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Structure and Expression of Several Putative Cdc42-Interacting Proteins in Magnaporthe grisea

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Structure and Expression of Several Putative Cdc42-Interacting Proteins in Magnaporthe grisea

    Structure and Expression of Several

    Putative Cdc42-Interacting Proteins in

    Magnaporthe grisea

    Availableonlineatwww.sciencedirect.com

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    StructureandExpressionofSeveralPutativeCdc42-InteractingProteinsin grtsea

    ZHENGWu,CHENJisheng,ZHENGShiqin,LUGuodongandWANGZonghua

    KeyLaboratoryofBio

    pesticideandChemistryBiology,MinistryofEducation~FunctionalGenomicsCenter,FujianAgricultureand

    ForestryUniversity,Fuzhou350002,P.R.China

    Abstract

    MgCdc42(Cdc42inMagnaporthegrisea),withhighhomologytoScCdc42(Cdc42inSaccharomycescerevisiae),has

    beendemonstratedtoinvolveinthemorphogenesisandinfectionprocess.Tofurtherunderstandthesignalingnetwork,

    theputativeMgCdc42interactingproteinswereanalyzed.ScCdc42

    interactingproteinsequenceswerefirstusedto

    BLASTagainsttheM.griseagenomedatabasetoretrievetheircorrespondinganalogs.Subsequently,conserved

    domainsoftheseproteinswerecomparedandexpressionpatternsoftheirencodinggenesindifferentMgCdc42mutation

    stateswereanalyzedbysemiquantitativeRTPCR.A1lretrievedanalogsofScCdc42

    interactingproteinsfromtheM.

    griseadatabasehaveconserveddomainsasthoseinS.cerevisiae.Expressionoftheirencodinggenesincreasedin

    MgCdc42CAmutantanddecreasedinMgCdc42KOmutant.However,MgBeml,Chml,andMgGiclinMgCdc42DN

    mutanthadthesameexpressionlevelasthatinthewildtype,althoughMgBem4,MgBoi2,MgCdc24,MgGic2,MgRgal,

    andMst20haddecreasedexpressionlevel,asexpected.Overall,itisconcludedthattheremayexistasimilarCdc42signal

    pathwayinM.griseaasinS.cerevisiaeandMgCdc42playsakeyroleinthepathway. Keywords:Cdc42,Magnaporthegrisea,guaninenucleotideexchangefactor,GTPase

    activatingprotein

    lNTRODUCTlON

    Cdc42(celldivisioncycle42).aRhofamilyGTPbind

    ingprotein(GTPase),functionsasaversatilemolecu-

    larswitchinnumeroussignaltransductionpathways

    andvariouscytoskeletonmediatedcellularprocesses

    ineukaryotes.Cdc42wasfirstdiscoveredinSaccha

    romycescerevisiaeasacelldivisioncontrolgenethatis

    requiredforpolarizationofmothercellsandbuddingof

    daughtercellsfAdamseta1.1990;JohnsonandPringle

    1990).Cdc42wasinitiallyshowntocontrolcellpolar-

    ityviaregulationoftheactincytoskeleton.However.it

    wasrecentlydemonstratedtocontrolawiderangeof

    cellularprocessesbymodulatingnotonlytheactincy-

    toskeletonbutalsothemicrotubulecytoskeleton,the subcellularlocalizationofsignalingproteins,andgene expressionfEtienneManneville2004).

    TheabilityofCdc42toparticipateinvarioussignal

    ingpathwaysandtocontroldiversecellularprocesses liesinitscapacitytoactivatemanyfunctionallydistinct effectorproteinsandbeactivatedbYmultiPle mechanisms.Itiswellknownthatsignalingmoduleof smallGTPasessuchasCdc42consistsofregulators oftheguaninenucleotideboundstateofGTPase,ie

    guaninenucleotideexchangefactors(GEFs),guanine nucleotidedissociationinhibitorsfGDIs),andGTPase

    activatingproteins(GAPs)(Johnson1999).Thesig

    nalsareturnedonandoffbetweenactiveGTPbound

    andinactiveGDPboundCdc42.

