An RNA Virus-Encoded Zinc-Finger

ThePlantCell,Vol.25:960–973,March2013,http://wendang.chazidian.comã2013AmericanSocietyofPlantBiologists.Allrightsreserved.

AnRNAVirus-EncodedZinc-FingerProteinActsasaPlantTranscriptionFactorandInducesaRegulatorofCellSizeandProliferationinTwoTobacco

NinaI.Lukhovitskaya,aAnnaD.Solovieva,bSantoshK.Boddeti,aSrinivasThaduri,aAndreyG.Solovyev,c,dandEugeneI.Savenkova,1

ofPlantBiologyandForestGenetics,UppsalaBioCenter,SwedishUniversityofAgriculturalSciencesandLinnean

CenterforPlantBiology,SE-75007Uppsala,Sweden

bDepartmentofVirology,BiologicalFaculty,MoscowStateUniversity,Moscow119992,Russia

cA.N.BelozerskyInstituteofPhysico-ChemicalBiology,MoscowStateUniversity,119992Moscow,RussiadInstituteofAgriculturalBiotechnology,RussianAcademyofAgriculturalSciences,127550Moscow,Russia

aDepartment

Plantvirusescauseavarietyofdiseasesinsusceptiblehosts.Thediseasesymptomsoftenincludeleafmalformationsandotherdevelopmentalabnormalities,suggestingthatvirusescanaffectplantdevelopment.However,littleisknownaboutthemechanismsunderlyingvirusinterferencewithplantmorphogenesis.Here,weshowthataC-4typezinc-?nger(ZF)protein,p12,encodedbyacarlavirus(chrysanthemumvirusB)caninducecellproliferation,whichresultsinhyperplasiaandsevereleafmalformation.Wedemonstratethatthep12proteinactivatesexpressionofaregulatorofcellsizeandproliferation,designatedupp-L(upregulatedbyp12),whichencodesatranscriptionfactorofthebasic/helix-loop-helixfamilysuf?cienttocausehyperplasia.Theinductionofupp-Lrequirestranslocationofthep12proteinintothenucleusandZF-dependentspeci?cinteractionwiththeconservedregulatoryregionintheupp-Lpromoter.Ourresultsestablishtheroleofthep12proteininmodulationofhostcellmorphogenesis.ItislikelythatothermembersoftheconservedC-4typeZFfamilyofviralproteinsinstigatereprogrammingofplantdevelopmentbymimickingeukaryotictranscriptionalactivators.

INTRODUCTION

Plantinnateimmunityhasevolvedasamultilayereddefensesystemtorecognizeandresistinvadingpathogens(JonesandDangl,2006).Asacounterdefensestrategytoovercomethe?rstlayerofdefense,pathogensdelivervariousvirulencedetermi-nantsintotheplantcell.Thesevirulencefactorshavebeentermedeffectors(BentandMackey,2007).Effectorshijackcellularmachinerytosuppressimmuneresponsesandenhancevirulence(DoddsandRathjen,2010).Gram-negativebacteriaemployatypeIIIsecretionsystemtoinjecteffectorsintohostcells(GalánandWolf-Watz,2006).PlantpathogenicbacteriaofthegenusXanthomonasdelivertranscriptionactivator-likeef-fector(TALe)proteins,whichactastranscriptionfactors(TFs)andactivatehostpromotersthroughdirectbindingtoUPT(upregulatedbyTALe)promoterboxes(Kayetal.,2007;Whiteetal.,2009;Römeretal.,2010).TherebyXanthomonasTALes,dependingoncropcultivar,caneitherpromotediseasethroughhypertrophyandhyperplasiadevelopmentorinduceplantde-fenseifthecorrespondingUPTboxislocatedinthepromoterof

1Address

correspondencetoeugene.savenkov@slu.se.

Theauthorresponsiblefordistributionofmaterialsintegraltothe?ndingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(http://wendang.chazidian.com)is:EugeneI.Savenkov(eugene.savenkov@slu.se).Some?guresinthisarticlearedisplayedincoloronlinebutinblackandwhiteintheprintedition.OnlineversioncontainsWeb-onlydata.

http://wendang.chazidian.com/cgi/doi/10.1105/tpc.112.106476

aresistancegene(Römeretal.,2007).Interestingly,anAvrBs3TALeofXanthomonasspppromotestissuegrowthafterin-?ltrationonleavesofSolanaceaespeciesand,inpepper(Cap-sicumannuum),isknowntoinduceacellsizeregulator,basic/helix-loop-helix(bHLH)-typeTFUPA20,suf?cienttocausetis-suegrowth(Kayetal.,2007).

