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TheRoleofRNASilencinginSatelliteRNA．mediatedAttenuationofVirusSymptomsinPlantsADissertationSubmittedtothecommitteeofSouthwestUniversityinPartialFulfillmentoftheRequirementfortheDegreeofDoctorofPhilosophyinPlantPathologyByPh．D．Candidate：WanxiaShenSuperVisor：Prof．ChangyongZhouCo—supervisor：Prof．Ming-boWangMajor：PlantPathologyField：MolecularPlantPathologySouthwestUniversity，Chongqing，P．R．ChinaMay，2013 独创性声明学位论文题目：擅堑痘盎里垦丛叁王拉基塑助痘壹塾痘性鲍金量扭弹本人提交的学位论文是在导师指导下进行的研究工作及取得的研究成果。论文中引用他人已经发表或出版过的研究成果，文中已加了特别标注。对本研究及学位论文撰写曾做出贡献的老师、朋友、同仁在文中作了明确说明并表示衷心感谢。学雠文慨＼中雌签字魄≯纠多年占月“学位论文版权使用授权书本学位论文作者完全了解西南大学有关保留、使用学位论文的规定，有权保留并向国家有关部门或机构送交论文的复印件和磁盘，允许论文被查阅和借阅。本人授权西南大学研究生院(筹)可以将学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫描等复制手段保存、汇编学位论文。(保密的学位论文在解密后适用本授权书，本论文：口不保密，口保密期限至年月止)。学位论文作者张哼哗签字日期：2p，弓年Z月易日导师张州．签字日期：知呜年占月‘日 谨以此论文献给我的恩师Jl常勇研究员和王明波研究员及我的家人 本研究承蒙国家留学基金委(2010699015)教育部创新团队发展计划(IRT0976)农业部行业公益专项(201203076)国家科技支撑计划专项(2007BAD47803)资助Thestudywassupportedinpartby●一一一ChinaScholarshipCouncil(2010699015)ProgramforChangjiangScholarsandInnovativeResearchTeamUniversity(IRT0976)MONsPublicBenefitResearchFoundationofChina(201203076)andNationalKeyTechnologyR&DProgram(2007BAD47803) ContentsAbstract⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯I中文摘要⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．VIIChapter1Literaturereview⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯．111．1Virusandvirussymptoms⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．111．2Smallparasitesofplantviruses⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯．121．2．1PlantsatRNAs⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯131．2．2Plantsatellitevirlls⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．141．2．3DIRNAs⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．141．2．4TherelationshipbetweenthesatRNAandtheplantvirus．⋯．⋯⋯⋯．⋯⋯⋯⋯⋯．⋯⋯⋯．151．2．5ThetraditionalexplanationofsatRNA—mediatedsymptomattenuation⋯⋯⋯．．．⋯⋯．．181．3RNAsilencing⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯．191．3．1RNAsilencingpathwaysinplants．⋯．⋯⋯．．．．．．⋯．．．．．．．．．．．．．．⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯．．．⋯⋯．．．201．3．2DistinctionsbetweenmiRNAsandsiRNAs．⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯。241．3．3AGOs⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．251．4RNAsilencingandplant—virusinteractions⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．261．4．1ViralcounterdefencemechanismsagainstRNAsilencing⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯271．4．2RNAsilencingandviraldiseasedevelopment⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．331．5Hypothesisandexperimentalstrategies．．．．．．．．．．．．⋯⋯⋯⋯⋯⋯⋯⋯⋯．．．，．．，，，⋯⋯⋯⋯⋯。。⋯⋯．⋯．361：5．1Hypothesis⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．361．5．2Experimentalstrategies⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯。⋯⋯⋯．．371．6Experimentsystem⋯⋯．⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．391．6．ICMVandCMVsatRNA⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．391．6．2j、【benthamianaandⅣtabacum⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．421．6．3TheP19andHcPro⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯．．．⋯⋯⋯⋯．⋯⋯．．．．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯．⋯⋯．42Chapter2Introduction⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．2I：；Chapter3Materialsandmethods⋯⋯⋯⋯⋯⋯⋯⋯．．．⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯．．⋯．⋯．⋯⋯⋯⋯⋯⋯⋯⋯．451I．1Plantmaterialsandgrowthconditions⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯：．．453．2RNAextraction⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．453．3DNasetreatmentofRNAsample⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯453．4Semi—quantitativeRT—PCR⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．46 3．5Ball-beatingDNAextractionandPCR⋯⋯⋯⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯463．6CloningandsequencingofPCRfragmentsforuseashybridizationprobes⋯⋯⋯⋯⋯⋯．．473．6．1Gel-purification⋯⋯．．．⋯⋯．．．．⋯⋯．．．⋯．．．．．⋯⋯．．．．⋯⋯．．．⋯⋯．⋯⋯⋯．．．．⋯⋯．．．．．．．⋯⋯．．．．．⋯⋯．473．6．2Ligation⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．⋯⋯．．．．⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯．⋯473．6．3Preparationofcompetentcells．．⋯．．⋯⋯⋯⋯．．．．．．，⋯．．．．．．．．．．⋯．⋯．．⋯⋯⋯．⋯⋯．⋯．⋯⋯⋯⋯．．473．6．4TransfolrmationofE．coli⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．483．6。5RecombinantplasmidDNAextraction．．⋯⋯．．．．，⋯⋯．．．．．．．⋯．．．．．．．．．⋯⋯．．．．．⋯⋯．．．．．．．．．⋯⋯493．6．6Insertdetectionandsequencing⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯503．7PlantexpressionvectorsandAgrobacteriumstrains⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯503．8Nicotianatransformation．⋯⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯．．．．⋯⋯⋯．．⋯⋯⋯．．⋯⋯⋯⋯．．⋯⋯⋯．．．．．⋯⋯503．8．1Constructs⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯．．503．8．2Triparentalmating．⋯⋯⋯．．．⋯⋯⋯．．⋯⋯⋯⋯⋯⋯．．．．⋯⋯⋯．．，，⋯⋯．．．．⋯⋯⋯⋯⋯⋯⋯⋯．．．．⋯．．513．8．3AnalysisofAgrobacteriumtransformantsbydiagnosticrestrictionenzymedigestion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯5l3．8．4LeafdiSCtransformation⋯⋯⋯．．．⋯⋯．．．⋯⋯．．．．．⋯⋯．．．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯．．523．9Virusinoculation⋯⋯⋯⋯⋯⋯．．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯．523．10Q．CMVstrain⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．523．11Q．CMVY．Satinoculurn⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．533．12Agrobacteriuminfiltrationassay，．．⋯．．．．⋯⋯⋯．⋯⋯⋯⋯⋯⋯．．．．⋯．．．．．．．⋯⋯．．．．．．．．，．⋯．．．．．．．．⋯⋯533．13GUShistochemicalstaining⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．533．14GUSMUGassayanalysis⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯一543．15Northernblothybridization⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯：．．553．15．1RNAgelelectrophoresis⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯553．15．2Hybridization⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯563．16SmallRNANorthernblothybridization⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．573．16．1SmallRNANorthernblot⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．573．16．2Hybridization⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯583．17RNAimmunoprecipitation⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．593．18Westernblot．．．．．．⋯⋯．．⋯⋯⋯．．．⋯⋯．．．⋯⋯．．．．．⋯⋯．⋯⋯．．⋯⋯．．．．．．⋯．．．．．．⋯⋯．．．．⋯⋯．⋯⋯⋯．．．⋯．．60Chapter4VSR-mediatedinhibitionofGUSsilencingcanbereversedbyY—Satinfection⋯．．．．614．1Introduction．⋯⋯．．．．，⋯⋯．．．．．⋯．⋯．．．⋯⋯．．．．⋯⋯．，⋯⋯．．．．．．．⋯．．．．．．．．⋯．．．．⋯⋯⋯．．⋯⋯．．．⋯⋯．．．．．．⋯．．614．2Theexperimentsystem⋯⋯⋯．⋯⋯⋯．．⋯⋯⋯．．⋯⋯．．⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯624．3Results．．⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯，．⋯．⋯．．．．⋯．．．．．．⋯⋯⋯．⋯⋯，．．．⋯⋯．．．．．，⋯．．．．．⋯．．．．．．．⋯⋯⋯．．62 4．3．1P19inhibitsGUSsilencinginuninfectedⅣbenthamiana⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯624．3．