrcV inserted mutant with different sites of the strain PXo99~A of Xoo and four hrpF and hrcV inserted mutant with different sites of the strain RS105 of Xooc;and 5 single(hrcV,hrpF,hrpE,hpaB,and hpal),4 double(hpaland hpa2,hpa1 and hrpF,hrpE and hrpF,and hpaB and hrpF),and 2 triple(hpaB,hpa1 and hrpF,and hpa2,hpa1 and hrpF) hrp knock-out mutants of Xooc have been constructed in this report.The results suggest that the established mutagenesis for the target inserted and nonmarker knock-out of single and multiple genes in Xooc mediated by the suicide vector pK18mobGⅡand pKMS1 provides a fundamental approach to the functional genomics of Xanthomonas oryzae and will facilitate the understanding of pathogenicity mechanisms of Xanthomonas oryzae in rice. Evidence indicates that each race of Xanthomonas oryzae contain more than 15 members of avrBs3/pthA family genes.They have almost identical the 5 ’and 3’ terminals. The difference is the repeat number of the 102 bp repeat unit among them.In this study,we use the 359 bp DNA fragment of 3’ terminal of the avrXa3 gene,a member of avrBs3/pthA family genes containing nuclear localization signals and acidic transcription activation domain,as the probe for genomic DNA Southern blot analysis of the Chinese Xoo and Xooc strains.The copies of avrBs3/pthA gene are different in races of these two pathogens and found some bands in all races and some of the bands are specific in certain races. Weaker virulent races contain fewer members of avrBs3/pthA family genes,and high virulent races keep more member of avrBs3/pthA genes with more repeat numbers of the 102 bp repeat unit.Using the same 359 bp fragment as the probe for colony in situ hybridization,we obtained the 35 positive clones from genomic library of Xooc strains RS105,then classified as 28 avrBs3/pthA genes after digestion and Southern blotting analysis.34 positive clones were introduced into PXO99~A strain of Xoo individually.The conjugants of PXO99A with individual avrBs3/pthA genes were inoculated into leaves of rice seedlings and adult rice, respectively,by needless syringes and leaf-clipping methods.We found that PXO99~A containing p6,p8,p9,p10 and p14 positive clones could trigger a specific HR (Hypersensitive response) in the rice eultivar IRBB10 containing a resistance gene Xa10. Sequencing showed that the genes in those positive clones were the members of the avrBs3/pthA family,thus named as avr/pth6,avr/pth8,avr/pth9,avr/pth10 and avr/pth14, respectively.Protein sequence comparison revealed that these five proteins at C-terminals lacked SGSVGGTI residues which are indispensable in AvrBs3/PthA members of Xoo.The flanking sequences comparison revealed that a specific GTTTCTG sequence existed in the promoter region of these avrBs3/pthA genes of Xooc but in that of Xoo.Interestingly,a direct and repeat sequence of a transferable transposon existed in the promoter and terminator regions in the avr/pth14.The BamHI fragment of the avr/pth6 and avr/pth8 genes replacing the corresponding BamHI fragment of the avrXa3 gene resulted in two new fusion genes,avrBre6 and avrBre8.These two fusion genes could retain avirulence to PXO99~A on IRBB10,respectively,suggested that avrBre6 and avrBre8 were the avirulence genes matching Xa10 in rice.This is the first report on avirulence genes in Xooc.The results in this study revealed that Xoo strain exist members of avrBs3/pthA genes,and probably some particular virulence factors inhibit avirulence activities of specific avrBs3/pthA genes in rice.We postulated that might be the reason that rice lacks major resistance gene against bacterial leaf streak.The hrcV gene in plant and animal pathogenic bacteria are highly conservative and the product of hrcV gene is the component protein of typeⅢsecretion system(T3SS), suggesting to form a ring structure in the inner membrane of bacteria.In this study we obtained inserted mutants,PXV268,PXV917,PXV106 and PXV452,in PXO99~A strain and RSV268,RSV917,RSV106 and RSV452 in RS105 strain,respectively,through single homologous exchange method,and a deleted mutant RS105△hrcV through double homologous exchange method.PXV268 and RSV268 mutants still triggered HR on nonhost and retained pathogenicity on rice.The rest of hrcV mutant,PXV917,PXV106, PXV452,RSV917,RSV106,RSV452,lost pathogenicity on Rice and the ability of triggering HR response on nonhost tobacco.The yeast two-hybrid results demonstrated that the HrcV protein interacted with T3SS component proteins,HrcN,HrcJ and HrcR,and T3SS effector,Avr/pth28,AvrXa3 and Avr/pth13,respectively.Truncated HrcV assays displayed that the N-terminal of the HrcV protein