PlantMolecularBiology53:383–397,2003.
?2003KluwerAcademicPublishers.PrintedintheNetherlands.
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MolecularcharacterizationofBrassicanapusNACdomaintranscriptionalactivatorsinducedinresponsetobioticandabioticstress
DwayneHegedus?,MinYu,DougBaldwin,MargaretGruber,AndrewSharpe,IsobelParkin,SteveWhitwillandDerekLydiate
MolecularGeneticsSection,AgricultureandAgri-FoodCanada,107SciencePlace,Saskatoon,S7N0X2Canada(?authorforcorrespondence;e-mailhegedusd@em.agr.ca)
Received22July2003;acceptedinrevisedform9October2003
Keywords:Brassicanapus,NACdomain,stress,transcriptionalactivation,yeast2-hybridAbstract
SubtractiveexpressedsequencetaganalysisandscreeningofcDNAlibrariesderivedfromBrassicanapusleavessubjectedtomechanicalwounding,?eabeetlefeedingorcoldtemperaturesrevealedeightgenesencodingNAC-domaintranscriptionfactors.Thegeneswerefoundtobedifferentiallyregulatedinresponsetobioticandabioticstressesincludingwounding,insectfeeding,Sclerotiniasclerotioruminfection,coldshockanddehydration.FiveBnNACproteinswereorthologoustoArabidopsisthalianaATAF1orATAF2andgaverisetodevelopmentalabnormalitiessimilartotheA.thaliananamandcucmutantswhenexpressedectopicallyinA.thaliana.Trans-geniclinesexpressingBnNAC14,exhibitedlargeleaves,thickenedstemsandhyper-developedlateralrootsystemssimilartothatobservedwithA.thalianaNAC1,butalsoweredelayedinboltingandlackedanapicaldominanttaproot.SeveraloftheBnNACproteinswerecapableofactivatinggeneexpressioninyeastandrecognizedanelementwithintheCaMV35Spromoter.Ayeasttwo-hybridscreenrevealedthatBnNAC14interactedwithotherselectBnNACproteinsinvitroandidenti?edanadditionalBnNACgene,BnNAC485.Theproteininteractionandtranscriptionalactivationdomainsweremappedbydeletionanalysis.
Introduction
Plantsareunderconstantthreatfromenvironmentalstressandattackfrompathogensandvertebrateandinvertebrateherbivores.Tocopewiththisonslaught,theyhaveevolvedelaboratemechanismstoperceivetheattackandaresponsivesignalinglanguagetotiethereceptionofinformationtotheinductionofappro-priatedefensestrategies.Thesemechanismsencom-passdirectphysicalandchemicaldefenses,aswellasindirectdefenses,suchasthereleaseofpredator-attractingvolatiles(DickeandVanPoecke,2002).Speci?cmetabolites,proteinsandcarbohydratesshedbythepathogenorinsect(Hammond-KosackandJones,1996;Bakeretal.,1997)andpolygalac-turonidefragmentsderivedfromdamagedplantcellwalls(Thainetal.,1990)interactwithspeci?cplantreceptors.Theseservetoinitiatesignalingcascadesleadingtolocalandsystemicdefenseresponsesthat
typicallyconsistofchangestoplantarchitecture(e.g.waxaccumulation,trichomeformation)andthein-ductionofdefenseproteins,hyperoxidizedchemicalsandsecondarymetabolites(Walling,2000).Oneoftheleastunderstoodresistancemechanismsistoler-ance,theabilitytocompensateforherbivoreattackbysustainingandre-growingdamagedtissue(StraussandAgrawal,1999;Stoweetal.,2000).Theplantaltersitsdevelopmentalpattern,establishinganeco-physiologicalequilibriumwiththeattackertolimitanydecreasein?tness(BaldwinandPreston,1999;KesslerandBaldwin,2002).Indeed,oneofthelargergroupsofgenesaffectedbyherbivoreandpathogeninteractionsarethoseinvolvedinplantprimarymeta-bolismandgrowth(Reymondetal.,2000;Stotzetal.,2000;Schenketal.,2000;Hermsmeieretal.,2001).Theregulationofdefensegeneexpressionislargelygovernedbyspeci?ctranscriptionfactors.A
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commonthemethathasemergedwiththesequen-cingofwholeplantgenomesisthatthesefactorsoftenbelongtolargegenefamiliesofmorethan100members.MembersoftheERF,bZIP,WRKY(Singhetal.,2002)andMYB(JinandMartin,1999)fam-iliesarethebestcharacterizedoftheplantdefenseresponseactivators.Whilesomemembersofthesefamiliesarespeci?callyinvolvedintheregulationofdefenseresponsestobioticandenvironmentalstress,othersappeartocoordinateplantdevelopmentalpath-ways(JinandMartin,1999;ChenandSingh,1999;RobatzekandSomssich,2001).