    ThispaperistranslatedfromitsChineseversioninScienfiaAgriculturaSinica.

    CorrespondenceLUGuodong.Email:lgd@fjau.edu.cn;WANGZonghua,E

    mail:wangzh@fjauedu.ca.'Theseauthorscontributedequallytothispaper

    02006,CAAS.Allnghtstasarvl~.PublishedbyElsevierLtd.

StructureandExpressionofSeveralPutativeCdc42

    InteractingProteinsinMagnaporthegrisea781 MultiplefunctionsofCdc42infilamentousfungiin' cludingplantpathogens(Scheffereta1.2005;Chenet a1.2006)havebeenreported.Giventheconserved functionofCdc42inthecontrolofcellpolarity,itwould beanticipatedthatCdc42playsapivotalroleinregula- tionofthefungalmorphogenesis.Ithasrecentlybeen demonstratedthatCdc42isinvolvedintheorganiza- tionofactinskeleton,polaritygrowth,andhyphal

    growthinSchizophyllumcommune(Webereta1.2005), Suillusbovines(Gorfereta1.2001),Wangiella (Exophiala)dermatitidis(YeandSzaniszlo2OOO),Peni- cilliummarneffei(Boyceeta1.2001),Claviceps purpurea(Scheffereta1.2005),andColletotrichum trifolii(Cheneta1.2006).Moreover,Cdc42isre- quiredforpathogenicityinClavicepspurpurea(Scheffer fa1.2005).

    TheascomyceteMagnaporthegriseaispathogenic toeconomicallyimportantcropssuchasrice,barley, wheat,andmillet.Ithasbeenusedasamodelsystem forstudyingfungus-plantinteractions(Valentand Chumley1991;Talbot2003).Thefungalinfectionisa sophisticatedinteractionprocesswithplant.Whenthe fungalconidialandonanda~achtoasuitablesurface, suchasariceleaf,agermtubearises.Subsequently, germ-tubeelongationceasesandthetipofthegerm tubeswellstoformanappressoriumsurroundedbya thickmelanizedcellwal1.Turgorpressurebuildsup insidetheappressorium,forcingapenetrationpeginto theplanttissues.Thisisthenfollowedbyinvasive growthofthefungus(Howardeta1.1991;deJonget a1.1997).Thus,thehostinvasionbythericeblast fungusinvolvesacomplexcellmorphogenesisrequir- ingdynamicchangesinpolarityinresponsetohost factors(Talbot2003).

    Severalsignaltransductionpathwaysincluding MAPKandcAMPcascadehavebeenreportedtoregu- latetheprocess.IntheMAPKcascade,PMK1

    (PathogenicityMAPKinase1),wasfirstreportedto

    regulateappressoriumformationandinfectioushyphal growthinM.grisea(XuandHamer1996).

    Subsequently,Mst20(thehomologofPAKkinaseinS. cerevisiae)andChml(thehomologofS.cerevisiae) werehypothesizedtointeractwithRho-GTPases(Liet a1.2004).TheseresultsindicatedthatCdc42maybe involvedintheinfectionprocessinM.griseabecause itwasshownjnS.cerevisiaethatCdc42actjvatedthe MAPKcascaderMarcuseta1.19951.andtheinterac. tionofCdc42andSte20regulatedtheHog1MAPK pathway(Raitteta1.2000)andisrequiredforefficient activityofthepheromoneresponsepathway(Ashet a1.2003).Andyeasttwo.hybridanalysisshowedthat theconstitutivelyactivatedCdc42interactedwithBeml, Bem4,Boi2,Cla4,Gicl,Gic2,Rgal,andSte20,whereas dominantnegativeCdc42interactedwithBem1.Bem4, andCdc24inS.cerevisiaerDteeseta1.20011. ItisthereforeconsideredthattheCdc42signaling networkinM.griseaissimilartothatinS.cerevisiae. Thus,furtherstudyonMgCdc42putativeinteracting proteinsandtheirexpressionwouldfacilitatetheanaly. sisoftheCdc42-interactingproteinmap,clarification ofthemechanismregulatingfungaldevelopmentand growth,aswellasanovelstrategytocontroltherice blastdisease.