interacted with HrcJ and HrcR proteins and the C-terminal did with HrcN.We proposed that the C-terminal deletion in the HrcV protein might affect the secretion of AvrBs3/pthA proteins into rice cells.T3SS effctors were secreted through the typeⅢsecretion system(T3SS) coding by the hrp genes of Xooc and then passed through the Hrp pilus and translocated into plant cells via HrpF translocon encoded by the hrpF gene,leading to resistance or susceptibility in rice.The hpal gene encodes a Harpin-like protein which elicits HR on non-host tobacco and the hpa2 gene is highly conserved in various Xanthomonas species and codes a protein similar to lysozyme.In this study we found that the single mutant of hrpF and hpa1 gene as well as the double mutant of hpa1 and hpa2 still triggered HR on tobacco and caused water soaking lesions on rice leaves but water soaking symptoms did not developed.The double mutant of the hrpF and hpa1 genes and the the triple mutant of the hrpF,hpa1 and hpa2 genes did not trigger HR on tobacco and form water soaking symptoms on rice.We also found that the hrpF mutant did not cause bacterial leaf streak symptom and the single mutant of the hpa1 gene and the double mutant of the hpa1 and hpa2 gene formed shorter leaf streak symptoms after inoculation by spraying the bacteria into the surface of rice leaves.Scanning electron microscope(SEM) revealed that the single mutant of the hpa1 or the hrpF gene infected rice through stomata,but the double mutant of the hpa1 and hrpF genes and the triple mutant of the hrpF,hpa1 and hpa2 genes lost the ability to infect rice through stomata and to multiply around stomata.The SEM also showed that the single mutant of the hrpF or hpalgene,the doubl mutant of the hpa1 and hpa2 genes interacted with rice callus cells,but the double mutant of the hrpF and hpa1 genes and the triple mutant of the hrpF,hpa1 and hpa2 genes lost interacting capabilities with callus cells.The results above suggested that the synergies function of the hpa1 and hrpF gene is the reason for formation of water soaking symptoms caused by Xooc,and possibly HrpF and Hpa1 are the two major translocators for release of plant liquids out of cells.We postulated that the hrpF gene is the key in determining the expansion of water soakin symptoms in rice and the hrpF and hpa1 genes control the bacteria to infect rice through stomata,but mutations in thoses genes would affect interactions of other T3SS effects with rice leading to reduction in development of bacterial leaf streak symptoms and nutrition parasitic relationships between Xooc and rice.Therefore,the elucidation of the function of the hrpF and hpa1 genes surely facilitate our understanding molecular mechanisms of formation of water-soaking symptoms in rice by the pathogen.The hpaB and hrpE genes are necessary for pathogenicity on rice and HR induction on tobacco.Double mutant of the hpaB and hrpF genes retained HR induction on tobacco but lost pathogenicity on Rice.The triple mutant of the hrpF,hpaB and hpa1 genes lost the pathogenicity on rice and HR induction on tobacco.The interactions between those mutants and rice callus showed:the single mutant of the hpaB gene and the double mutant of the hrpF and hpaB gene interacted with rice callus cells,but the triple mutant of the hrpF, hpaB and hpa1 genes and the mutant of the hrpE gene lost the ability to interact with rice callus cells.Scanning electronic microscope results showed that the hrpE mutation made the pathogen lose pathogenicity and not be capable to form Hrp pilus apparatus.Putatively, the HpaB protein specifically controlled the secretion of effectors which cause water soaking symptoms in rice.Some T3SS effectors,inhibiting the HR induction on tobacco by the pathogen,might be secreted in HpaB-independent manner,or translocated in HrpF-dependent manner into plant cells.The Hpa1(Harpin) protein might not be secreted into plant cells through the HrpF translocon.Three hrpF mutant,PXF322.PXF410 and PXF744,of Xoo strain PXO99~A were constructed in this study.The mutants still kept the ability to cause water soaking lesions in rice after they were inoculated into rice seedlings by an injection method and to elicit HR response on tobacco,but lost pathogenicity in adult rice when they were inoculated in rice by leaf-clip method.The hrpF gene could restore the ability of foaming bacterial blight symtoms in rice to the hrpF mutant.These results confirmed that the hrpF gene of Xoo is a critial gene in pathogenicity of the pathogen in rice.However,the mutation in the hrpF gene does not completely block the secretion of T3SS effectors into plant cells.更多还原