2Y-SatinfectionreversestheP19-mediatedsuppressionofGUSsilencing⋯⋯⋯⋯⋯．664．3．3Y-SatinfectionaffectsCMV2b-mediatedsuppressionofhpGUS—inducedsilencing⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯734．4Discussionandconclusion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．774．4．1Discussion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯774．4．2Conclusion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．78Chapter5TheinterferenceofVSRisduetosequestrationofVSRproteinbyY—SatsiRNAs．．795．tIntroduction⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯．．．．．⋯⋯．．．．⋯．．⋯．⋯⋯．．．⋯⋯．⋯⋯．⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯．795．2Results⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．805．2．1StrongerGUSsilencinginthepresenceofY-SatisnotduetOanincreaseinhpGUSsiRNAaccumulation⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．805．2．2Y．Sat．derivedsiRN触bindtoP19toinhibititsfunction⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯835．3Discussionandconclusion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯．．．．⋯⋯．．865．3．1Discussion．⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．865．3．2Conclusion⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．⋯⋯．⋯⋯⋯．⋯⋯⋯．87Chapter6Y·SatinfectionreversestheeffectsofhelpervirusVSRonmiRl68expression⋯⋯896．1Introduction．⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．896．2Results⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯．．．⋯．．⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．906．2．1VirusinfectionandsymptomsinⅣbenthamiana⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．906．2。2miR68expressioninplantsinfectedwithsubgroupⅡCM-V⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯916．2．3RNA4alevelmQ．CMV．infectedⅣbenthamianaplants⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯926．2．4miRl68expressioninN．benthamianaplantsinfectedwithsubgroupICMV⋯⋯⋯．936．2．5miRl68expressioninP1／HcProtransgenicⅣtabacumplants⋯⋯⋯⋯⋯⋯⋯⋯．946．3Discussionandconclusion⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．976．3．1Discussion⋯⋯⋯．．．．⋯．．．．．⋯．．．．．．⋯．．．．．．⋯⋯．．．⋯．⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．976．3．2Conclusion⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯．．99Chapter7Generaldiscusfionandconclusion⋯⋯⋯⋯⋯⋯⋯⋯⋯。⋯．．．．。。⋯．．．．⋯．．．⋯．．⋯⋯⋯．⋯⋯．．．．1017．1Generaldiscussion⋯⋯．．⋯⋯．．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．．．．．．⋯．．．．．⋯．．⋯．⋯．．．．．⋯⋯．．⋯．．1017．1．1satRNAsinterferewiththesuppressorfunctionofVSRsonhostsmallRNA—directedsilencing⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯．．⋯⋯⋯．．⋯⋯．⋯．⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯。1017．1．2Y-Sat．mediatedsymptomattenuationisbyreducingVSRcausedinterferingwithmiRNA⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯．．⋯⋯⋯．⋯⋯．⋯⋯⋯⋯⋯⋯102 7．2Conclusion⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．1047．3Futureprospectives．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯·106Appendix1SequencesofprobesusedinNorthernblothybridization⋯．．．．，⋯⋯．．．．⋯．⋯⋯·．．⋯···129Appendix2Abbreviationsofvirus⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯131Appendix3Abbreviations⋯⋯⋯⋯⋯⋯⋯⋯．⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯。。133PublicatioIls⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．135Acknowledgements⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．137附录I全文概要⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯．．141 AbstractTheRoleofRNASilencinginSatelliteRNA··mediatedAttenuationofVirusSymptomsinPlantsMRjor：PlantPathologyDoctoralCandidate：WanxiaShenSupervisors：Prof．ChangyongZhouandProf．Ming-boWangPlantRNAvirusesareoftenassociatedWithsubviralRNAagentsknownassatelliteRNAs(satRNAs)，whicharesmallnon-codingRNAsandsharelittleornosequencehomologywiththeirassociatedvims(helpervirus)．ThegenomesofsatRNAsarearound200—1500nucleotides(nt)，whichdependontheirhelpervirusesforreplication，encapsidation，systemicmovementandtransmission．ThemajontyofsatRNAsattenuatesymptomsinducedbythehelperviruses，whileonlyafewsatRNAshavebeenshowntoexacerbatediseasephenotypesbyinducingtheirownsymptomsininfectedhostplants．However，howsatRNAsaRenuatesymptomsremainsunclear．IthasbeenproposedthatsatRNAsmayreducehelperviresaccumulationbycompetingforRNAreplicaseorincreasingantiviralsilencingagainstthehelpervirus，therebyreducingsymptomsinthehostplant．However,symptomreductionbysatRNAsisoftennotassociatedwithacorrespondingreductioninhelpervirusaccumulation．Thissuggeststhatsomeotherfactorsormechanismsmaybeinvolved．RNAsilencingisasequence-specificRNAdegradationprocessinducedbydouble-strandedRNA(dsRNA)orself-complementaryhair；linRNA(hpRNA)．ThisdsRNAorhpRNAisprocessedbyDicer，anRNaseIII-likeendoribonuclease，togeneratesmallRNAs(sRNAs)，whichaleloadedontoArgonaute(AGO)proteinstoformtheRNA-inducedsilencingcomplex(RISC)．RISCisguidedbythesesRNAstobindanddegradecognatemRNAsorothersingle—strandedRNAs．RNAsilencinginplantshasbeenwellestablishedasananti-viraldefencemechanism．Viralinfectionisassociatedwiththeaccumulationofvirus-derivedsmallinterferingRNAs(siRNAs)，whichinturndirectthede酉adationofviralgenomeRNAs．Toovercomethishost SouthwestUniversityPhDThesisdefencemechanism，viruseshaveevolvedacounter-defensestrategybyencodingsuppressorsofRNAsilencing(VSRs)．ApredominantmodeofactionbyVS凡istobinddouble·strandedsRNAs，therebypreventingtheformationofRISC，whichisessentialforthesilencingofviralgenomesbythehost．VSRsarekeysymptomdeterminantsofplantviruses，possiblybecause也eyinterferewiththehostsRNApathwaysespeciallythemicroRNA(miRNA)pathway．miRNAsplaycriticalrolesinsuchprocessingascelldivision，leafformationandflowerdevelopmentinplants．VSRsaffecttheexpressionandfunctionofmiRNAs，therebyhavethepotentialtocausedevelopmentalabnormalitiesandhencedisease—associatedsymptoms．AtypicalexampleistheenhancedaccumulationofmiRl68causedbyviralinfectionthroughthefunctionofVSRs．111emiRl68negativelyregulatestheexpressionofAGO1．akeycomponentofRISC．neinductionofmiRl68reducestheexpressionofAGO1protein，whichCansubsequentlyinterferewithnormalplantdevelopmentanddecreaseantiviralImAsilencing，bothofwhichCanincreaseviraldiseasesymptoms．ArecentlyobservedcharacteristicofsatRNAsisthattheirreplicationisassociatedwi也extremelyhighamountofsatRNA-derivedsiRNAs(sat-siRNAs)．Thischaracteristic，togetherwiththeevidencethatVSRsinterferewithhostsmallRNAfunctionandhenceplantdevelopment，showsthatsat-siRNAsandVSRsmaybeinvolvedinthesymptomattenuationmechanism．Inthisthesis，aRNAsilencing-basedhypothesisforsatRNA-mediatedsymptomaaenuationwasproposedandvalidated．ItisbasedonthatsymptomreductionbysatRNAsisduetosequestrationofVSRsbysat-siRNAs，whichminimizestheVSR’SinterferencewithhostmiRNAfunction．Firstly,whethersatRNAinfectionwouldminimizetheeffectofVSRsonhostsiRNA-directedgenesilencingwastested．Usingap—glucuronidase(GUS)reportgenesysteminconjunctionwithAgrobacterium—infiltration(Agro-infiltration)assays，theinteractionbetweentwoVSRs(P19and2b)andY-satelliteRNAⅣ一Sat)ofCucumbermosaicvirus(CMV)wasexamined．