AnothersuchfamilyoftranscriptionfactorsaretheNAC-domainproteins.ThenamederivesfromaconserveddomainoriginallyassociatedwiththeNOAPICALMERISTEM(NAM)geneinPetunia(Soueretal.,1996)andtheATAF1,ATAF2andCUP-SHAPEDCOTYLEDON(CUC)genesofArabidopsisthaliana(Aidaetal.,1997).NAC-domainproteinsareuniquetoplantsandcompriselargegenefamilies(Kikuchietal.,2000);103membersarepresentwithintheA.thalianagenomeandhavebeenimplicatedinvariousaspectsofplantdevelopment.InPetunia,NAMwasexpressedattheprimordiaandmeristemboundar-iesandmutantsfailedtodevelopapicalshoots(Soueretal.,1996).However,additionalNAMgeneswerebe-lievedtobeinvolvedsinceco-suppressionstudiesgaverisetoplantswithfurtherdefectsincludingthickerstems,largerleavesandtheabsenceofaxillarymer-istems(Soueretal.,1998).Similarly,A.thalianacuc1andcuc2double-mutantlineslackedshootapicalmeristemdevelopmentandalsoexhibitedfusedcoty-ledons,sepalsandstamens(Aidaetal.,1997,1999).NAC1,anotherA.thalianaNAC-domaingene,wasfoundtobeinvolvedintheauxin-dependentformationofthelateralrootsystem(Xieetal.,2000).Morere-cently,NAC-domaingeneshavebeenimplicatedintheplantdefenseresponseasthepotatoStNACgeneandA.thalianaATAF1andATAF2geneswereinducedbypathogenattackandwounding(CollingeandBoller,2001).Moreover,thegeminivirusDNAreplicationprotein,RepA,wasfoundtointeractwithtwoNAC-domainproteins,GRAB1andGRAB2;however,theireffectonviralDNAreplicationorplantdevelopmenthasnotbeendetermined(Xieetal.,1999).
AllNAC-domainproteinshaveacommoncon?g-urationconsistingofaconservedamino-terminalNACdomainregionproceededbyahighlyvariablecarboxyterminus.TwolinesofevidencesupportthenotionthatNACproteinsareinvolvedintranscriptionalreg-ulation.First,A.thalianalinesover-expressingNAC1
exhibitedup-regulationoftheauxin-responsivegenes,AIR3andDBP,andacorrespondingdecreasewasob-servedinanti-senselines.NAC1alsolocalizedtothenucleus(Xieetal.,2000).Second,earlyreportsindic-atedthatATAF1andATAF2wereabletotrans-activatetheCaMV35Spromoter(Hirt,originalGenBankannotation).ThiswassubstantiatedbyDuvaletal.(2002)whoshowedthattheA.thalianaNAMNACdomainrecognizeda7bpregionwithintheCaMV35Sas-1(activationsequence1)element.
Inaneffortofbetterunderstandplantdefensesandregulationofsuchweconductedsubtractiveex-pressedsequencetaganalysisofB.napussubjectedtomechanicalwounding,?eabeetlefeedingorcoldtemperatures.Herewereporttheisolationandchar-acterizationofninemembersoftheB.napusNAC(BnNAC)genefamilyandshowthattheyarediffer-entiallyregulatedinresponsetobioticandabioticstress,formheterodimersandgiverisetodevelop-mentalabnormalitieswhenexpressedectopicallyinA.thaliana.