Whereaspathogen-inducedexpressionofspeci?cgenesandtranscriptionalreprogrammingofhostgeneexpressionhasbeenlinkedtohyperplasiaandothermorphologicaldefects,especiallyforsomemembersoftwogeneraofgeminiviruses(Lathametal.,1997;Pirouxetal.,2007;Ascencio-Ibáñezetal.,2008;Mills-LujanandDeom,2010),noTF-likeproteins,whichwoulddirectlybindtohostpromoters,havebeenreportedforphytoviruses.TheC4proteinsoftwomonopartitemembersofthegenusBegomovirusandCurtovirusinducehyperplasiaprobablyduetointerferencewiththebrassinosteroidpathwayandnotasdirectTFs(Lathametal.,1997;Pirouxetal.,2007).AlthoughtheC4proteinofbeetcurlytopvirus(genusCurtovirus,familyGeminiviridae)induceslocalizedhyperplasiaininfectedphloemtissueandtumorogenicgrowthintransgenicplants,theC4proteinhasnotbeenlinkedtoactivationofhostpromotersthroughdirectbindingtospeci?cpromoterelements(Pirouxetal.,2007;Mills-LujanandDeom,2010).Onthecontrary,theC4proteinofbeetcurlytopvirusislocalizedtotheplasmamembrane,interactswithplantSHAGGY-relatedproteinkinases,andseemstoactthroughthedisruptionofbrassinosteroidphytohormonalnetworksandsignaling(Pirouxetal.,2007).Forothergeminiviruses,C4andthehomologousAC4/AL4havebeenreportedtobesilencingsuppressors(Vanitharanietal.,2004).

Recently,wefoundthatthep12proteinofchrysanthemumvirusB(CVB)isimplicatedinhostgeneinductionasapatho-genicityandvirulencedeterminant(Lukhovitskayaetal.,2009).CVBbelongstothegenusCarlavirus,whichcomprises35de-?nitivespeciesand29tentativememberswithconservedge-nomeorganization(LevayandZavriev,1991;Adamsetal.,2004;Martellietal.,2007).Theirpositive-strandedgenomicRNAencodessixgenes,ofwhichthegeneforp12occupiestheextreme39-terminalposition(seeSupplementalFigure1Aon-line;Adamsetal.,2004;Martellietal.,2007).CVBp12anditshomologsinothercarlavirusesareCys-richproteinscharac-terizedbyacentralC-4typezinc?ngerde?nedbyitsRCxRCxRxxPx6-8CDxxxCmotifandanadjacentnuclearlocali-zationsignal(NLS;seeSupplementalFigure1Conline;Gramstatetal.,1990;Kooninetal.,1991;Lukhovitskayaetal.,2009;notethatthecarlavirusC-4superfamilyshouldnotbeconfusedwiththeC4proteinsofgeminiviruses).Thep12proteinbindsbothRNAandDNAbutexhibitsapreferenceforDNAinthepresenceofZn2+anditspathogeniceffectsdependontranslocationintothenucleus(Gramstatetal.,1990;Lukhovitskayaetal.,2009).Sincecarlavirusesarepositive-strandedRNAvirusesandrepli-cateinthecytoplasm,theroleofthep12proteinnuclearlocali-zationandDNAbindinginthevirus–plantinteractionshasbeenenigmatic.Wehypothesizedthatthep12mightactasatran-scriptionalactivatorlocalizedtotheplantnucleus(Lukhovitskayaetal.,2009).