Tofacilitatetheexperiments，asatRNA-freeCMVisolate(Q-CMV)andtransgenicNicotiana．benthamianaplantsresistanttoY—Sat-inducedyellowingsymptomsweredeveloped．Resultsshowedthati1GUSexpressionWaseffectivelysilencedbyahpmqAconstruct(hpGUS)butthissilencingwassuppressedbyCO·-Agro·-infiltrationwithP19or2bconstructs；ii)UponY·-Satinfection，stronghpGUS-inducedGUSsilencingoccurreddespitethepresenceofP19 Abstractor2b，indicatingthatthefunctionoftheVSRswasinterferedWi也byY—Satinfection；iii)Agro—infiltratedGUSunderwentsenseCO—suppression，whichWaSinhibitedbyP19or2bCO-infiltration,butuponY-Satinfection，theCO—suppressionwasenhanceddespitethepresenceofP19or2b，againindicatingthatthefunctionoftheVSRswasinterferedwithbyY—Satinfection．Takentogether，theseresultsindicatedthatsatRNAinfectioninterfereswiththefunctionofVSRsandreducestheirsuppressoreffectonsmallRNA-inducedsilencingofthehost．ThisisconsistentwiththehypothesisthatsatRNAsreducehelpervirus--causedsymptomsbyminimizingtheeffectofhelpervirus--encodedVSRsonhostmiRNAorsiRNA·mediatedgeneregulation．InordertoruleoutthepossibilityiftheincreasedGUSsilencingbyY—SatinfectioninthepresenceofVSRswasduetoincreasedamountsofGUSsiRNAs，theeffectofY-SatinfectionontheaccumulationofhpGUS—derivedsiRNAaccumulationWasconductedbyNorthernblothybridization．ResultsshowedthatY—SatinfectiondidnotaffecttheprocessingofhpGUSRNAortheaccumulationofhpGUSsiI汛A，whichindicatedthattheeffectofY·SatinfectiononGUSsilencingwasduetointerferencewitlltheVSRfunction．TodemonstratethatsatRNA-derivedsiRNAsindeedbindVS黜andsequestertheVSRsfrominterferingwitllhostsRNAfunction，RNA—immunoprecipitationwasdeployedbyusingP19antibodiestoexaminethesiRNAsassociatedwithP19inthepresenceorabsenceofY-Satinfection．ItshowedthatY-SatinfectionreducedtheamountofhpGUS-derivedsiRNAboundtotheP19VSR，andinstead，P19wasboundwithabundantY．Sat-deftvedsiRNAS．ThisresultindicatedthattheincreasedhpGUS·-inducedsilencingbyY--SatinfectioninthepresenceofVSRswasduetosaturationoftheVS风byY—SatsiRNAs，releasingthehpGUS-derivedsiRNAsavailablefordirectingGUSsilencing．TheseresultssupportedthehypothesisthatsatRNAsreducetheeffectofhelpervirus-encodedVSRsonhostsRNA-mediatedgeneregulationbysequesteringtheVSRswithY-SatsiRNAs．AnultimatedemonstrationofthehypothesisistoshowthattheinterferenceofhostmiRNAorsiRNAfunctionissolelyorpredominantlyresponsibleforhelpervirus-inducedsymptoms，andthisinterferenceofhostsRNAfunctionisdiminisheduponsatRNAsinfection．Forthispurpose，theeffectofY-SatinfectionontheaccumulationofmiRl68wasexamined．miRl68isaprimaryregulatorinthehost SouthwestUniversiWPhDlhesismiPmAandsilVApathwaysandakeyindicatorofVSRfunction．TheresultsshowedthatinfectionofⅣbenthamianabybothsubgroupIffny-CMV、andsubgroupII(Q-CMV)CMVstrainsinducedmRl68accumulation，andthisinductionwasdownregulatedinthepresenceofY—Sat．TheseresultsindicatedthatY-SatreducesthehelpervirusCMV2b—causedinductionofmiRl68．Interestingly，thelevelofmiRl68inductionwascorrelatedwiththeseverityofCMV-inducedsymptoms，whichimpliesthattheinterferenceofhostsRNApathwaysbyVSRsareindeedresponsibleforvirus—causedsymptoms，andY-Sat-mediatedsymptomaaenuationisindeedduetominimizedinterferenceofhostsRNApathwaysbyVSRs．InadditiontOCMV-infectedⅣbenthamianaplants，theeffectofY—SatinfectionOilmiRl68accumulationinP1／HcPro(VSRofTobaccoetchvirus．TEV)transgenicⅣtabacumplantswerealsoinvestigated．TheresultsshowedthatP1／HcProstronglyinducedmiRl68expression，andthisinductionWaslargelyreverseduponCMVY-Satinfection．Moreover，thetiltedphenotypeassociatedwithtransgenicP1／HcProplantswasalsolargelycorrectedbyCMVY-Satinfection．TheseresultssuggestedthatsatRNAinfectiongenerallyminimizestheVSR—causedinterferenceofhostmiRNAandsiRNAfunctioninplants，andthisdiminishedeffectonhostsRNAfunctionaccountsforreducedsymptomscausedbyhelperviruses．Insummary，thisthesisprovidesseveralpiecesofexperimentalevidencetodemonstrateanRNAsilencing-basedhypothesisproposedtoanswersuchalongstandingquestionashowviralsatRNAsattenuatediseasesymptomscausedbytheirhelpervirusesinplants：i、Agrobacteriuminfiltrationofviral—infectedⅣbenthamianaleavesshowedthattheTombusvirus．encodedP19VSRandtheCMV-encoded2bVSRbotheffectivelysuppressedhpRNA-inducedsilencing，butthissuppressionwasreleaseduponinfectionwiththeCMvY—Sat；ii、RNAimmunoprecipitationshowedthat，inthepresenceofY—Sat，P19wassaturatedwithY—Sat-derivedsiRNAs，resultinginreducedamountofhpRNA—derivedGUSsiRNAsboundtoP19；iii、NorthemblothybridizationanalysesofCMV—infectedⅣbenthamianaplantsorP1／HcPro-expressingtransgenicⅣtabacumplantsshowedthatY—SatinfectionminimizedtheinductionofmiRl68expressioncausedeitherbyCMVinfectionorbyoverexpressionofP1／HcProVSR．Takentogether，theseresultssupportthehypothesisthatsatRNAsattenuatesymptomsbysequesteringhelpervirus-encodedV AbstraetVSRsthroughabundantsatRNA—derivedsiRNAs，preventingtheVSRsfrominterferingwithhostsiRNAormiRNA-directedgeneregulationandhenceimprovingplantdevelopment．Keywords：virus，satelliteRNA，symptomattenuation，RNAsilencing，smallRNA，viralsuppressorofRNAsilencingV 中文摘要植物病毒卫星RNA干扰其辅助病毒致病性的分子机理植物病理学专业博士研究生申晚霞指导教师周常勇研究员王明波研究员植物RNA病毒常常伴随有小的卫星RNA。卫星RNA是一类小的非编码RNA，基因组大小为200．1500nt，通常不编码蛋白，完全依赖于辅助病毒来完成复制、包被、移动和传播，且和其辅助病毒的基因组不存在序列同源性。部分卫星RNA可以影响辅助病毒在寄主植物上诱发的症状，多数为减轻，少数会加重寄主症状。传统理论认为卫星RNA是通过与辅助病毒竞争复制酶，减少了辅助病毒的复制，从而减轻了寄主症状。然而，传统理论不能解释卫星砌妊．在减轻辅助病毒诱导症状的同时，部分辅助病毒复制量不受影响的现象。由此推测，卫星RNA可能存在其它途径来减轻辅助病毒诱导的症状。RNA沉默(或称为I矾Ai)是一种广泛存在于真核生物中，由小RNA介导的、以序列特异性的方式抑制或破坏靶标基因表达，在DNA或转录水平上的调控机制。RNA沉默可以调控基因的表达、修饰DNA和特异染色体区域组蛋白的表观遗传、防御转座子和病毒等核酸的入侵。病毒为了成功侵染寄主，也进化了相应的反防卫机制。大部分是通过编码基因沉默抑制子(viralsuppressorofRNAsilencing，VSR)蛋白结合病毒的siRNA，阻止RNA沉默复合体(RNA．inducedsilencingcomplex，RJSC)的形成，抑制病毒基因组的降解。基于RNA沉默具有调控植物正常生长发育和防御病毒的功能、VSR具有干扰RNA沉默反防卫机制、多数卫星RNA在复制过程中伴随着巨量的卫星RNAsiRNA产生的三个依据，本文提出了以下假说来解释卫星RNA减轻病毒对寄主植物所诱导的症状。1)病毒通过其产生的VSR干扰寄主植物的miRNA代谢途径，从而致病；2)卫星RNA通过其产生的巨量siRNA捕获并饱和大部分的VSR，减少VSR干扰寄主植物的miRNA代谢途径，减轻病毒对寄主植物诱导的症状。本文通过对烟草、辅助病毒CMV和Y．卫星RNA(Y．SatelliteRNA，Y．Sat)组成的模式系统进行研究，验证了以上假说。模式系统选用CMV亚组Ⅱ的病毒株Q．CMV作为负对照。Y．Sat被选用于本研究，是因为卫星RNA的研究中以对 SouthwestUniversityPhDhesisCMV卫星RNA研究最多，而Y．Sat在CMV卫星RNA中具有代表性，可以导致本氏烟和普通烟等的斑驳黄花，方便了对病毒侵染过程的检测。验证实验分为两个方面，1)应用GUS报告基因监测在有无卫星RNA伴随侵染时P19和2b两种VSR的活性；2)应用miRl68表达量易受VSR诱导的特性，监测在有无卫星RNA伴随侵染时VSR对miRl68的影响。验证实验获得了以下几个方面的结果：1．hpGUS可以有效的沉默GUS，而TBSV的P19和SD．CMV编码的2b两个VSR构件都可以干扰hpGUS介导的GUS沉默。实验采用农杆菌浸润法将GUS+hpGUS、GUS+hpGUS+P19或2b等构件转入本氏烟中进行瞬时表达，比较有无VSR共浸润时GUS蛋白量的变化。并通过GUS染色和GUS的相对酶活性测定分析了P19和2b的活性。实验成功模拟了植物内源的小RNA代谢途径，检验了P19和2b对hpGUS介导的GUS沉默的影响；实验结果与以前报道的VSR可以干扰hpmqA介导RNA沉默的实验结果相吻合。2．Y．Sat影响了VSR干扰RNA沉默的功能。在野生型本氏烟上进行Q．CMV和Q．CMV+Y．Sat感染实验，然后瞬时表达GUS、hpGUS和P19或2b等构件。在只有Q．CMV感染时，P19和2b都可以干扰hpGUS介导的GUS沉默；在Q．CMV+Y．Sat感染的样品中，P19和2b对GUS的沉默却被削弱了；在只有GUS构件时，存在GUS基因的共抑制现象(另一种RNA沉默形式)；在GUS+P19或GUS+2b共浸润的样品中，共抑制现象明显减少，而在有Y．Sat感染时，即使有P19或2b构件的存在，共抑制水平仍然很高。通过对GUSmRNA的Northern印迹分析，GUSmRNA的量与GUS蛋白的量正相关，证实了沉默发生在RNA水平。上述结果证明了Y．Sat能影响VSR干扰RNA沉默的功能，说明VSR对植物内源小RNA介导的沉默途径的影响能被卫星RNA削弱。3．Y-Sat导致的斑驳黄化对实验体系无明显影响。