MaterialsandmethodsPlant,fungalandinsectmaterials
BrassicanapuslineDH12075andArabidopsisthali-anaecotypeColumbiaweregrowninsoilundera16?hphotoperiodwithadaytimetemperatureof22Candanighttemperatureof16?C.Rootde-velopmentintransgenicA.thalianalineswasas-sessedinMSmediumwith3%sucroseaccordingtoXieetal.(2000).A.thalianaColumbialineSALK030702.55.50.XcontainingaT-DNAinser-tionintheAt5g63790locusencodingATAF2wasobtainedfromtheArabidopsisBiologicalResourceCenter(Columbus,OH).Insertionmutantinform-ationwasobtainedfromtheSIGNALwebsiteathttp://signal.salk.edu.Theinsertionwasveri?edbyPCR??usingtheATAF2-speci?cprimerSALK030702(5-TGGCGTTGTACGGTGAGAAAG-3??)andeitheroneoftwoT-DNAspeci?cprimers,LBa1(5????-TGGTTCACGTAGTGGGCCATCG-3)orLBb1(5??-CGCTGGACCGCTTGCTGCAACT-3??)aswellasbySouthernblotting.Alinehomozygousfortheinsertionwasusedforphenotypicassessment.
Sclerotiniasclerotiorumisolate‘100’wasorigin-allycollectedonB.napus.Fungalmycelia?weregrownonpotato-dextroseagar(PDA)at20Candstoredat?80?Cin25%glycerol.
Adult?eabeetleswerecollectedinsweepnetsfromawintercanola?eld(B.napuscv.Casino)attheSaskatoonResearchCentrefarm.Beetlesweremaintainedonadietofcabbageleavesinmeshcagesinacontrolledenvironmentchamber(23?C,16hphotoperiodwith?uorescentlighting).Beetleswerestarvedfor48hpriortoconductingthefeedingbioas-says.
ConstructionofnormalandsubtractivecDNAlibraries
TotalRNAwasextractedfrom8-weekoldB.napusDH12075leaves2haftercrushingwithforceps,after30hofconstant?eabeetlefeedingasdescribedbeloworfromplantsthathadbeengrownat5?Cuntiltheyreachedtheexpandedsix-leafstage.Poly(A)+RNAwasisolatedwithOligotex(Qiagen)followingthemanufacturer’sinstructions.cDNAsubtractionwasperformedwiththePCR-SelectcDNASubtractionKit(Clontech).ThePCRproductswereinserteddirectlyintopGEM-TEasy(Promega)andca.1500expressedsequencetags(EST)generatedfromeachlibrary.Nor-malcDNAlibrarieswereconstructedwiththeZAPIIcDNAsynthesiskit(Stratagene).AnESTcorres-pondingtotheamino-terminusofB.napusNAC1-1(BnNAC1-1)wasusedtoscreenthecDNAlibrariestoisolateadditionalfull-lengthBnNACcDNAs.TheBnNACcDNAswereexcisedinvivofromthelambdaZAPExpressvectoraspBluescriptSKphagemidsusingExAssisthelperphage(Stratagene).Expressionanalysis
B.napusleaveswereinfectedwithS.sclerotiorumaccordingtothemethodofLietal.(2003).Tosimu-latewounding,leavesweremechanicallydamagedbycrushingwithsterileforceps.For?eabeetlefeeding,plantswereplacedincageswithstarved?eabeetlesatadensityof100–150insectsperplant.Toexaminetheeffectoflowtemperatureanddehydration,8-weekoldplantswereplacedat5?Cfor24horhadmoisturewithhelduntilthepointofwilting.
