水稻细菌性条斑病菌中受DSF调控的鞭毛基因flgD、flgE的功能分析  

Analysis of the flgD and flgE genes regulated by diffusible signal factor in Xanthomonas oryzae pv.oryzicola

作　　者：殷芳群[1,2] 赵延存[1,2] 刘春晖[1,2] 钱国良[1,2] 范加勤[1,2] 胡白石[1,2] 刘凤权[1,2] 

机构地区：[1]南京农业大学植物保护学院 [2]农作物生物灾害综合治理教育部重点实验室,南京210095

出　　处：《微生物学报》2011年第7期891-897,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31071657;30971894);国家公益行业(农业)科研专项(3-23)~~

摘　　要：【目的】为了阐明可扩散性信号分子(diffusible signal factor,DSF)调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)Rs105的致病性等方面的影响。【方法】采用PCR的方法克隆靶标基因flgDxoc和flgExoc,用同源重组法构建缺失突变体,测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型,用反转录PCR(RT-PCR)的方法验证Rs105和ΔrpfFxoc(rpfFxoc基因的缺失突变体,不产生DSF)中flgDxoc、flgExoc表达量的差异。【结果】从Rs105基因组中克隆到flgDxoc、flgExoc基因,并证实这两个基因在基因组中均为单拷贝。PCR和Southern杂交结果显示,flgDxoc、flgExoc基因被成功敲除。与野生型相比,突变体的鞭毛产生能力丧失,游动性和趋化性能力减弱,接种水稻叶片显示其致病性部分减弱,基因互补可使其恢复。生长能力和对烟草叶片的致敏性无明显改变。RT-PCR结果显示,flgDxoc、flgExoc基因在ΔrpfFxoc中的转录水平明显降低。【结论】本试验表明:FlgD、FlgE是水稻细菌性条斑病菌鞭毛形成所必需的因子;进一步证明了DSF通过调控flgDxoc、flgExoc基因表达,从而影响条斑病菌的致病性等表型。为深入认识DSF对细菌性条斑病菌鞭毛基因簇的调控提供了科学依据。[Objective] To investigate functions of flgDxoc and flgExoc genes regulated by diffusible signal factor（DSF） in Xanthomonas oryzae pv.oryzicola（Xoc）Rs105.[Methods] The flgDxoc and flgExoc genes were amplified by PCR.We constructed ΔflgDxoc and ΔflgExoc,the deletion mutants from Rs105 by using double crossover method,and determined cell morphology,motility,pathogenicity in host rice and hypersensitive response（HR） in nonhost tobacco.We tested the differential expression of flgDxoc and flgExoc gene by reverse transcriptional polymerase chain reaction（RT-PCR） between the wide type and ΔrpfFxoc（the deletion mutant of rpfFxoc gene,which could not produce DSF）.[Results] We cloned flgDxoc and flgExoc from genomic DNA of Rs105.PCR and Southern blot analysis demonstrated that the flgDxoc and flgExoc genes were knocked out successfully.Both mutants were non-flagellated and significantly attenuated motility on the 0.3% semi-solid medium.The pathogenicity on rice were obviously attenuated in ΔflgDxoc and ΔflgExoc compared to the wild type.All the changes in mutant could be restored through the complementation.However,there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant（ΔflgDxoc or ΔflgExoc） and the wild type.In addition,the results of RT-PCR demonstrated that the transcription level of flgDxoc and flgExoc were down-regulated in ΔrpfFxoc.[Conclusion] This study showed that expressions of flgDxoc and flgExoc were positively regulated by DSF,and necessary for flagellar hook assembly and flagellar structure in Xoc.Meanwhile,FlgD and FlgE contributed to pathogen＇s virulence,motility and chemotaxis,but no differences at growth rate in MMX medium and HR in nonhost.In addition,our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen＇s virulence might be,at least partially,dependent on bacterial flagellar in Xoc.

关 键 词：水稻细菌性条斑病菌 鞭毛 flgD flgE DSF 

分 类 号：S435.111[农业科学—农业昆虫与害虫防治]