CMV+Y．Sat共同侵染的本氏烟植株出现了斑驳黄化症状，影响了植物的正常光合作用，也可能对整个实验体系产生干扰。为了排除Y-Sat导致的斑驳黄化对实验体系的影响，实验用CHLI突变体mCHLI转化本氏烟，获得不再出现斑驳黄化植株。应用mCHLI转基因本氏烟F2代，进行了Q．CMV或O—CMV+Y．Sat的侵染，然后瞬时表达GUS、hpGUS和P19或2b、GFP等构件。实验结果表明，VSR影响了hpGUS介导的GUS沉默，在有O．CMV+Y．Sat共侵染的样品中，Y．Sat干扰了VSR的功能。实验结果与在野生型本氏烟中所得结果一致，说明了Y．Sat导致的斑驳黄化对实验体系无明显影响。 中文捅要4．在Y-Sat感染且有GUS+hpGUS+VSR共浸润的样品中，GUS仍被高度沉默的现象不是由于hpGUS产生更多siRNA所致。在用GUS、hpOUS和P19或2b进行共浸润时，Q．CMV+Y．Sat共同感染比Q．CMV单独感染的样品产生了更高水平的GUS沉默。这种现象的出现有可能是由于Y-Sat干扰了VSR的功能，也有可能是由于hpOUS产生了更多的siRNA，从而介导了更多的GUS沉默。为了排除后一种可能性，实验设计了hpGUSsiRNA的Northern印迹检测分析。结果显示，在感染Q—CMV+Y。Sat样品中，hpGUSsiRNA的量并没有增加，排除了hpGUS产生更多siRNA介导更多的GUS沉默的可能性。5．Y．卫星RNAsiRNA捕获了VSR，削弱VSR对hpGUS介导的GUS沉默途径的影响。实验应用P19抗体进行RNA．免疫捕获分析，先对植株感染Q·CMV或Q．CMV+Y．Sat处理，再进行GUS、hpGUS、P19共浸润，后采用P19抗体沉淀P19蛋白复合体，并从沉淀复合体中分离RNA，通过Northern印迹分析与P19结合的hpGUSsLRNA、Y．SatsiRNA量。结果显示，在Q．CMV感染的样品中，有部分hpGUSsiRNA与P19结合；而在Q．CMV+Y．Sat感染的样品中，与P19结合的hpGUSsiRNA较少，大部分的P19和Y-SatsiRNA结合在一起。结果说明P19是通过结合hpGUSsiRNA来干扰hpGUS介导的GUS沉默；Y．SatsiRNA通过饱和VSR，增加游离的hpOUSsiR．NA，介导了较高水平的GUS沉默。实验结果进一步表明卫星RNA是通过产生的大量siRNA捕获VSR，从而削弱VSR对寄主小RNA介导的对内源基因调控的干扰。6．Y-Sat可以削弱CMV编码的2b对miRl68的诱导。miRNA在植物生长发育过程中起着至关重要的作用。有报道证实了VSR可以诱导miRl68的表达量，miRl68负调控AG01mRNA，影响了植物miRNA代谢途径中关键蛋白AG01的量。为了证实VSR对植物内源miRNA的影响是病毒致病的主导因素，且卫星RNA可以减少VSR对miRNA代谢的影响，本实验以miRl68的表达量为指示，检测了感染CMV亚组II(Q—CMV)和CMV亚组I(Fny．CMV)及与Y—Sat的本氏烟样品中miRl68的表达量。结果显示，Q．CMV和Fny．CMV都可以诱导miRl68的表达量；当有Y．Sat共同感染时，miRl68的表达量明显减少，感染植株症状减轻；Fny-CMV可以引起相对于Q．CMV而言更高的miRl68累积，感染植株也产生了更严重的症状。结果说明Y．Sat可以削弱CMV编码的2b对miRl68的诱导，且miRl68的表达量高低与植物发病程度紧密相关。实验用半定量RT-PCR检测CMVRNA4a，发现Y．Sat在削弱2b对miRl68诱导的同时，编码2b蛋白CMVRNA4a的量有轻微的减少，导致miRl68表达量减少的原因部分可能是由于Y-Sat共同感染时2b量的减少。X SouthwestUniversiWPhDThesis7．Y-Sat显著的抑制了P1／llcPro对miRl68的诱导。本文应用了由烟草蚀纹病毒(Tobaccoetchvirus，TEV)编码的VSRP1／HcPro转基因普通烟(Ⅳtabacum)体系对miRl68的表达量进行了检测分析。实验结果显示，在P1／HcPro转基因普通烟中，miRl68被明显的诱导，同时出现茎基部弯曲生长的表现型；在Q．CMV+Y．Sat共同感染转基因普通烟中，miRl68的表达水平显著降低，茎基部弯曲生长的表现型基本消失。实验结果说明，植物病毒VSR通过影响植物内源的miRNA代谢途径而致病，而卫星RNA通过干扰VSR，削弱病毒的致病性，减轻病毒诱导的症状。以上7个结论，从小RNA的水平验证了植物病毒卫星RNA弱化辅助病毒致病机理的假说，证明了1)病毒通过其产生的VSR干扰寄主植物的miRNA代谢途径，从而致病；2)卫星RNA通过其产生的巨量siRNA捕获并饱和大部分的VSR，减少VSR干扰寄主植物的miRNA代谢途径，减轻病毒对寄主植物诱导的症状。卫星RNA主要存在于植物病毒，DIRNA主要存在于动物病毒。假说在植物和病毒卫星RNA上面得到了部分验证，作者推断假说还适用于动物和病毒DIRNA。部分动物病毒编码的VSR，也具有结合小RNA的特征，动物病毒的DIRNA，在复制过程可能也会产生大量的siRNA，饱和VSR，干扰VSR的致病性。关键词：病毒，卫星RNA，减轻症状，基因沉默，小RNA，病毒RNA沉默抑制子X Chapter1Literaturereview1Literaturereview1．1VirusandvirussymptomsVirusesareparasiticinlivingcellsandcauseawidespectrumofdiseasestoalltypesoflivingorganisms，fromsingle-celledtohigherplantsandanimals．Someattackhumanbeings，andcausediseasessuchasFlu，Smallpox，ChickenpoxandAcquiredimmunodeficiencysyndrome(AIDS)，someattackplants，andsomeothersCanevenattackmicroorganismssuchasbacteriaandfungi【1，21．Virusesconsistofnucleicacidandprotein，withtheproteinwrappedaroundthenucleicacid。¨．NucleicacidofavirusiseitherRNAorDNA，andgenerallyencodeasmallnumberofproteininmostvirusesDJ．Mostofplantvirusesarepositive-senseRNAviruses，withsingle-strandedRNAgenomeofthesameorientationasproteincodinggenes[11．Virusesdependontheirinfectedhostcellsforreproduction【3】．Inplants，theyinduceadiversityofsymptoms，suchasmosaic，mottling，chlorosis，veincleating，leafrolling，andstuntingt31．Viralsymptomsareimportantdeterminantsinthediscoveryofaviraldisease，aswellastheidentificationofcausalagent，andsometimesthesymptomsareregardedasdecorative．Virus-inducedsymptomsinplantswereobservedbeforeviruseswereknowntoexist【31．OneofthefamousstoriesofviralsymptomsinplantsisassociatedwithTutipbreakingvirus(TBV)．TBV—infectedtulipproducedflowerswithflame-likestreaks(asshowninFig．1．1)，whichatonetimeinthe17thcenturybecameextremelyexpensiveintheNetherlandscausingaf'mancialcrisis【jJ．Itwasnotuntilthelate1880sthatviruseshadbeenrecognizedastheagentofdiseases【41．Cun'ently，over2000virusesareknown，approximatelyhalfoftheseattackandcausediseasesinplants【21．Theyinducedamagetoanyorallpartsoftheplant，whichmayreducetheyieldandqualityofcropproducts，andcausedetrimentaleconomiclosses．SometinylittledamageCanevenoccurwithoutanyvisiblesymptoms．Virusesareregardedasbeingsecondonlytofungiinhierarchyasplantpathogens[41．11 SouthwestUniversityPhDThesisFig．1．1Flame．1ikestreal【sintulipflowersinducedbyTulipbreakingvirus．Still-lifeofFlowersbyAmbrosiusBosschaert(1573—1621)oftheDutchGoldenAge(http：／／flaggedrevs．1abs．wikimedia．org／wiki／Tulip_mania)．图1．1感染郁金香碎色病毒后郁金香花瓣呈现火焰状图案。图为著名画家AmbrosiusBosschaert在荷兰黄金时代(DuntchGoldenAge)所著的“Still-lifeofFlowers”。Someviruses．especiallyRNAplantviruses，areaccompaniedbysmallparasitessuchassatelliteRNAsandsatelliteviruses[5-81．Interestingly，mostoftheparasitesreducethesymptomscausedbytheirassociatedvirus(helpervirus)，andsomeofthemhavebeenusedtocontroltheirhelperviruses，suchasCucumbermosaicvirus(CMV)[9-13]．However，todate，thereisnoreasonablemolecularexplanationforthissymptomattenuation．ThischapterfocusesonthecⅢ了entunderstandingofplantviralsatelliteRNAs(satRNAs)，andhowtheymayreducethesymptomscausedbyhelperviruses．AsatRNAmediatedviralattenuationmodelisproposedbasedonthecharacteristicsofsatRNAsandtheprocessofRNAsilencingpathway．SomeofthecurrentknowledgeonparasitesofplantvirusesandonRNAsilencingmechanismsrelevanttothescopeofthisthesisiSalsocovered．1．2SmallparasitesofplantvirusesSmallparasitesofplantvirusesarebecomingafascinationofmanyvirologistsbecauseoftheirprofoundmodulationt。theirhelpervims—inducedsymptoms[3,6,14,15]，whicharedependentontheirhelpervirusforreplication【6]，butdispensableforthe12 ChapterlLiteraturereviewhelperviruses【8】．ThreemajortypesofsmallparasitesaresatelliteI斟A(satRNAs)，satellitevirusesanddefectiveinterferingI斟As(DIRNAs)o孓引．Theclassificationisbasedontheiroriginsandthedependenceon也eirhelperviruses(Table1．1)Lo,／J．PlantDNAvirusesalsohavesatellites，whichhaveE}NAgenomes【o’引，butthisthesisisfocusingonsatRNAsassociatedwithRNAviruses．Table1．1Propertiesdis血guishingsmallparasitesofplantRNAviruses表1．1区分植物RNA病毒所伴生小寄生物的标准Criteri。nSatelliteRNAsSatellitevirus慧盏gRNAreplicationdependenceonhelpervirus+Encapsidationinhelper-viruscoatprotein+-+Interferencewithreplicationofhelpervirus+8+脚塑竺翌垒曼P苎垡垡!垒￡塾竺生竺堂兰g望竺望竺：±一Notes：+，yes；·，no；‘，mostofthem．1．2．1PlantsatI矾AsThesatRNAsaresmallnon．codingRNAsof220-1500nucleotides(nt)insize嘲whichsharenoorverylittlehomologywiththeirhelpervimsgenome(Table1。1)[5,16,17]．Asdiscussed，theyrelyonthehelpervirusforreplicationandencapsidation[5,S,16,17]．Functionalopenreadingflames(ORFs)foundinthelargersatRNAs(normallylargethan300nt)，butwithnofunctionalproducts【61．Generally,satRNAscompletelydependedontheirhelpervirusesforreplication，encapcidation，movementandtransmission【6，161．ThesatRNAsaretransmittedtogetherwiththehelpervirus，e．g．satRNAofCMVispackagedinthecoatproteinofCMVtogetherwiththeirhelpervirusesgenome，andtransmittedbyaphids[18-20]．ThesatRNAsarehighlystable．invivo，satRNAofQ-CMVstraincouldsurvivemorethan10dayswithoutahelpervirus【2¨．invitro，somesatRNAsofCMVCansurvivebyitselfforeighthrinabufferextractofhealthyleaves[22]。