    MATERIALSANDMETHODS

    HomologsearchofScCdc42-interactingproteins AminoacidsequencesofBem1rCAA45320).Bem4 (AAB47774),Boi2(BAA07427),Cdc24(NP_009359), Cla4(CAA578791,Gic1rNP0119281,Gic2

(NP010595),Rga1(CAA62445),andSte20

    (NP0118561fromcerevisiaewereusedasqueries tosearchfortheirhomologsingriseadatabaserhttp:/ /www.broad.mit.edu/annOtatiOn/fungi/magnaporthe/ index.html1byBLASTPprogramwithE.valuesetas le+0.MultiplealignmentwasconductedwithDNAstar ClusterV.Theconserveddomainswereanalyzedby Pfamrhttp://www.sanger.ac.uk/Software/Pfam/ search.shtm1).

    solationoftotaIRNA

    The^griseastrain.70.15.waskindlyprovidedby Dr.XuJinrongrPurdueUniversity,USA).and Cdc42KO(MgCdc42knock-out),Cdc42CA(MgCdc42 constitutivelv.activated).andCdc42DN(MgCdc42 dominantnegative1mutantswerepreviouslyconstructed bytheauthorsofthisstudy.CulturesforRNAisola. tionweregrownonliquidcompletemediumrCM:0.6% yeastextract.0.6%caseinhydrolase.1%sucrose)in ~2006.CAAS.Allrighisrese.Publi,.~edElsevierLtd.

782ZHENGWueta1.

    130r/minshakeratroomtemperaturefor34days.

    Themyceliawerecollectedbyfiltrationandplacedin theliquidnitrogenimmediately.Themixturewasthen groundtopowderforRNAisolation.110talRNAwas isolatedusingSVtotalRNAisolationsystemrPromega Company,USA)fromvegetativelygrowinghyphae followingthesystemprotoco1.ThetotalRNAwas quantifiedbyUVspectrometer(GeneQuantPro,

AmershamBiosciences,Inc.)anddenaturinggelelec

    trophoresisrSambrookandRussell2002). RT?PCRandPCR

    ThefirststrandcDNAforreversetranscriptionalPCR (RTPCR)wassynthesizedbyfollowingtheprotocol ofSuperScriptfirststrandsynthesissystem

    (Invitrogen,USA)with3PgoftotalRNAasthe template.Then,2pLofcDNAwasusedinfurther

    tubu PCRanalysis.PrimersforamplificationofBeta

    linandMgCdc42putativeinteractingproteinswerede

    signedusingDNAstarsoftware(Table)andsynthesized byBeijingSunbiotechCo.,Ltd,China.Amplification wasquantifiedbycomparingthebrightnessof10pL RTPCRproductsafterelectrophoresison1.5%agar

    osege1.

    RESULTS

    ThehomologsofScCdc42?interactingproteins inMgrisea

    Tostudythestructureandexpressionpatternsof MgCdc42-interactingproteininM.grisea.firsttheM. griseadatabasewassearchedusingtheprogram BLASTPbasedontheaminoacidsequencesOf. cerevisiaeCdc42interactingproteins.Thecorrespond

    inghomologsofBeml,Bem4,Boi2,Cdc24,C1a4,Gic1, Gic2,Rgal,andSte20wereretrievedfromM.grisea databaseandwereidenticaltothoseofS.cerevisiae at24.0.11.7,16.9,17.4,34.8,12.1,12.6,17.0,and 32.1%.respectively.TheywerethennamedasMgBeml (MG01702.4),MgBem4(MG00879.4),MgBoi2 (MG07310.4),MgCdc24(MG09697.4),Chm1

    (AAL15449)byLieta1.(2004),MgGicl(MG07663.4), MgGic2(MG01025.4),MgRgal(MG04186.4),and Mst20rAAP93639)alsobyLietaZ.(2004), respectively.

    Furtheranalysiswasconductedtodetectthecon- serveddomainsofMgCdc42interactingproteins.