Thep12-likeproteinofanothercarlavirus,potatovirusM,hasadifferentfunctionandactsinconcertwiththeTGB1(forTripleGeneBlock)proteinasasilencingsuppressortoenhanceviralRNAaccumulation,cell-to-cellmovement,andsystemictransport(Senshuetal.,2011).Ontheotherhand,wedemonstratedex-plicitlythatCVBp12didnotsuppressRNAinterferenceand,moreover,didnotsuppressmicroRNA-mediatedmRNAcleavage(Lukhovitskayaetal.,2005,2009).Theseresultssuggestthattherearesomefundamentaldifferencesevenamonghomologousproteinsencodedbythevirusesofthesamegenus.Indeed,inthisarticle,characterizationofthehostresponsestothep12proteinexpressionrevealedadditionalunexpectedlevelsofcomplexityduringvirus–hostinteractions.Wedemonstratedthatthep12proteinactsasaeukaryoticTFtoupregulateabHLH-TF–encodinggene,whichinturnstimulatesectopiccellproliferationandmod-ulatestissuegrowthinthemesophyllofinfectedleaves.

RESULTSANDDISCUSSION

p12ExpressionCausesSevereLeafMalformationandHyperplasia

Chrysanthemum(Chrysanthemummorifolium)plantsinfectedwithCVBoftenshowcurlingoftheupperleaves.CrosssectionsandlightmicroscopyrevealedpronouncedcellproliferationinCVB-infectedchrysanthemumleavescomparedwiththemock-inoculatedplants(Figure1A;seeSupplementalFigure2Aon-line).Thisaberranttissuestructureisreferredtohereafterashyperplasia(i.e.,disorganizedcelldivision).Interestingly,Agro-bacteriumtumefaciens–launchedCVBp12expressionresultedinhyperplasia(seeSupplementalFigure2Bonline),althoughit

RNAVirus-EncodedTranscriptionFactor961

wasnotaspronouncedasduringthevirusinfection,probablyowingtolowlevelsofp12proteinaccumulationuponagro-in?ltrationaswasrevealedbyimmunoblotting(seeSupplementalFigure2Conline).Therefore,toachieveproteinexpressionlevelscomparabletothoseduringCVBinfectionandtoinvestigatethehostresponsesspeci?callytop12intheabsenceofothercar-laviralproteins,weexpressedp12fromtwounrelatedvirusvec-tors(toavoidvector-relatedbias),namely,theTobaccomosaicvirus(TMV)vector(TMV-p12construct)andthePotatovirusX(PVX)vector(PVX-p12;seeSupplementalFigure1Bonline).Re-gardlessofthevectorused,weobservedstrongphenotypesmanifestedassevereleafmalformation(Figure1B;seeSupplementalFigures2Dand2Eonline)inthesusceptibleNico-tianatabacumcvSamsun.Lightmicroscopyrevealedpronouncedcellproliferation(increaseincellnumber)inthep12-expressingleaftissue,withpalisadeandspongymesophyllcellsbeingstronglyreducedinsizebutmoreabundantinnumbercom-paredwiththoseintissueofthecontrol(seeSupplementalFigure2Fonline).Thus,p12convertedtypicalmosaicsymptomsofTMVandPVXintoleafmalformationbecauseofhyperplasiaoftheplanttissue.

SevereleafmalformationandhyperplasiawereobservedinNicotianaoccidentalisssphesperisplants,commonlyknownasnativetobacco,infectedwithCVB(Figures1Cand1D),dem-onstratingthatour?ndingsre?ectactualeventsduringcarlavi-rusinfectionnotonlyinchrysanthemumbutalsoinoneofthetobaccospecies,whichiscommonlyusedasindicatorhostforCVB.AdditionalphenotypesassociatedwithCVBinfectioninnativetobaccoincludedhypersensitiveresponse(HR)inductionatlaterstagesofvirusinfection(seeSupplementalFigure2Gonline)consistentwiththepreviouslyreportedroleofp12inHRinduction(Lukhovitskayaetal.,2009).

Thep12-inducedhyperplasiamightaccountfortheleafmal-formationsymptomsobservedininfectionscausedbysomeothercarlaviruses(HakkaartandMaat,1974;Dijkstraetal.,1985;Larsenetal.,2009;Tangetal.,2010).Althoughleafmalformationisaquitecommonsymptominducedbymanycarlaviruses,tothebestofourknowledge,noneofthehost-carlaviruspathosystemsotherthanCVBhavebeeninvestigatedforhyperplasiainductionandthisawaitscharacterization.p12InducesExpressionofabHLHTF