ThesatRNAsofTomatoblackringvirus(TBRV)couldsurvivemorethanfourdaysininoculatedleavesbythemselvesintheabsenceofthehelpervirus[161．ThestabilityandsimplicityofsatRNAsattactedattentiontodetailedanalysisoftheirstructure【16I，whichwereshowntopossesshighlyorderedsecondarystructures【6J．Thishighlyorderedstructureprobablyexplainedtheirhighstabilityinvitroandinvivo[22,231．Indeed，amorerecentstudyshowedthattheCMVY．satelliteRNAfY—Sat)sequencewasresistanttosmallRNA-directedRNAdegradation【241．1穹 SouthwestUniversityPhDThesis1．2．2PlantsatellitevirusSatellitevirusesaredifferentfromsatRNAsin也atmeyencodetheirowncoatprotein【5，16'17】bmyetareunabletomultiplywithouttheassistanceofthehelpervirus．LikesatR．NAs，theysharenosequencehomologywiththehelpervirusgenome吵Thesesatellitevirusesarethesmallestknownplantviruses，thediametersofwhicharearound17nm，andtheirgenomeencodepolypeptidesofabout17—21kiloDalton∞)【31．Fewsatelliteviruseshavebeenreported，andthosereportedincludethesatelliteTobaccomosaicvirus(TMV)(Fig．1．2)，satelliteTobacconecrosisvirus(TNV)，satellitePanicummosaicvirus(PMV)andMaizewhite-linemosaicvirus(MWLMV)【3，16，25矧．SatellitevirusesoRenreducedtheabilityoftheirhelpervirusestoreplicate，andtoreducehelpervirus．inducedsymptoms[3，2刀．Fig．1．2SatelliteTobaccomosaicvirusclusteredaroundarod-likeparticleofTobaccomosaicvirus【251．Scalebar=50nm．图1．2烟草花叶病毒的卫星病毒聚集在烟草花叶病毒棒状粒体周围，标尺长50Sln。1．2．3DIRNAsDIRNAsaretruncatedorrearrangedfragmentsoftheviralgenome，andaretheproductsofprematureterminationandreinitiationoftheirhelpervirusgenome【8】．DIRNAscallencodenormalviralstructuralproteins，buttheyreproduceonlyinthepresenceofahelpervirus[7,28-30]．DIRNAsaremorecommonlyassociatedwithanimalvirusesthanplantviruses；theyareassociatedwithnearlyallanimalviruses【8】butonly14 Chapter1LiteraturereVleWfoundforsomeplantviruses，suchas彳蜘驰mosaicatfalmovirus(alMV)，CMV，Broadbeanmottlevirus(BBMV)andTomatobushystuntvirus(TBSv)t3U．IncontrasttosatRNAsandsatelliteviruses，theyarealmostexclusivelyassociatedwithplantsviruses【8】，butsimilartOsatRNAs，DIRNAsdependonthehelpervirusformultiplication．movementandencapcidation【8】．SomeDIRNAsdramaticallyreducedthehelpervirusaccumulationandresultedinattenuationofhelpervirus-inducedsymptoms【7】．1．2．4TherelationshipbetweenthesatRNAandtheplantvirusOneofthedramaticandinterestingeffectsofplantsmallviralparasitesistochangethehelpervires．inducedsymptoms【6，J．ThesatRNAswereoncetakenaspureparasitesofthehelperviruses[6，321．However，recentstudiesindicatethattherelationshipbetweensmallparasitesandthehelpervirusesismorethanparasitic；theycouldbeofmutualandbenefit．alsowithpotentialbenefittothehostplant吵ThesatRNA-causedsymptomeffectsvariedwiththehostplant，virusandsatR_NAs【6J．ThesamesatRNAcouldreducethesymptomscausedbythehelpervirusinonehost，andexacerbatethesymptomsinanother(Table1，2)【6'331．Forexample，thepresenceofsatRNAofCMVinChenopodiumquinoa，CucurbitamaximaDuch．，CapsicumfrutescensL．andZeamaysL．attenuatedCMV-inducedsymptoms；whilethesamevariantsatRNAofCMVincreasedtheseverityofsymptomsintomatoplants[341．Moreover，thesamesatRNAcallintensifyorattenuatethesymptomsinthesamehost，dependingonthehelpervirusstrains．Intobacco，satRNAstrainB5ofCMVinduceschlorosiswhenassociatedwiththeCMVsubgroupIIstrain，butattenuatessymptomscausedbyaCMVsubgroupIstrain【35】。ThissuggeststhatthepathogenicitydependsonsatRNA．virus-hostinteractions，resultingineithersymptomsreductionorexacerbation【6】1．2．4．1SymptomsattenuationofplantsatRNAsAlthoughsatRNAscanmodulatehelpervirus-inducedsymptomsinbothdirections，inmostcasestheyamelioratethesymptoms【6】．TherelationshipbetweenCMVanditssatRNAshasbeenwellstudied。MostoftheCMVsatRNAsattenuateCMV-inducedsymptoms．Forexample，satRNAsfromtheQ—CMVstraineitherreducedorhadnocleareffectonthosesymptomscausedbythehelpervirusintomatoandsome15 SouthwestUniversiWPhDThesisNicotianaplants[231．OthersimilarexamplesarediscussedbelowandsummarizedinTable1．2．Tobaccoringspotvirus(TobRV)isabipartite，singlestrandedRNANepovirus，whichhasbeenoRenassociatedwithasmallsatR_NA【17】．SatRNAsofTobRVeitherhadnoeffectorreducedtheTobRV．causedsymptoms【361．VariantsatRNABreducesthesizeofthelesionscausedbyTobRVincowpea(Vignasinensis)‘纠o；presenceofeitherNC·-62orbudlight··satRNAstrainsdramaticallyreducedtheseveresymptomcausedbytheTobRVbudblightstrainincowpeas(矿unguiculatacv．)[38】．Tomatoblackringvirus(TBRV)isanotherNepovirus【161．ThesatRNAsofTBRV[6】reducedthenumberoflesionscausedbyTBRVinC口maranticolor【391．Peanufstuntvirus(PSV)belongstotheCucumovirusfamily【401．PSVisaneconomicallyimportantpathogenoflegumesaroundtheworld．VariantsG·andWC-satRNAsofPSVbothreducethehelpervirus．inducedsymptoms【6’411．UponCO．infectionwithPSVandeitherofthetwosatRNAstrains，bothsatRNAandvirusRNAswererestrictedintheinoculatedarea．resultinginsymptomattenuationintobacco【加】．Accompaniedwithsymptomattenuation．alargeaccumulationofds-satRNAofPSVwasobservedinthesymptomattenuatedtissues，aswellaSthereductionofthePSVtitter【22]．1．2．4．2ThesatRNAsexacerbatehelpervirus—inducedsymptomsFewsatRNAsintensifiedthehelpervirus．inducedsymptoms[6'23，34，421．AlthoughmostoftheCMVsatRNAsreducedthehelpervirus—causedsymptomsinalmostalltestedhosts，afewsatRNAstrainsexacerbatedsymptomsinoneortwohosts(Table1．2)【61．Forexample．satRNAoftheS-CMVstraininducesalethalnecroticdiseaseintomato(Lycopersiconesculentum)，otherthanthecholorosisand‘'fernleaf’symptomsnormallyinducedbytheS-CMVstrain[421．Theseedsyieldofsoybearl(Glycinemax)plantsCO—infectedbysatRNABandTobRVBBstrainisdecreasedcomparedwiththatinfectedbyTobRVBBalone【16】．Arabismosaicvirus(AMY、hopstraininducesnettleheadsymptomsinhop；thesesymptomsappearedevenmorepronounceduponCO．infectionofsatRNAandAMV【431．ThissuggeststhatthesatRNAintensifiestheAMV—inducedsymptoms．ThesatRNACofTurnipcrinklevirus(TCV)exacerbatedthenormallymildsymptomscausedbyTCVinturnipcultivarJustRight，BrassicarapaandArabidopsisthalianaM．However．virionaccumulationofTCVWasreducedupon16 ChapterlLiteraturereviewsatRNACinfectionintheseplants【“'45】．InTCV-toleranthosts，TCVvirionaccumulationwasthesameasinsymptomatichosts；hosttolerancetoTCVismaintainedinthepresenceofsatRNAC．ThissuggeststhattheintensifiedsymptomswerenotduetoincreasedTCVaccumulation．Table1．2SummaryofsatelliteRNAsdiscussedinthisreview表1．2综述中出现的卫星RNAHelpervirusSatelliteGenomeORFEffectonRNAsizesymptomsCMV-SC^n，-、VrCM=、，-、^呵CM：、，PeanlIfStltntvirusTobaccotlngspotvirusffobRV)TobRVlCasertasquash(CucurbitaJmaximaDueh)1Tabascopepper(Capsicumltrutescens)、-Balltalnsweetcoill(Zeamay$L．1TobRV-BBB359×+Soybean(Glycinemax)”。知胁砌蝴嘲g诎懈／1375、『．Cagtlara^ticolor∞1Note：e侬娥on卿toms：．，meliorationofsymptoms；0，noeffect；+，exacerbation．ORF，√，Yes；。，No．／，nOnallle·17 SouthwestUniversiWPhDIhesisHowsatRNAsexacerbatehelpervirus．inducedsymptomsstillremainsullkllown，asdiscussedinsection1．4．2．allRN．Asilencing—basedmodelhasrecentlybeenproposed．StudiesonthemechanismofCMVY·—satRNA--causedyellowingsymptomsinNicotianaspeciesdemons仃atedthatthesymptomswerearesultofhostchlorophyllbiosynthesisgenesilencinginducedbysatRNA．