Tissueswerecollectedatvarioustimeintervalsandimmediatelyfrozeninliquidnitrogenuponre-moval.TotalRNAwasisolatedbydispensing200mg(wetweight)ofgroundplanttissueintoa1.5mlmicrocentrifugetubecontaining1mlTrizolReagent(Invitrogen)andRNAextractedaccordingtotheman-ufacturer’sprotocol.TheRNApelletwaswashedwith70%ethanol,driedfor5minandre-suspendedin
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50μlRNase-freedouble-distilledH2O.TodetectBn-NACmRNA,20μgoftotalRNAwasseparatedon1.2%agarosegelsin10mMphosphatebuffer(pH7.0)andblottedontoanylonmembranewith10×SSC.Hybridizationswereconductedin0.5Mphosphatebuffer(pH7.2)with7%SDS,1%bovineserumalbu-minand1mMEDTAat65?Covernight.Membraneswerewashedtwicewith2×SSCat65?Cfor10min,twicewith1×SSC/0.5%SDSat65?Cfor20minand?nallywith0.1×SSCat20?Cfor10min.DNAprobesconsistedofPCR-ampli?edfragmentscorres-pondingtoeithertheconservedamino-terminalregionofBnNAC1-1orcarboxyl-terminalregionsuniquetoeachBnNACandlabeledwith[α-32P]dCTPwiththePrime-A-GeneLabelingSystem(Invitrogen).Computationalanalysis
ESTsequenceswereannotatedaccordingtoBLASTnucleotideandproteinalignments(www.ncbi.nlm.nih.gov).Multiplepeptidesequencealignmentswereper-formedwithClustalW1.2and100permutationsofthemultiplealignmentgeneratedusingSeqboot.Distancematriceswerecalculatedfromthepermutatedalign-mentwiththePAMmethodinProtdist.Unrooted,neighbor-joinedtreesweregeneratedwithNeighborandaconsensustreewasdeterminedwithConsense.Sequenceassembly,analysisandphylogenetictoolswereaccessedthroughtheCanadianBioinformaticsResource(www.cbr.nrc.ca).Proteinsecondarystruc-turewasdeterminedbySSPRO,PSIpredversion2.4,PFRMATSSandPHDsecavailableinthePredictPro-teinserver(cubic.bioc.columbia.edu/predictprotein)andCOILS(www.ch.embnet.org/software/COILS).DeterminationofnuclearlocalizationsequenceswasperformedbyPredictNLS(maple.bioc.columbia.edu/predictNLS)withthemethoddescribedbyCokoletal.(2000).Peptidesequencescommontoproteinsthataretargetedforrapidturnoverwereidenti?edwithPEST-FIND(www.at.embnet.org/embnet/tools/bio/PEST?nd).Planttransformation
TheentirecodingregionsofsixBnNACcDNAs(1-1,5-1,5-7,5-8,5-11and14)wereampli?edbyPCRwithPWODNApolymerase(Stratagene)andoligo-nucleotideprimerscontainingrestrictionenzymesitessuitableforcloningintopBI121(Clontech)down-streamoftheCaMV35Spromoter.Senseandanti-senseBnNAC5-1wereinsertedintotheBamHI/SacIsites,BnNAC5-7,5?8and5?11intotheXbaI/SacI
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sitesandBnNAC1-1and14intotheBamHI/EcoICRIsites.Planttransformationwascarriedoutaccordingtothe?oraldipprocedure(CloughandBent,1998).MappingofBnNACtranscriptionactivationdomainsTheentirecodingregionsofnineBnNACcDNAs(Bn-NAC1-1,3,485,5-1,5-7,5-8,5-11,8and14)wereampli?edbyPCRwithPWODNApolymeraseandinsertedintotheSalIandNotIsitesofpDBLeu(In-vitrogen)whichfusedtheBnNACopenreadingframetotheGAL4DNA-bindingdomain(DB).Eachcon-structwasintroducedintoSaccharomycescerevisiaeMaV203andtestedinaone-hybridsystemfortheabilitytoactivatetranscriptionoftheLacZreporter.TheBnNACgenesweredividedintoregionsencod-ing?veNACsubdomainsaccordingtotheschemeofDuvaletal.(2002)andtwocarboxyl-terminalsub-domains.VariouscombinationsofthesubdomainsderivedfromBnNAC14,BnNAC5-8andBnNAC485wereampli?edbyPCR,insertedintopDBLeuandexaminedfortranscriptionalactivationoftheLacZreportergene.
