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Regulationofcyclicpeptidebiosynthesisinaplantpathogenicfungusbyanovelt


AbbreviationsGAL4GAL4DNAbindingdomainUTRuntranslatedregionbZIPbasicleucinezipperTowhomreprintrequestsshouldbeaddressedE-mailwaltonmsueduThepublicationcostsofthisarticleweredefrayedinpartbypagechargepa

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Document on Subject : "Regulationofcyclicpeptidebiosynthesisinaplantpathogenicfungusbyanovelt"— Transcript:

1 Regulationofcyclicpeptidebiosynthesisina
RegulationofcyclicpeptidebiosynthesisinaplantpathogenicfungusbyanoveltranscriptionfactorKerryF.PedleyandJonathanD.Walton*DepartmentofEnergy-PlantResearchLaboratory,MichiganStateUniversity,EastLansing,MI48824CommunicatedbyHansJ.Kende,MichiganStateUniversity,EastLansing,MI,September18,2001(receivedforreviewAugust5,2001)StrainsoftheÞlamentousfungusCochlioboluscarbonumproducethehost-selectivecompoundHC-toxin,acyclictetrapep-tide,arehighlyvirulentoncertaingenotypesofmaize(ZeamaysL.).ProductionofHC-toxinisunderthecontrolofacomplexlocus,,whichiscomposedofatleastsevenlinkedandduplicatedgenesthatarepresentonlyintoxin-producingstrainsofC.car-.Oneofthesegenes,,wasearliershowntoberequiredfortheexpressionoftheothergenes.TOXEhasfourankyrinrepeatsandabasicregionsimilartothosefoundinbasicleucinezipper(bZIP)proteins,butlacksanyapparentleucine Abbreviations:GAL4,GAL4DNAbindingdomain;UTR,untranslatedregion;bZIP,basicleucinezipper.*Towhomreprintrequestsshouldbeaddressed.E-mail:walton@msu.edu.Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked“”inaccordancewith18U.S.C.§1734solelytoindicatethisfact.14174Ð14179November20,2001vol.98no.24www.pnas.org RVorRIfragmentfrompKP10clonedintopQE30,respectively.Forexpression,theE.coliM15containingpREP4(Qiagen)wastransformedwithpKP10.Asampleofanovernightculture(250l)wasusedtoinoculate5mlof2YTmedium(16g/ltryptone10g/lyeastextractg/lNaCl,pH7.5)containingampicillinandkanamycin.Isopro--thiogalactopyranoside(IPTG;0.1mM)wasaddedafter2.5hofgrowth,andthecellsweregrownforanadditional30minbeforeharvest.DeterminationofTranscriptionalStartSites.RandomamplificationofcDNAends(RACE),usingakitfromLifeTechnologies(Rockville,MD),wasusedtodeterminethelengthofthe5untranslatedregions(5-UTRs)of(17,23).SouthwesternBlotAnalysis.E.colicellpelletswereresuspendedinsamplebuffer[0.25MTrisHCl,pH6.820%2-mercaptoethanol8%SDS(wt/vol)30%sucrose(wt/vol)0.01%bromo-phenolblue]andboiledfor5min.ThedenaturedproteinextractswerefractionatedbySDSPAGE(10%acrylamide).Afterequilibrationwithblottingbuffer[25mMTris192mM20%methanol(vol/vol)]theproteinsweretransferredtonitrocellulose(Schleicher&Schuell)byusingaMinicell(Bio-Rad).Themembranewasincubatedinrenaturationbuffer(100mMHepes,pH7.5100mMKCl1mMDTTmMEDTA10mMMgCl5%nonfatmilk)at4Cfor18h.AfterrinsingwithTNE-50buffer(10mMTris,pH7.550mM1mMEDTA1mMDTT),theblotwasincubatedinthesamebuffercontaininga[P]-labeledDNAprobe(10andnonspecificcompetitorDNA(10mgmlshearedsalmonspermDNA)for6hat25C.TheblotwasthenwashedtwotimeswithTNE-50at25Cfor15minfollowedbyautoradiography.ConstructionandLabelingofProbesforSouthwesternBlotting.promoterfragmentswereamplifiedbyPCR,usingspecificprimersforeachofthepromoters.Primerswere1826ntinlength(sequencesavailableonrequest).PCRconditionswere35cycles(94Cfor1min;55Cfor2min;72Cfor1min)afteraninitialdenaturationfor2minat94C,followedbyafinalextensionof5minat72C.Reactionproductswerepurifiedbygelfiltrationchromatographyandlabeledwithith32P]dCTPbyrandompriming(24).Tomakedouble-strandedprobes,com-plementaryoligonucleotidesJDW-831(5-AACACAATCT-CACGTAAGTCTGCAG-3)andJDW-832(5GACTTACGTGAGATTGTGT-3)representingaTOXErecognitionelement(tox-box)fromthepromoter,andJDW-833(5-AACACAGCAGGACGTAAGTCTGCAG-3andJDW-834(5-CCTGCAGACTTACGTCCTGCTGTGT-),representingamutantversionofthissite,wereannealedbeforeuse.Double-strandedoligonucleotideswereend-labeledwithith32P]ATPbyusingpolynucleotidekinase(24).YeastStrainsandMethods.Standardmethodswereused(25).YeaststrainMG106(MATaade2–1can1–100his3–1115leu2–3112trp1–1ura3–1;ref.26)wasusedfortestingDNAbinding,andY190MATalys2–801trp1–901URA;CLONTECH)fortestingTOXEtranscriptionalactivationfunction.AnalysisofDNABindinginYeast.Complementaryoligonucleotidescontainingeitherfourtande