derivedsmall对姨s(sRNAs)[46’471。ThiSsuggestedthatat1eastsomecasesofthesatR_NA．causedsymptomexacerbationwereduetOsatRNAsinailRNA．directedhostgenesilencing．1．2。5ThetraditionalexplanationofsatRNA-mediatedsymptomattenuationUsually,aRenuationofsymptomsisaccompaniedbyadecreasedvirusaccumulation[6,33,48,49]．ThisphenomenonhasledtothenotionthatcompetitionfortllelimitedreplicaseenzymebetweensatRNAsandhelperviruscontributestosymptomattenuation【6，321．ThisistheoverwhelmingviewofsatRNA-causedsymptomattenuation．From1970sto1980s，thegenome—componentanalysisinCMVsatRNAs—mediatedsymptomeffectshowedthatthereplicationofsatRNAsdecreasedthe1evelofCMVRNAs(RNAs1，2and4)，whichcorrelatedwithreducedCMV．causedsymptoms[23,50-54]．ThesatRNA．causedameliorationofsymptomsbyTBSVinCamaranticolowasalsoaccompaniedbylessviralaccumulation【55J．Morerecently，thepresenceofaCMVsatRNAinCMV．infectedplantshasbeenfoundtoreducetheaccumulationofRNA4aaswellasitsencoded2bproteinL3．3J,aCMV-encodedviralsuppressorofRNAsilencing(vsg)[561．TheauthorsproposedthatthisreducedVSRaccumulationmightincreaseanti-viralsilencingagainsttheCMVhelpervirusleadingtoreducedviralaccumulation，whichmaybeaccountedforthesymptomattenuation例．Traditionally,itiSalsobelievedthattheDIRNA．mediatedsymptomaRenuationwasmainlyduetocompetitionwiththehelpervirusforthelimitedsupplyoftrans—actingfactorsrequiredforreplication咧．However，symptomattenuationinsomesatRNA-helperviruscombinationswasnotnecessarilvassociatedwithareductionofvirustiter[6,39,57]．hlthetransgenictobaccothatexpressedthesatRNAofCMV，bothCMVandTamatoaspermyvirus(TAV)(alsoamemberoftheCucumovirusfamily)infectionsshowedsuppressedsymptomsoftheviruscomparedtowildtypeplants．ExpressionlevelsofsatRNAsweresimilarinplantsinfectedbVbothviruses．However，whiletheviralRNAlevelwas18 Chapter1LiteraturereviewgreatlyreducedwithCMVinfection。itremainedalmostunchangedwithTAVinfection[39,58]．whichindicatedthatthereductionofTAV-causedsymptomswasnotduetoreducedviraltiter．AnotherreportonTAValsoshowedthatuponCO-infectionwithsixvariantsofCMVsatRNA．thesymptomswereeitherattenuatedorunaffectedintobaccoplants．However．thepresenceofCMVsatRNAsdidnotaffecttheyieldofTAVvirionspurifiedfromtobacco．Amongt11esi)【satRNAvariants，thosethatattenuatedthesymptomscausedbyCMValsoattenuatedthesymptomsinducedbybothTAV-1andTALV．Vstrainsintomato．ComparedwiththesatRNA—freeTAVinfection，t11e姐eldofvirionswasnotaffectedwiththeappearanceofthesatRNAp“．ThesereportssuggestedthatsymptomattenuationbysatRNAprobablyinvolvedsomeotllermechaniSmsotherthancompetitionforreplicationalone【6，39，581．Todate，itwaSbelievedthatthevirussymptomsandvirestitterarepositivelycorrelated例．However．highlevelofvirusaccumulationwasnotessentialfortheseveresymptoms．Forinstance．asmentionedaboveinsection1．2．4．2，theintensifiedTCVsymptomscausedbysatRNACwereaccompaniedwithreducedamountofvirions．Thus，howsatRNAsattenuatesymptomsremainsunclearatthemolecularlevel．sa倦NAscouldaccumulatetohighlevelsinthehost，especiallyintobacco[54’60，611，andatthesametimea1argeamountofdoublestrandRNA(dsRNAs)areassociatedwithsatRNAreplication[52,53]．Moreinterestingly，hi曲levelsofsatRNA-derivedsmallinterferingRNAs(sat．siRNAs)areassociatedwiththereplicationofsatRNAsM⋯．Asdiscussedbelow，siRNAisakeycomponentofRNAsilencing，anaturalantiviraldefencemechanisminplants．TheassociationofhighlyabundantsiRNAswithsatRNAinfectionininfectedhostplantsmakesitpossibleforthatsomeaspectsofRNAsilencingcanbeinvolvedinsymptomattenuation．1．3RNAsilencingRNAsilencingemergedinthelastcentury[56,62-66]，andhasbeenbecomingahottopicsincethen．ThefirstreportofRNAsilencingtypeofphenom．enonwasin1990inaplanttransgenicexperiment，inwhichboththetransgeneandhomologousendogenousgeneweresilenced【671．Inordertoincreasethepurplecolourofthepetuniahybridflowers，achalconesynthasetransgene(CHS)Wastransformatedintopettmiato19 SouthwestUniversityPhDThesisoverexpressCHS，whichcontrolsthesynthesisofthecolorpigment．However,theflowersinmanyofthemodifiedplantsexpressedapatternofpurpleandwhite，insteadofmeintensepurplecolor．MolecularanalysisofthesetransgenicpetuniashowedthatboththetransformedandendogenousC邯geneweresilenced[40,67]ObservationsofsuchphenomenatriggeredRNAsilencingresearch．Soonafterthat，RNAwasidentifiedastheinducerofgenesilencinginthestudyingofthepathogen。mediatedvlnlsresistance[68】．andin1998，dsKNAwasfoundtobeacommoninducerofRNAsilencinginplantsandanimals[66,69]．RNAsilencingisanessentialregulatorymechanisminmosteukaryotes【701．Inplants，RNAsilencingplaysmultiplerolesincludingregulationofgeneexpressionepigeneticmodificationofDNAandhistonesatspecificchromosomaldomains，anddefenceagainstinvadingnucleicacidssuchastransposonsandviruses．RNAsilencingisbasedonanucleotidesequence．specificmechanism[71-74]．1．3．1RNAsilencingpathwaysinplants髓ebausicprocedureofRNAsilencinginplantsiswellillustrated[71,75-77]．dsRNAor11DRNAiscleavedbyDicer．1ike(DCL)protein(s)，atypeofRNaseIIIenzyme，into21．24ntsRNAduplex．Thestrandwithits5’terminusatthelessthermodynamicallystableendofthesRNAduplexisselectedastheguidestrandI硝J，withtheotherstrandbeingdegraded。TheguidesRNAisloadedtooneofArgonaute(aoo)proteinstoform丑RNA．inducedsilencingcomplex(PdSC)，whichisthenguidedbythesRNAtodirectRNAdegradation，translationalrepression，orDNAmethylationofhomologoustargetgenes【79]．ThreebasicRNAsilencingpathwayswerefoundinplants(Fig．1．3)，includingthemicroRNA(nliRNA)pathway，thesiRNA-directedRNAdegradationpamwayandtheRNA．directedDNAmethylation(RdDM)pathway[71,80]TheysharetheCOreprocessofRNAsilencingbutmaintaintheplantsystemindistinctmanners·1．3．1．1ThemiRNApathwayThemiRNApathwayplaysacriticalroleinplantdevelopmentalprocesses，suchascelldivision，leafformationandflowerdevelopment【81'82】．PlantmiRNAspredominantlynegativelyregulatetargetmRNAbydirectingcleavageofthecodingregions，andcanalsocause仃anslationalrepressionastheanimalmiRNAsdo[831．20 Chapter1Literaturereview(：》心。：，、I’”h重；”知葛匡h≥“占Fh妻吾h卜21．m_zu。餐仨骠怒扯团蛊^∞oo¨v淞mgII是∞瞩罂圈塔。娆般餐蟮《蚕窨埒醐茫⋯廿霉璎，_圈一∞oo¨一飞≈∞_8_意幽uo专∞∞(I鼍lJIpo量№B≥Q．Il量■∞=≈IdIIl∞爸≥暑B(I譬弓_互Is蚕2露o8三．L，．【篁函丸嚣参lI苌盘苫QI'篮一：n，，¨，，】】——，1，吼-=v，-=r，1，，3一．m．∞{吓E虐1hhEE崖l上●难hh{越淫h，n——J】】1_——--—．．．—_．m．n—JJ】】1，__—-_．——I_，m、m．n．m、n朗厂}。一．n，"了J—哪，，，1，，1，，，哪舢1，吼l'．=-．西赫仁正}FEI唯EJ正蔓n^矗譬_暑嚣盘《Z笛馕-n_工u、m--．-II一--_---P、nh．zu．m衲EHuCCrCCCF．n、n、m、n．mhCCCCCCF．n衲七匕EFEEEF．n土一；∈们∞ao—>J们]山1)3Z≯露参_暑嚣盘《Z笛o．Dlu_h_工uh，￡fH)lCEI-CC-Enm0n]D嗄、-在}．叮，哩；q7笆}．!^《Z芷一≥-ILA SouthwestUniversityPhDhesisInthemiRNApathwayofplants(Fig．1．3left)，primarymiRNA(pri。miRNA)transcriptisgeneratedfromthetranscriptionofMIRNAgenesbyRNApolymeraseIIfPolII)【84】．Single—strandedpri．miRNAisfoldbackintostem—loopstructurebetweenthecomplementarysequences．Thesestem．100psrangefrom50to250ntinlength恻．TheyareprocessedbyDCLl，whichisoneofthefourDCLsinArabidops&，intoprecursormiRNA(pre．miRNA)[86】，ashorthpRNAstructure．DCLl，assistedbyHyponasticLeaves1(HYLl)，alsoknownasdouble—s仃andedRNA—bindingprotein1(DRB1)[87-89]，cutsthetailandtheloopofthemiRNAprecursortoliberatethemature21—24ntmiRNA／miRNA*duplex【84]．