    MgBem1containsSH3.PX.andPB1domains.Its PB1domainsadoptabetagraspfold.similartodo

    TablePrimersequences,theircorrespondingannealingtemperature,andtimeforextensionu

    sedinRTPCRtodetecttheencodinggene

    expressionofputativeMgCDC42interactingproteinsinM.grisea

    o2oo8.CAASAll岣?reserved.PuNishedbyElsevierLtd.

StructureandExpressionofSeveralPutativeCdc42

    InteractingProteinsinMagnaporthegrisea783 mainsforubiquitinandRasbinding,andPXdomaias

    areimportantphosphoinositide?-bindingmodulesofvari?- ouslipidbindingspecificities(Fig.1).MgBem4has MutSI.II,III,IV,andVdomainsfoundin

    MutSfamilyproteins(forDNAmismatchrepair). MgBoi2consistsofSH3.SAM,andPHdomains.Sterile alphamotiffSAM)areknowntoinvolveindiversepro

    tein.proteininteractionsassociatedwithbothSAMcon

    tainingandnonSAMcontainingproteinpathways.111e pleckstrinhomology(PH)domainisadomainofabout l00residuesthatoccursinawiderangeofproteins thatareinvolvedinintracellularsignalingorthatfunc

    tionascytoskeletonconstituents.MgCdc24hasCdc24 calponin.RhoGEF,andPB1domaias.Cdc24calponin

isacalponinhomologousdomainandguaninenucle

    otideexchangefactorrGEF)forRho/Rac/Cdc42like

    GTPases,theregulatorofRhoGTPasesthatprompts RhoGTPasestoGTPboundfromGDPbound.

    MgGic1containspseudoU——

    syntdomains,whichwere

    initiallyidentifiedintRNApseudOuridinesynthase. MgRgalhasLIMandRhoGAPdomains.RhoGAPisa domaininRho/Rac/Cdc42..1ikeguanine..activatedpro.- teinsfoundinawidevarietyofmultifuncfionaiproteins. LIMdomain.bindingwithtwozincions,actsasan interfaceforproteinproteininteraction.Mst20and

    SH3PXPB1

    ChmlcontainaconservedCterminalserine/threonine

    catalytickinasedomainandaP2lRhobindingdomain

    (PBD,attheNterminalnoncatalyticregion.PBDisa domainthatbindsCdc42p..and/orRho..1ikesmall GTPases.whichisalsoknownasCdc42/Racinterac

    tivebindingfCRIB)domain.Furthermore.PHdomain alsoexistsinChml(Fig.1,.

    Geneexpressionpattert~sofMgCdc42-

    interactingproteinsindifferentmutants TounderstandtherelationshipbetweenMgCdc42and ltsinteractingproteins,expressionpatternsofencod

    inggenesoftheseputativeMgCdc42?-interactingpro?- teinsweredetectedinwildtypeM.griseaCdc42KO,

    Cdc42CA,andCdc42DNstraiasbysemiquantitative RTPCR(Fig.21.AcomparisonofinternalcontrolBeta

    tubulinamplificationshowedthattherewasnoobvious differenceintheamountoftotalRNAloadedfordif-

ferentstrains.InMgCdc42KOmutant,geneexpres

    sionofalltheputativeMgCdc42interactingproteins decreasedalthoughtheycouldstillbedetected.In MgCdc42CAmutant,expressionofMgBeml,MgBem4, MgBoi2,MgCdc24,Chml,MgGicl,MgGic2,MgRgal, andMst2oincreasedconstantlY.However.in MgBem1

    MutslMutsIIMutsIllMuts_

    lVMuts

    V

    SH3

    M

    SAMPH

    MgBem4

    CalponinRhoGEF

    MgBoi2

    PBl

    PseudoUsyntPseudoUsynt

    MgGic1

    RhoGAP

    MgCdc24

    PBDPKinase

    PHPBDPKinase

    Fig.1StructureoftheputativeregulatorsandeffectorsofMgCdc42

    Mst20

    Chm1

    MgRga1

    ~2006.CMS.Allnghbrened.PrOWledbyElsevierLtd.

784ZHENGWtlf

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