Identi?cationofBnNAC-interactingproteinswithayeast2-hybridscreen
Ayeasttwo-hybridcDNAlibrarywasconstruc-tedfrompoly(A)mRNAisolatedfromtheabove-groundpartsoffour-leafstageseedlingsofB.napusDH12075andclonedintotheGAL4activationdo-main(AD)vector,pPC86,usingtheSuperScriptPlasmidSystem(Invitrogen).Theentirecodingre-gionsoftheBnNAC5-1andBnNAC14,whichencodedtheonlytwoBnNACproteinsthatdonotactivatetranscriptiondirectly,wereusedasabaittoscreentheB.napuscDNAlibrarywiththePROQUESTTwo-HybridSystem(Invitrogen).About1.8×106S.cerevisiaetransformantsweresubjectedtotwo-hybridselectiononsyntheticcomplete(SC)mediumlackingleucine,tryptophanandhistidinebutcon-taining15mM3-amino-1,2,4-triazole(3AT).PutativeHis+(3AT-resistant)transformantsweretestedfortheinductionoftwootherreportergenes,URA3andLacZ,andreassessedbyasecondtransformationintoyeast.RegionsinvolvedinproteininteractionweremappedbyfusingtheNACorcarboxyl-terminaldo-mainsofBnNAC5-7,BnNAC5-8andBnNAC485totheGAL4ADinpPC86.TheseweretestedforinteractionwithwholeBnNAC14orconstructsen-codingvariouscombinationsofBnNAC14NACandcarboxyl-terminaldomainsinpDBLeu.
InteractionofBnNACproteinswithCaMV35Spromoterelements
PlasmidpFL759-1containingtheCaMV35Spro-moter,andresidentas-1element,fusedtoβ-glucurondiase(GUS)wasobtainedfromP.Fobert(NRCC,Saskatoon,Canada)andhasbeende-scribedbyStonehouse(2002).ThreeindependentS.cerevisiaeMaV203coloniestransformedwithpD-BLeuorpDBLeuBnNACandpFL759-1wereas-sessed.TotalproteinineachsamplewasdeterminedusingBradfordreagent(BioRadLaboratories)andGUSspeci?cactivityexpressedinunitsequivalenttopmolumbelliferylreleasedperμgproteinperhour.Thedatawerenormalizedbylog(x+1.0)trans-formation,analysisofvariancewasperformedwithgenerallinearmodels(ProcGLM)andthedifferencesbetweenmeansweredeterminedwiththeWaller-DuncanK-ratiot-test(P<0.05)(SASInstituteInc.2000,version8.2.0).
Results
IsolationofB.napusNACdomain-containinggenesAbout1500ESTsweregeneratedfromeachofthreesubtractivecDNAlibrariesdevelopedfromplantsthathadbeengrownat5?C,mechanicallywoundedorsubjectedtoherbivoryby?eabeetles.About5%oftheESTsencodedproteinsinvolvedinsignaltransductionpathways(G-proteins,MAPkinasesandtranscriptionfactors)orcontaineddomainsindicativeofDNAinter-action(helix-turn-helixandzinc?nger).Proteinscon-tainingaNACdomainsimilartothewound-inducibleATAF1andATAF2wereencodedbyasmallpro-portionoftheseandwereinvestigatedfurther.SincethePCR-basedsubtractivelibraryproceduregeneratedonlysmallcDNAfragments,BnNAC1-1wasusedtoscreennormalcDNAlibrariesderivedfromB.napusleaftissuetreatedasabove.cDNAsencodingeightdistincttypesofBnNACproteinsranginginsizefrom252to285aminoacidswereidenti?edinthisman-neranddenotedBnNAC1-1,3,5-1,5-7,5-8,5-11,8and14.Eachproteinpossesseda160aminoacidamino-terminalNACdomainconsistingof?vehighlyconservedregions,denotedA–EasperKikuchietal.(2000),whichwereinterspersedbyshort,variable,spacers(Figure1A).TheregionencodingsubdomainCandextendingthroughthe?rsthalfofsubdomainDcontainedahighproportionofconservedbasic