ItisbelievedthatallthesestepsoccurinthenucleuS．sinceDCLlandHYLlonlyexistinthenucleus【90，9l】．ThemiRNAduplexesareexportedtothecytoplasmbytheRNAExporterHasty(HST)，amemberofnecleo-cytoplasmictransproters[92】．Thetwonucleotide3’overhangsoftheduplexaremethylatedbyHuaEnhancer1(HENl)，amethyltransferaseenzyme，butwhetherthemethylationhappensinthenucleusorinthecytoplasmisstillunclear[90J．ThismethylationprocessisalsoinvolvedinsiRNAbiogenesis，anditappearstoprotectallsRNAspeciesfrompolyuridylationanddegradation【93】．Thematuresingle’strandedmiRNAisloadedontoAG01，oneofthetenAGOsinArabidopsis，toformRISC．RISCisguidedbythemiRNAtorecognizetheperfectornearperfectcomplementarytargetRNAsequence，inducingcleavage，orrepressingtranslationofthetargetmRNA[71，76，94】1．3．1．2ThesiRNApathwayThesecondpathwayisthecytoplasmicsiRNAsilencingpathwayL95JThispathwayisconsideredasthecentralcomponentofRNAsilencinginallofthesilencingsvstemscharacterizedtodate[94,95]．DiversesiRNApathwaysareclassifiedaccordingtothedifferentoriginsofthesiRNAs，suchasnaturalantisensesiRNA(nat—siRNA)andtrans-actingsiRNA(ta—siRNA)[76】．Theprimaryprecursorofta—siRNAislongnoncodingRNA，whilethatofnat．siRNAisRNAtranscriptcomplementarytOmRNA[761．Bothta—siRNAsandnat．siRNAsareimportantinplantdevelopmentandbioticandabioticstressresponse[96-98]．However，thesiRNA．mediatedRNAdegradationpathwaynlnctionsDredominantlyinplantantiviraldefence[79】．22 ChapterlLiteraturel'evlewInthesiRNAsilencingpathway(Fig．1．3，middle)，endogenousorexogenous(includingtransposons，仃ansgenesandviruses)singles仃andedRNA(ssRNA)iscopiedintodsRNAbyRNA．dependentRNAPolymerase6(RDR6)and／orRDRl[79'991．twoofsixRDRsinArabidopsis【1001．ThedsRNAisthencleavedbyDCL4andDCL2into21and22ntsiRNAduplexes，respectively．ThesesiRNAduplexesaresubsequentlymethylated，andmaturesiRNAisloadedintoAGO1orAG07toformRISC，andguidetheRISCtoinducecleavageoftheRNA，whicharecomplementarytothesiRNAsequence【97]．Oneof也efn-stdiscoveredandwellstudiedfunctionsofthesiRNApathwayISviral．derivedsiRNA(vsiRNA)．mediatedviralRNAdegradation[711．vsiRNA-medimedviralRNAsilencingisassociatedwithhighlevelofvsiRNAaccumulation[95,101-105】．Moreimportantly，thesevsiRNAsareassociatedwithAGO1，thecentralcomponentinthesliceractivityofplantRNAsilencing[106’1071．ThevsiRNAsilencingpathwayhasbeendividedintothreesteps：1)recognitionofviralRNAs，conveningthemintotemporarydsRNA；2)vsiRNAsformation，amplificationandaccumulation；3)vsiRNA．AGORISCformationandviralRNAtargeting【1081．Inplants，primaryandsecondaryvsiRNAshavebeenidentified．PrimaryvsiRNAsresultfromDCL-dependentcleavageofinitialviraldsRNAorhpRNAformedbyannealingofplusandminus·-strandviralRNAsorfold-·backofcomplementaryregionswithinsingle-strandedviralRNAs；thebiogenesisofsecondaryvsiRNAishostRDR-dependent[100,101,109-III]．DCL4andDCL2processds．orhp．viralRNAintovsiRNA，21and22nt，respectively[101,112]．AGO1andAG07areinvolvedinthevsiRNAsilencingpathway，toallowforefficientclearanceofviralRNAs．AG07appearstosen，easabackupslicerintheabsenceofAGO1[108,113]．MaturedvsiRNAbindstoAGO1orAG07toformRISC，andguidestheRISCtodegradeviralRNA．ThecleavedviralRNAsfragmentsCanbeconvertedintodsRNAbyhostRDRs，whichCanbeprocessedintosecondaryvsiRNAbyDCLtomediatethesubsequentviralRNAdegradation【1111．ThesecondaryvsiRNAsareimportantformeRNAsilencing-mediatedanti．viraldefensetodirectmorepotentialantiviralsilencing[III,114]． SouthwestUniversityPhDThesis1．3．1．3TheRclDMpathwayThethirdpathwayofRNAsilencingistheRdDMpathway，whichisuniquetoplants[71,115]．Itisallepigeneticmechamsminducingthesilencingofatransgeneoranendogenousgenebytheinactivationoftheirpromotersequencesandeventhecodingsequences[116,1171．Itnotonlyinducestransgenesrepression[115]，butalsoplaysakeyroleinmaintaininggenomestabilityandintegrity[118,119]．Also，RdDMseemstobeadefencemechanismagainstDNAvirusesbyinducingDNAmethylationandtranscriptionalrepressioninDNAvirusgenome【791．InthecurrentmodelfortheRdDMpathway(Fig．1．3right)，methylatedDNAisthetemplateforthetranscriptionofaberrantRNA，andthistranscriptionismediatedbyPolIV(aplantspecificRNApolymerase)[120]．TheaberrantRNAisconvertedintodsRNAbyRDR2．RDR2-transcribeddsRNA，whichcanalsobeatemplatetoproduceadditionalaberrantRNAbyPolWinaself-perpetuatingloopL121|．ThedsRNAiscleavedbyDCL3into24一ntsiRNAs，whicharethenmethylatedbyHEN1andloadedontoAG04toformRISCanddirectDNAmethylation[122,123]．TheRdDMpathwayismediatedbythecombinedactionsoffollowingproteins：theSNF2一likechromatin．remodelingprotein[124]。DefectiveinRNA．directedDNAMethylation1(DRD1)，PolIV，PolV(anotherplantspecificRNApolymerase)，andtheprimarydenoYoDNAmethyltransferase，DomainsRearrangedMethylase2(DRM2)[79,122,125]．Onceestablished，themethylationcanbemaintainedbytheDNAmethyltransferases，includingMethyltransferase1(METl)andChromomethylase3(CMT3)，inasequencecontext．dependentmanner【761．1．3．2DistinctionsbetweenmiRNAsandsiRNAsAsmentionedpreviously，bothsiRNAsandmiRNAsare21-24ntsRNAs，andtheyaresimilarintheirchemicalstructure，centralbiogenesisandmechanismofaction[7t,76,81,82]．Theirmaincharacteristicssuchasthetypeofprecursor，evolutionaryconservationandthetargetCanbeconsideredasstrictrulesforclassification[126]．First，miRNAsarederivedfromgeneloci，whereassiRNAsareoftenoriginatedfromtransposons，repeatsandviruses．Secondly，miRNAsareprocessedfromsingle-strandedtranscripts，whichcanfoldbacktoformhairpinstructure，whereassiRNAsaremainlyprocessedfromlongdsRNA．Thirdly，eachmiRNAprecursorgenerateasingle，4 Chapter1LiteraturereviewmiRNA／miRNA木duplex，whereasonesiRNAprecursorprocessesmanydifferentsiRNAs．ThedifferentfeaturesofprecursorhavebeenusedtoverifythepredictedsRNAsindeepsequencingdatabase【1271．Fourthly．thesequenceandfunctionofmiRNAsarehighlyconservedacrosskingdoms[128】，whereassiRNAsarerarelyconservedandseemtoberandomlyprocessedfromdsRNAsbyDCLs．Fifthly，miRNAsspecify“trans—silencing”，i．e．theslicedgenes，aredifferentfromtheirprecursorgenes，whereasendogenoussiR．NAstypicallyspecify“auto—silencing”，namely，theslicedgenesaresharingthesameorsimilarlociwiththeirprecursorgenes【81]1．3．3AGOsAnumberofassociatedfactorshavebeenidentifiedthatfunctioninplantRNAsilencingpathways[711．ThesefactorssuchasDCLs，RDRs，AGOproteinfamily，HENl，andRNApolymerases，reflectthefunctionaldiversityofthedifferentpathways【1291．Manyofthesefactorsarelocalizedinthenucleus[130]，indicatingtheimportanceofthenucleusintheinitiationofsilencinginplants[1291．InallRNAsilencingpathwayswithinplantsandanimals，maturedsRNAwith5’phosphategroupsand2-nt3'-overhangsatthetermini[131-133]．isassembledwithanAGOproteintoformRISC[71,76,78,134]．AGOproteinsconferthesliceractivity，andareessentialcomponentsintheRNAsilencingsystem．AGO1inA．thalia咒口isthefirstidentifiedAGOprotein【135】：theotherAGOmembersweredefinedbythepresenceofPAZ(Piwi-Argonaute—Zwille)andPIWIdomainsasinAGO1[136,137]．Basedoncrystalstructuralstudies，thePAZdomainhasbeenidentifiedasanRNA．