Physical and Functional Interactions between Patho

PhysicalandFunctionalInteractionsbetweenPathogen-InducedArabidopsisWRKY18,WRKY40,andWRKY60TranscriptionFactorsWXinpingXu,a,1ChunhongChen,b,2BaofangFan,aandZhixiangChena,3aDepartmentofBotanyandPlantPathology,PurdueUniversity,WestLafayette,Indiana47907-2054bDepartmentofMicrobiology,MolecularBiology,andBiochemistry,UniversityofIdaho,Moscow,Idaho83844-3052LimitedinformationisavailableabouttherolesofspecificWRKYtranscriptionfactorsinplantdefense.Wereportphysicalandfunctionalinteractionsbetweenstructurallyrelatedandpathogen-inducedWRKY18,WRKY40,andWRKY60transcrip-tionfactorsinArabidopsisthaliana.ThethreeWRKYproteinsformedbothhomocomplexesandheterocomplexesandDNAbindingactivitiesweresignificantlyshifteddependingonwhichWRKYproteinswerepresentinthesecomplexes.SingleWRKYmutantsexhibitednoorsmallalterationsinresponsetothehemibiotrophicbacterialpathogenPseudomonassyringaeandthenecrotrophicfungalpathogenBotrytiscinerea.However,wrky18wrky40andwrky18wrky60doublemutantsandthewrky18wrky40wrky60triplemutantweresubstantiallymoreresistanttoP.syringaebutmoresusceptibletoB.cinereathanwild-typeplants.Thus,thethreeWRKYproteinshavepartiallyredundantrolesinplantresponsestothetwodistincttypesofpathogens,withWRKY18playingamoreimportantrolethantheothertwo.ThecontrastingresponsesoftheseWRKYmutantstothetwopathogenscorrelatedwithoppositeeffectsonpathogen-inducedexpressionofsalicylicacid–regulatedPATHOGENESIS-RELATED1andjasmonicacid–regulatedPDF1.2.WhileconstitutiveexpressionofWRKY18enhancedresistancetoP.syringae,itscoexpressionwithWRKY40orWRKY60madeplantsmoresusceptibletobothP.syringaeandB.cinerea.TheseresultsindicatethatthethreeWRKYproteinsinteractbothphysicallyandfunctionallyinacomplexpatternofoverlapping,antagonistic,anddistinctrolesinplantresponsestodifferenttypesofmicrobialpathogens.INTRODUCTIONPlantsareconstantlyexposedtoavarietyofmicrobialpatho-gensandthroughevolutionhavedevelopedabatteryofactivedefensemechanismstoprotectthemselves.Inresistance(R)gene–mediatedresistance,directorindirectrecognitionofrace-specificelicitors(avirulenceproteins)bytheplantRgeneprod-uctcanoftenleadtorapidactivationofhypersensitiveresponse(HR)thatconfersspecificandeffectiveresistanceagainsttheracesofthepathogen(DanglandJones,2001).Pathogen-inducedHRisoftenassociatedwithactivationofsalicylicacid(SA)–regulateddefensemechanismsinthesurroundingorevendistalpartsoftheplants,leadingtodevelopmentofsystemicacquiredresistance(SAR).HRandassociatedSA-dependentsignalingpathwaysgenerallyprovidedefenseeffectiveagainstbiotrophicpathogensthatfeedonlivinghosttissues.Ethylene(ET)-andjasmonate(JA)–mediatedsignalingpathways,ontheotherhand,oftenmediateplantdefenseagainstnecrotrophicpathogensthatpromotehostcelldeathatearlystagesofinfection(Glazebrook,2004).IthasbeenwelldocumentedthatthereisanextensiveantagonismbetweenSA-mediatedandJA/ET-mediateddefensesignalingpathways(KunkelandBrooks,2002).Asaresult,blockingSAaccumulationinpathogen-infectedplantscanpromoteJAsignaling(Spoeletal.,2003),andmutationsofimportantregulatorsofJAsignaling,suchasMPK4andCOI1,leadtoenhancedSAaccumulationandsignalinginpathogen-infectedplants(Petersenetal.,2000;Kloeketal.,2001).Plantdefenseresponsesareassociatedwiththetranscrip-tionalactivationofalargenumberofplanthostgenesafterpathogeninfection(RushtonandSomssich,1998).Inductionofcertainpathogenesis-related(PR)proteinsisdependentuponSA-mediateddefensesignalingpathwaysandisassociatedwithresistancetobiotrophicandhemibiotrophicpathogens.TheexpressionofArabidopsisthalianaPDF1.2,whichencodesaproteinwithantimicrobialactivity,isregulatedbyJA/ETdefensesignalingpathwaysandoftencorrelatedwithresistancetonecrotrophicpathogens(Penninckxetal.,1998;Thommaetal.,1998).Otherpathogen-inducedgenesencodeenzymesinvolvedinthebiosynthesisofantimicrobialcompounds(phytoalexins).Pathogen-inducedplantgenesalsoincludethoseencodingregulatoryproteinsinvolvedinsignaltransductionofplant1Currentaddress:KeyLaboratoryofGeneEngineeringoftheMinistryofEducation,BiotechnologyResearchCenter,ZhongshanUniversity,Guangzhou510275,People’sRepublicofChina.2Currentaddress:AgResearchLimited,TennentDrive,PrivateBag11008,PalmerstonNorth,[e-mail];fax[phone].TheauthorresponsiblefordistributionofmaterialsintegraltothefindingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors()is:ZhixiangChen([e-mail]).WOnlineversioncontainsWeb-onlydata.Article,publicationdate,andcitationinformationcanbefoundat–1326,May2006,ficrecognitionofcis-actingpromoterelemen