【作者】 武晓敏；

【导师】 陈功友；

【作者基本信息】 南京农业大学 ， 植物病理学， 2006， 硕士

【摘要】 稻黄单胞菌种下的水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae，Xoo)和水稻细菌性条斑病菌(X.oryzae pv.oryzicola,Xooc)，分别引起水稻白叶枯病和水稻条斑病，在生产上造成较大危害。水稻黄单胞菌—水稻互作系统是研究植物—病原物互作的理想模式系统。白叶枯病菌与水稻互作符合基因对基因关系，但基因-基因互作特异性的本质并不十分不明确。本研究以avrBs3基因家族中高度保守的酸性转录激活域的359 bp EcoRI-BamHI DNA片断为探针，扫描日本系统Ⅲ小种JXOⅢ和菲律宾系统6号小种PXO99~A菌株基因组，发现JXOⅢ菌株特有5.0 kb和1.8 kb的avrBs3／PthA家族基因。菌落原位杂交JXOⅢ菌株的基因文库，经限制性酶切分析和Southern杂交归类，获得了共含有17个avrBs3／PthA家族基因的7个克隆，众多avrBs3／PthA基因在基因组中单独存在，或者2个和2个以上串联排列。将获得的阳性克隆导入PXO99~A菌株中，成株期致病性测定结果显示，JXOⅢ菌株中可能存在与Xa2、Xa3、xa5、xa8等匹配的avr基因，p54克隆中含有1.8 kb的avrBs3／PthA基因，在IBBB3水稻品种上显示为无毒性。比对avrBs3家族无毒基因重复单元中的12-13位氨基酸残基，初步揭示Xoo基因组中avr／pth基因的重组特征。本研究还提示，作为水稻近等基因系轮回亲本的IR24，可能存在多个R基因。avrBs3／PthA家族基因的获得以及与水稻互作的对应关系为加快水稻抗白叶枯病R基因克隆提供了科学线索。水稻黄单胞菌的avrBs2、pga和popC基因的启动子区域存在PIP-box，推测其表达受HrpX调控，然而其在致病性中的作用还不清楚。本研究根据植物病原黄单胞菌中报道的相应基因序列设计兼并引物，PCR方法从水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae, Xoo)和水稻条斑病菌(X.oryzae pv.oryzicola, Xooc)基因组中分别克隆了avrBs2、pga和popC基因。序列比较发现，从Xoo中克隆的该3个基因与已知基因组测序的菌株中的基因相同。从Xooc中克隆该3个基因尚属首次。蛋白质水平上比较发现，水稻黄单胞菌中的该3个蛋白与其它黄单胞菌中的相应蛋白具有74％—98％的同源性。依据同源交换原理，本研究分别对水稻黄单胞菌的该3个基因进行了突变。水稻苗期water soaking、成株期致病性和非寄主烟草过敏反应测定发现，avrBs2、pga和popC突变体与野生菌一样，并不改变在水稻上的water soaking症状和在烟草上激发过敏反应。有意义的是，Xooc的pga突变体在水稻中的繁殖能力和致病性得到了加强。这提示，编码多聚半乳糖醛酸酶的pga基因可能是致病性的抑制因子。更多还原

【Abstract】 The gram-negative plant-pathogenic bacteria Xanthomonas oryzae pv. oryzae （Xoo）and Xanthomonas oryzicola pv. oryzicola （Xooc） are the causative agents of bacterialblight and bacteria leaf streak in rice, respectively, which lead to economicallyimportant yield losses. These pathosystems have become well-established models forunderstading molecular interactions of plant-pathogens.The interactions between Xanthomonas oryzae pv.oryzae and rice are controlledin a gene-for-gene manner. However, the basis of the gene-for-gene specificity is notwell understood. In this study, a DNA fragment （359 bp） of the avrXa3 genecontaining three nuclear localization signal （NLS） motifs present in all members ofavrBs3/pthA family was used as a probe to screen the genomic library of JXOⅢstrainof Xoo. Our results demonstrated that diverse members of the family existed in thepathogen genome. The avrBs3/PthA （avr/pth） genes could individually or collectivelyreside in one locus. The positive avr gene clones were transferred into the compatiblerecipient PXO99^A. Pathogenicity tests in near isogenic lines of rice confirmed thatthere were four R genes （Xa2, Xa3, xa5 and xa8） matching the four avr genes （avrXa2,avrXa3, avrxa5 and avrxa8） genes in the genome of Xoo strain JXOⅢ. TheavrBs3/PthA gene （1.7 kb） present in p54 could be an avirulence gene specificallyinteracting with Xa3 gene present in IRBB3, hereby nominated as avr/pth3. Thesequence indicated that there are only 1.5 repeat numbers of the 102 bp repeat unit inavr/pth3. Alignments of 12^th-13^th amino acids together in the repetitive regionsencoded by this gene with those by other representatives in the AvrBs3 familyrevealed unique repeat arrangement which might reflect varieties of avirulence genesin Xoo. Furthermore, the recurrent parent rice line IR24 could contain several R genesresistant to Xoo bacterial blight. Some avr/pth genes and their functions identified inthis study shed new light on the mechanisms of gene for gene specificity in the Xoo-rice system, which will be definitely useful for further identifications of unknownR genes in rice against the bacterial blight.avrBs2、pga and popC in Xanthomonas oryzae contain a conserved sequencemotif in their promoter region （plant-inducible promoter, PIP-box,TTCGC-N15-TTCGC）. It is speculated that HrpX controls their expression bybinding to this promoter element. However, the function of these three genes inpathogenicity is not being understood, avrBs2、pga and popC genes were PCR-generated by using the degenerated primers which were designed on the basic ofknown relative genes in other xanthomonads. Sequencing confirmed that the genesfrom Xoo in this study were as same as those reported in the genome sequence ofMAFF311018. avrBs2、pga and popC genes were first confirmed existing in Xooc.Comparison of the putative proteins with those of xanthomonads loaded in thedatabase of the NCBI revealed that the proteins of X. oryzae had 74%-98% identitiesto those in other Xanthomonas species. Based on the strategy for mutation byhomology recombination, the avrBs2, pga and popC mutants of two pathovars ofX.oryzae were generated and the single cross event in mutation was confirmed byPCR and Southern hybridization. The ability to cause water soaking symptoms in riceseedlings, the lesion lengths in adult rice, and the induction of hypersensitive responsein tobacco by those mutants were tested individually and respectively. The resultsdemonstrated that the mutation of the avrBs2, pga and popC genes, either in Xoo or inXooc, did not lost the ability to induce hypersensitive response in nonhost tobaccoand kept the same lesion lengths in rice as their parental wild strains. However, themutation in the avrBs2 gene reduced the ability to form water soaking symptoms indee seedlings compared with those by the wild type strains of Xoo and Xooc.Interestingly, the growing ability of and the water soaking symptom by the pgamutant of Xooc in rice were enhanced when compared with those by the wild typestrains. It is suggested that the polygalacturonase encoded by the pga gene of Xoocwas of the inhibitor for the innate immunity in rice.更多还原