【作者】 陈磊；

【导师】 刘凤权；

【作者基本信息】 南京农业大学 ， 植物病理学， 2008， 硕士

【摘要】 水稻黄单胞菌包含两个致病变种Xanthomonas orzae pv.oryzae（Xoo）和X.oryzaepv.oryzicola（Xooc）,在水稻上分别引起白叶枯病和细菌性条斑病。在本研究中,我们鉴定了水稻白叶枯病菌PXO99菌株和水稻条斑病菌RsGD42菌株的双精氨酸运输（Tat）途径,明确了Tat系统对这两种植物病原菌在游动性,趋化性,胞外多糖和毒力方面的影响。（一）根据GeneBank数据库中Xoo KACC10331和MAFF311018的tat基因序列设计引物,采用PCR方法从Xoo PXO99中克隆了tat同源基因,命名为tat_Xoo。通过同源重组的方式成功构建了PXO99菌株tatB和tatC基因的插入突变体,经PCR和Southern杂交验证确认。野生型菌株PXO99细胞在0.3%NA软琼脂平板上具有强游动性。而tatB和tatC基因突变体游动性削弱。而且,tatB和tatC基因突变体细胞缺失了鞭毛,并且在有氧条件下都不能对葡萄糖产生趋化应答。这表明,Tat分泌系统与鞭毛的形成和趋化性应答相关。同时tatB和tatC基因突变体减少了胞外多糖的分泌量。另外,与野生型菌株PXO99相比,tatB和tatC基因突变体都极大削弱了在水稻上的毒力。然而,两种突变体仍然能激发非寄主烟草的过敏性反应。结果显示,Tat系统对水稻白叶枯病菌在水稻上的致病力起重要作用。（二）根据水稻黄单胞菌tat基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌中克隆了tat同源基因,命名为tat_Xooc。通过同源重组构建了RsGD42的tatC基因突变体,经PCR和Southern杂交验证确认。与野生型菌株RsGD42相比,tatC基因突变体并不影响正常生长,并且仍可激发非寄主烟草的过敏性反应。有趣的是,tatC基因突变体缺失鞭毛,极大减弱了游动性。此外,在有氧条件下,该突变体对葡萄糖几乎不产生趋化性应答。明显地,与野生型菌株相比,tatC突变体减少了胞外多糖的分泌量,并且显著削弱了在成熟期水稻上的毒力。结果显示,水稻条斑病菌的Tat系统对其在水稻上的致病力起重要作用。更多还原

【Abstract】 Xanthomonas oryzae pv.oryzae（Xoo） and X.oryzae pv.oryzicola（Xooc） cause leaf blight and leaf streak on rice respectively.In this study,we identified and characterized the contribution of the twin-arginine translocation（Tat） pathway to motility,chemotaxis, extracellular polysaccharide（EPS） and virulence of the phytopathogens X.oryzae pv. oryzae strain PXO99 and X.oryzae pv.oryzicola strain RsGD42.（Ⅰ） A tat homologue,designated tat_Xoo,was cloned from Xanthomonas oryzae pv. oryzae strain PXO99,with degenerated primers by polymerase amplification reaction （PCR）.tatB and tatC disruption mutants of strain PXO99 were successfully generated by a single cross-over event,and confirmed by PCR and Southem blotting.Wild type strain PXO99 cells were highly motile in NYGA 0.3%soft agar medium.In contrast,the tatB and tatC mutation impaired motility.Furthermore,the cells of tatB and tatC mutants lacked detectable flagella and both mutants exhibited almost no chemotaxis toward glucose under aerobic conditions indicating this secretion pathway contributes to flagellar biogenesis and chemotactic responses.It was also obersved that the tatB and tatC mutants exhibited reductive production of extracellular polysaccharide（EPS）.In addition,both tatB and tatC mutants of strain PXO99 had significant reduction of virulence on rice when compared with the wild type.However,tatB and tatC mutants still could elicit the hypersensitive response（HR） in nonhost tobacco.Our findings indicated that the Tat system of X.oryzae pv.oryzae played an important role in virulence on rice.（Ⅱ） A tat homologue,designated tat_Xooc,was cloned from Xanthornonas oryzae pv. oryzicola,with degenerated primers by polymerase amplification reaction（PCR）.A tatC disruption mutant of strain RsGD42 was constructed by a single cross-over event,and confirmed by PCR and Southern blotting.The tatC mutant was not affected in in vitro growth and induction of hypersensitive response（HR） in nonhost tobacco compared to wild type strain RsGD42.Interestingly,the tatC mutant lacked detectable flagella and highly impaired in motility.Furthermore,the tatC mutant displayed almost no chemotaxis toward glucose under aerobic conditions.Remarkably,it was observed that the tatC mutant exhibited reductive production of extracellular polysaccharide（EPS） and significant reduction of virulence on adult rice plants when compared to wild type strain.Our findings indicated that the Tat system of X.oryzae pv.oryzicola played an important role in virulence on rice.更多还原