Sincetobaccospeciesrepresentamoregeneticallytractablemodelthanchrysanthemum,andbothreactwithhyperplasiatop12expression,allsubsequentexperimentswereperformedintobacco.Thephenotypescausedbyp12resembledleafmalfor-mationinducedbyCaUPA20TFafterin?ltrationontobaccoleaves(Kayetal.,2007).Therefore,wewantedtoanalyzewhetheratobaccoorthologofthepepperUPA20isupregulatedinthep12-expressingtissues.Sincethegenesequencewasnotavail-able,weperformedtobaccoESTdatabasesearchesandidenti?edanESTencodingapolypeptidehighlysimilartotheUPA20proteinfrompepper.Real-timeRT-PCRexperimentsperformedwithpri-mersdesignedbasedontheESTsequencerevealedupregulationofthetobaccogeneinbothTMV-p12andPVX-p12–infectedplants(Figure1E).Therefore,theupregulatedtobaccogenewastermedupp(forupregulatedbyp12).

962ThePlantCell

Figure1.CVBInducesHyperplasiainChrysanthemumandTobaccoLeaves,andp12UpregulatesExpressionofabHLHTF.

(A)Crosssectionsandlightmicroscopyofchrysanthemumupperleavesfrommock-inoculatedplantsandfromplantssystemicallyinfectedwithCVB.

(B)AppearanceofthesymptomsinducedbyemptyTMVvectorandTMV-p12intobacco.

(C)LeafmalformationsymptomsinducedbyCVBinsystemicallyinfectedleavesofN.occidentalisssphesperis.

(D)CrosssectionsandlightmicroscopyofN.occidentalisssphesperisleavesfrommock-inoculatedplantsandfromplantssystemicallyinfectedwithCVB.

(E)ExpressionlevelsofuppweredeterminedbyquantitativeRT-PCR.Valuesontheyaxisrepresentnormalizedfoldexpression(n=6);theerrorbarsshowSD;*P<0.05,**P<0.01,and***P<0.001.

(F)Structureofupp-Landupp-Sintobacco.Boxesandlinesindicateexonsandintrons,respectively.

(G)AlignmentofN-terminalsequencesofUpp/Upa20andbHLH137fromArabidopsis.ThetranslationinitiationcodonforNtupp-Sisunderlined.(H)PhylodendrogramofUpp/Upa-likeTFsfrom18plantspecies.ArabidopsisAt1g59640BIGPRTALpbHLHTFwasusedasanoutgroup.Forplantspeciesabbreviationsandotherdetails,seeSupplementalTable1online.NumbersrepresentGenBankaccessionnumbersforthecorrespondinggenes.ThesequencealignmentusedforthisanalysisisavailableasSupplementalDataSet1online.

(I)Expressionlevelsofupp-Landupp-SweredeterminedbyquantitativeRT-PCR.Themean22DDCTvalues6SDfromthreeindependentexperimentsareshown(n=9);**P<0.01and***P<0.001.cDNAinputswerenormalizedtomRNAofthreereferencegenes(NtEF-1a,Ntb-actin,andNtcdc2).(J)N.occidentalisssphesperisupp-LisupregulatedduringCVBinfectionasanalyzedbyRT-PCR.

Barsin(A)and(D)=100µm.

[Seeonlinearticleforcolorversionofthis?gure.]

Weisolatedthefull-lengthcDNAoftheuppgenesfromN.

tabacumandN.occidentalisssphesperisbyrapidampli?cation

ofcDNAends(RACE)anddeterminedthecorrespondingge-

nomicsequencefromclonesgeneratedbyPCRandgenome

walking.Itappearedthatthetobaccogenomeencodestwoupp

paralogs,designatedNtupp-LandNtupp-S(Figure1F),whereasthegenomeofnativetobaccoencodesonlythelongergene(http://wendang.chazidian.comparedwiththeupp-Lgenes,Ntupp-Shasatwonucleotidedeletioninthebeginningofthecodingregionthatpreventsinitiationonthe?rstAUGcodon(Figure1G)andresultsinashorterprotein.Ntupp-LandNtupp-Sgenesbothcompriseeightexonsandsevenintrons(the6thintronis

longer

内容需要下载文档才能查看

RNAVirus-EncodedTranscriptionFactor963

Figure2.InducibilityoftheNtupp-LandNtupp-SPromotersandPromoterFragments.