bindingmodule[137-139]，whichspecificallyrecognizedthe3’ssRNAsoverhangs[138-140]．ThisRNA．bindingmoduleisalsocommontoDCLenzymes，whichformsaspecificbindingpocketforthe3’endofthesmallRNA【1411．ThestructureofthePIWIdomainisfolded1ikeanRNaseH，allendoribonuclease，whichcleavestheRNAstrandofanRNA．DNAhybrid[141,142]．The5’phosphategroupsofsiRNAsandmiRNAs，whichresultfromtheirmechanismofbiogenesis，arecrucialfortheefficientassemblyofthesesmallRNAsintoRISC[131,133,143,144]．DistinctsRNAsarepreferentiallyassociatedwithdifferent SouthwestUniversityPhDThesisAGOproteinsindifferentsilencingpathways；thispreferentialsRNA—AGOrecognitionisbasedontheidentityofthe5，nucleotideofmesI矾A[145,146]．A．tha／／a，zaAGO1preferstobindwithsRNAswithauridineat5’ends，whereasAG02recruitssRNAswitha5，adenosine．end[141】．AGOmembershavebeenreportedindifferentorganismswithdifferentnumbers，suchasoneinthefissionyeast(Schizosaccharomycespombe)，fiveinDrosophila[147]，eightinHumansbeings[1481，teninArabidopsisand27inthenematodeworm(Caenorhabditiselegans)‘149·1511．IntheRNAsilencing-mediatedviralimmunityinplants，AGO1isthecriticalcomponent[113]．InductionoftheAG01mRNAexpressionbyviresinfectionisananti-viraldefencereactionofhostplants，whichconsequentlyenhancesAGO1proteinlevelp08]．TheincreasedlevelofAG01proteinfacilitatestheformationofvsiRNA-AGO1RISC，resultinginmoreeffectivedegradationofviralRNAs．However，inordertosurvive，viruseshaveevolvedadiversesuiteoffactorstocountertheantiviralRNAsilencing008]．Thefollowingsectionfocusesonthecounter．defencereactionofplantviruses．1．4RNAsilencingandplant-virusinteractionsRNAsilencingisessentialforplantdevelopmentandformaintenanceofgenomeintegrity，butitisalsoallantiviraldefencemechanism[79,152,153]．ViralRNAsilencingisconsideredanaturalantiviraldefencemechanismsinceviraldsRNAareformedduringvirusreplicationbyviralorhost．encodedRDRenzymes[154]．Antiviralfunctionwasthefirstdiscoveredandwell．studiedoftheRNAsilencingmechanism[95,101-103,108,154]．Fortheplantperspective，uponvirusinfection，viralRNAsalesensed，processedintovsiRNAs，loadedintoanAGO1orAG07toformRISC，whichthemsguidedbyvsiR_NAtoinduceviralRNAdegradation[155]．Fromtheviralperspective，virusesaretryingtheirbesttoinfectandinvadeplants，ensuringtheirsurvivalsandmultiplications．Indeed，plantvirusesareefficientpathogens，andtheycallalwayssuccessfullyinvadeoneormorehostplantspecies．ThissuggeststhatviruseshaveevolvedefficientcounterdefencestrategiesagainstantiviralRNAsilencing．26 Chapter1Literaturereview1．4．1ViralcounterdefencemechanismsagainstRNAsilencingAsacounter-defencestrategy，manyplantvirusesencodeproteinsinterferingwithRNAsilencingatthedifferentstageoftheprocedure[71,129]．Amongtheproteins，VSRsareconsideredtobetheoutcomeofevolutionaryprocesses[96,97,108,156]．VSRswerefirstreportedin1998№641，andsubsequentlymanyVSRshavebeenidentified(Table1．2)．Theyarediversewithinandacrossviralkingdoms，sharingnoobvioussequencehomology【108】．WhiledifferenttypesofVSRsoftenfunctionindifferentmodes，togethertheyareabletointerferewithalmostallfactorsalongtheRNAsilencingpathway,includingsensingofviralRNA，dicingofdsviralRNA，formationofRISC，targeting,andsRNAaccumulation．Greateffortshavebeenmadesincethefn-stdiscoveryoftheVSRstounderstandtheirstructuresandtoillustratemolecularmechanismsassociatedwithRNAsilencingsuppression[96,108]．MostidentifiedVSRsaremultifunctional(Table1．3)，andbesidesbeingsilencingsuppressor，theyareidentifiedaspathogenicityfactor，coatprotein，movementprotein，replicaseortranscriptionalactivator[108]．1．4．1．1sRNAbindingtoinhibitRISCformationAsshownm也eTable1．3，acommoncharacteristicformostVSRsisRNAorsRNAbinding．Theseincludesomewell．studiedVSRs：P19ofTombusvirus[157]，CMV2b[15s]，TAV2b[159]，乃6口ccDetchvirus(TEV)HcPro[160]，andsomeotherVSRslikeTurnipCrinkleVirus(TCⅥP38[160,161]，TMVP122／P130[162]，P14qfPothoslatentvirus(PoLV)【163】，P15ofPeanutclumpvirus(PCV)[163]，andV2ofTomatoyellowleafcurlvirus(TyLCⅥ[164-167]．DetailedmolecularstudiesandstructureanalysishavedemonstratedthatsiRNAsequestrationisthebasicsilencingsuppressionmechanismofVSRs，evolvedbyseveralviralgenera，suchasP19[157,168-172]，HcPro[173，1741，and2bD58,175]．ThesiRNAduplexsequestrationpreventsef!ficientassemblingofRISC．ThesecompletelyunrelatedVSRssharethesamemechanismofRNAsilencingsuppression，suggestingtheirindependentevolutionindifferentviralgenera[108]．ThebestknownVSRisP19ofTombusvirus．ItWasfirstdemonstratedasasymptomdeterminant【1761．andwassubsequentlyfoundtosuppressRNAsilencing[157,168,177,178]．P19ofCarnationItalianringspotvirus(CIRV)hasbeenusedtoillustrateitscrystalstructure【1791．ItssRNAbindingabilitywasprovenbothinbiochemicalandinvivo27 SouthwestUniversityPhDIhesisassays[179】．P19bindswithdssiI：NAinahead-to．tailhomodimer．whichfunctionedasamoleculecalipermeasuring21ntsiRNAduplexandbindedwiththeduplexinasequence．independentmanner[179,180]．This21ntsiI{NAbindingaffinitysuggestedagreatereffectonthemRNAdegradationpathwaythanontheothersiRNAsilencingpathways【1791．The2-nt3’overhangsofsRNAwerenotnecessaryforhighaffinitybindingwithP19．butme5’phosphategroupenhancesthebindingaffinity【¨圳．ThesesiRNAssequestrationbyP19preventssiRNAfromloadingintoAGOproteintoformRISC．whicheventuallyinhibitsvsiRNA．mediatedviralRNAdegradation．CMV2b，anotherwellstudiedsuppressor，hasbeenshowntobindWithdssRNAsandlongdsRNAs[158,1811．The2bVSRofasubgroupICMVstrain(CM95R)iscapableofbindinginvitrosynthesizedsiRNAsandevenlongdsRNAs．However，the2bproteinofthemildstrainCM95，whichhadasingleaminoaciddifferencewiththatofCM95R，showedalowersiRNAbindingaffinity．ItwasproposedthatthereducedsiRNAbindingbytheCM952b，ledtomorefreevsiRNAstoinducesilencingofviralRNAs，resultinginattenuatedsymptoms[1581．Inplanta．CMV2bformsinclusionswerelocalizedinthenucleuswhichmadetheis01ationof2bmuchharderthanP19[182]．Thisnucleuslocalizationmighthavearoleinitssilencingsuppressionfunction[183】．However,Gonz{ilezandtheircolleagueshavedemonstratedthatRNAbindingby2bismuchmoreimportantforsuppressionofRNAsilencingthanitsnucleuslocalization[181]．Inaddition，a2bproteinofCucumovirusstructurewasanalyzed．Inthisstudy，2bproteinhasbeendemonstratedtogenerateahook-likedimertorecognizeandbindwith2lntand33ntsiRNAduplexesinasequence—independentmanner；this2bcanalsobinddsRNAsuponchangingofitssecondarystructure[159]．The廿lirdwellillustratedVSRsuppressedsilencingbysiRNAsduplexsequestrationisthehelpercomponentproteinase(HcPro)ofthepotyvirusgroup[173J．HcProwasoneofthefirstidentifiedVSRs【■SeveraldifferentmodelsfortheHcProsilencingsuppressionmechanismshavebeenproposed[56,168,173]．Amongthem，siRNAduplexsequestrationhasmoredetailsofthemolecularbasis．Accordingthestructuralanalysis，HcProcontainsRNAbindingdomains[227,228]．Subsequently,HcProbindingwithsiRNAduplexeswasdemonstratedbothinvitroandinvivo．DifferentfromP19，the2一nt3'overhangsstructurewasrequiredforHcProbinding．LikeP19and2b，HcPro28 价NAl_>11u≈o∞ooIloc《7T_Z口=傅“II_ocI|o囊熬=011再口醇．I雠u口一oo
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