(A)to(C)AnalysisofGFP?uorescence3DAIshowsp12inducibilityoftheNtupp-L(A)andNtupp-S(B)promoterscomparedwiththeconstitutiveactin2promoter(C).TheNtupp-L,Ntupp-S,andactin2promoterswereclonedupstreamofGFPandtestedbycodeliverywithp12(righthalfoftheleafin[A]to[C])oremptyT-DNA(lefthalfoftheleaves).

(D)TheGFPproteinlevelsuponGFPcodeliverywithp12andemptyT-DNA.Coomassiebluestainingofthemembraneispresentedasaloadingcontrol.

(E)FragmentsoftheNtupp-LandNtupp-Spromoterstestedforp12inducibility.PositionoftheUPAboxisshownasagrayrectangle.

(F)ResultsofGFP?uorometricanalysisoftheproteinextractsfromAgrobacterium-in?ltratedN.benthamianaleaves.Thepromoterfragmentsshownin(E)wereclonedupstreamofGFPandassayedasbefore(seeabove).Thebaselinerepresentsthelevelof?uorescenceoftheproteinextractfromtheleafexpressinganemptyT-DNA.Statisticalanalysiswasperformedinthreeindependentexperimentsbyttest,P<0.05.ErrorbarsdenoteSEofthemeasurements.

964ThePlantCell

inNtupp-L,438nucleotidesversus156nucleotidesinNtupp-S;Figure1F)andencodeputative362–and260–aminoacidbHLH-typeTFswithpredictedmolecularmassesof42.2and29.0kD,respectively.Asexpected,BLASTanalysesdemon-stratedthatNtupp-L,Nhupp-L,andNtupp-SarerelatedtoUPA20frompepper.TheonlyhomologofNtupp-LinArabi-dopsisthalianaisanuncharacterizedbHLH137TF(At5g50915;54%identityand65%similaritytoNtupp-L).ThecentralpartoftheUPA/upp-L–relatedproteinsiswellconservedandcom-prisesthebHLHmotif,whereastheNterminioftheproteinsshowverylittlesimilaritybetweentheUPA/upp-LproteinsandAtbHLH137(Figure1G).

AphylogeneticanalysisoftheUPA/upp-L–relatedproteinsshowedthattheyformedfourdistinctcladesinthephyloden-drogramwithahighstatisticalprobability(bootstrapvalues>70%),andthesecladeswerenamedgroups1to4,respectively(Figure1H;seeSupplementalTable1andSupplementalDataSet1online).Notably,group1includesthebHLHproteinsfromSolanaceaespecies,group3comprisesproteinsfrommono-cotyledons,andAtbHLH137fallsintogroup4andisdistantlyrelatedtogroup1(Figure1H).

Real-timeRT-PCRexperimentsemployingsetsofgene-speci?cprimers,whichareabletodiscriminatebetweenNtupp-LandNtupp-S,revealedupregulationofbothNtupp-LandNtupp-Sbyp12(Figure1I).Similarly,CVBinfectionactivatedtheNhupp-Lgene,theN.occidentalisssphesperisorthologoftobaccoupp-L(Figure1J).

Bothupp-Landupp-SPromotersArep12ResponsiveActivationoftheuppgenesbyp12mightbeeitherdirect(i.e.,involvinginteractionofp12withtheupppromotersand/orthepromoter-interactingTFs)orindirectviasignaltransductionpath-wayswherep12interactswithacellularreceptorandinitiatesasequenceofeventsresultingintheupregulationoftheuppgenes.Toelucidatethemechanismofuppactivation,westudiedtheinducibilityoftheupppromotersbyp12.WeisolatedtheNtupp-LandNtupp-Spromotersbygenomewalking.SequencecomparisonsoftheNtupp-L,Ntupp-S,andCaUPA20promotersrevealedthepresenceofapreviouslyidenti?edconservedmotifwiththeconsensussequenceTATATAAACCN2-3CCthatwastermedtheUPAbox(Kayetal.,2007,2009;seeSupplementalFigure3Aonline).ATATA-boxislocatedwithintheUPAbox33bpupstreamofthetranscriptionstartsite.

Toanalyzethep12inducibilityoftheupppromoters,weusedtheupp-Lpro:GFP(forgreen?uorescentprotein)andupp-Spro:GFPcassettesasreportersandactinpro:GFPasaconstitutivepromotercontrolinN.benthamianaafterAgrobacterium-mediatedtrans-formation.GFP?uorescenceandimmunoblotsrevealedthatbothupppromotersarep12responsive(Figure2;seeSupplementalFigures3Cto3Eonline).Engineeredpromoterdeletionsfrom

bothendsnarrowedthep12-responsiveregiontoa103-bpse-quence94%identicalinbothpromoters(sixvariablepositionsper103bp)(Figures2Eto2G).Notably,the103-bpfragmentsforbothpromoterspossessedtheUPAbox(http://wendang.chazidian.comparedwithupp/UPA,theAtbHLH137promoteralsohasanUPAbox,thoughslightlydeviatingfromtheconsensus(TATATAAACCNCC),andcouldbeactivatedbyp12(Figure2G,rightpanel).Nevertheless,Agrobacterium-mediatedAtbHLH137overexpressiondidnotinduceanyvisibletissueorcellularchanges.Concordantwiththeseobservations,transientexpressionofXanthomonasAvrBs3TALeinArabidopsissppdoesnotcauseanycellularchanges(Maroisetal.,2002),furtherreinforcingtheideathattheTFsofgroup1(Figure1H)mightdifferfromthoseofgroup4intheirroleinplantmorphogenesis.

Inaddition,thep12mutantsalteredintheNLSorzinc?nger(Lukhovitskayaetal.,2009)failedtoactivatetheupppromoters(seeSupplementalFigure4online).Thesedatafurthersup-portedourhypothesisofdirectpromoteractivationbyp12,asourpreviousstudyrevealedthatthep12ZFdomainisinvolvedDNAbindingandtheNLSisrequiredforp12translocationintothenucleus(Lukhovitskayaetal.,2009).p12AssociateswithuppPromoters

Weperformedachromatinimmunoprecipitation(ChIP)assaywithahemagglutinin(HA)epitope-speci?cantibodytodeterminewhethertheHAepitope–taggedp12associateswiththeupppro-motersinvivo.ChromatinwasisolatedfromplantsinfectedwithPVXorPVX-p12HA.PrecipitatedDNAswereextractedandam-pli?edbyPCRusingprimers?ankingthe103-bpp12-responsiveelement.p12precipitatedwiththeupppromoterscontainingtheUPAbox,butnotwithasequencelocated2.5kbdownstreamoftheTATA-box(Figures3Aand3B;seeSupplementalFigure5Aonline).Thisdemonstratesthattheinteractionofp12withDNAinvivoisspeci?c.Cloningandsequencingofthe103-nucleotidefragmentsthatprecipitatedwithp12revealedthepresenceofboththeNtupp-LandNtupp-Spromotersequences.

TheChIPdataweresubstantiatedbysubcellularfractionationandanti-HAimmunoblots.Thep12HAproteinwasdetectedinthenuclearP10fractionandintheprecipitatedchromatin(Fig-ures3Cand3D).Additionally,wefoundthatp12directlyboundtothe32P-labeledupppromoterDNAinelectrophoreticmobilityshiftassays(EMSAs)(http://wendang.chazidian.competitionexperimentsin-volvingtwoDNAsubstrates,aDNAfragmentoftheNtupp-LpromotercomprisingtheUPAboxandthesamefragmentwitharandomizedUPAboxsequence,revealedthattheunlabeledUPAboxfreedmoreofthelabeledUPAboxfromthecomplexthantherandomizedUPAboxsequence,suggestinghigheraf?nityofp12totheUPAbox–containingDNA(Figure3F).Acontrolcompetitionbindingwithp12lackingthezinc-?ngerdomainrevealednopreferencefortheUPAbox–containingDNA

Figure2.(continued).

(G)TheGFPproteinlevelsuponGFPcodeliverywithp12andemptyT-DNA.ThesamplesruninthegelincludedL0U0(1179bpupp-Lpromoterconstruct),L1U2(103bpupp-Lpromoterconstruct),S1U2(103bpupp-Spromoterconstruct),andAtbHLH137promoterconstruct.Coomassiebluestainingofthemembraneispresentedasaloadingcontrol.