2 mcopiesofthewild-typeTOXErecognitionsequ
mcopiesofthewild-typeTOXErecognitionsequence(tox-box)(JDW-825,5-tcgaATCT-CACGTAATCTCACGTAATCTCACGTAATCTCACGTA-,andJDW-823,tcgaTACGTGAGATTACGTGAGAT-TACGTGAGATTACGTG-3;lowercaselettersindicateoverhangsusedforcloning)ormutantversions(JDW-836,,andJDW-837,5)werean-nealedandligatedintotheIsiteofthereportervectorpBgl-lacZ(27).Theresultingreporterconstructswereinte-gratedintothe3locusofstrainMG106bytransformationandselectionforuracilprototrophy.TOXEwasexpressedinyeastbyusingthepG-1expressionvector(28)containingtheTOXEcDNAfrompAJ39modifiedbyPCRtocreaterestrictionsitesforcloning.TheresultingplasmidpKP40wastransformedintothereporterstrainsbyselectingfortryptophanprototrophy.AnalysisofTOXEActivationActivity.TotesttheactivationactivityoffragmentsofTOXEfusedwiththeGAL4DNAbindingdomain(GAL4Isites(underlined)wereintroducedatsitescorrespondingtoaminoacids1,167,and254byusingPCRprimersJDW-768(5GCACGACTTCCCCG-3),JDW-868(5ACTTGCGTTCTGGT-3),andJDW-1059ATTCAGTTATAGTAACCTC-),respectively.ThecarboxyterminiofthefragmentstestedforactivationactivitywerecreatedbyusingnativerestrictionsitesorreverseprimerJDW-1061(5-GGGGGTCGACTAGTCTTC-CTTTGGGCCATG-3)correspondingtoaminoacid289.TheresultingproductswereclonedintopAS21(CLONTECH)toexpressGAL:TOXEfusionproteins.Site-DirectedMutagenesis.Aminoacidsubstitutionswereintro-ducedintotheTOXEbasicregionbyusingPCRprimerscontainingthedesiredmutations(sequencesavailableonre-quest).TheproductswereclonedintopKP9byusingrestrictionsitespresentinthesequence.AllchangeswereconfirmedbyDNAsequencing.AminoacidchangesintheactivationregionofTOXEweremadebyusingthemutagenesisprotocol(29).AllchangeswereconfirmedbyDNAsequencing.MutatedcopiesofwereclonedintopAS21andusedtoexpressGAL4:TOXEfusionsinyeaststrainY190.-GalactosidaseAssay.Yeasttransformantsexpressingsidasewereassayedintwoways.Foraqualitativeassay,yeastcellsweregrownonSDplatesfor23daysat30Candoverlaidwithanitrocellulosemembranefilter(Schleicher&Schuell)for5min.Thenitrocellulosefilterwasfrozeninliquidnitrogenandplacedonapieceoffilterpapersaturatedwith50nM5-bromo--galactoside(X-Gal)dissolvedinZ-buffer(60mMNa40mMNaH10mMKCl1mM38mM-mercaptoethanol,pH7.0).ThefiltersweremonitoredfortheappearanceofbluecolorafterincubationatCfor1h.Foraquantitativeassay,yeastcellsfroma2-mlovernightcultureinSDmediumwereusedtoinoculate8mloffreshYPDmedium.Theculturesweregrownat28Cwithshaking(230250rpm)untilthecellsreachedamid-logphase0.5).Cells(1.5ml)wereharvestedbycentrifugationat14,000rpmfor30secandwashedinZ-buffer.Cellswereresuspendedin300lofZ-bufferanddisruptedwiththreefreezethawcycles.Aliquotsofthedisruptedcells(100l)were Fig.1.StructureofTOXE.TOXEhasamolecularmassof49kDaand441aa.ThebZIP-likebasicdomainisbetweenresidues19and34andthefourankyrinrepeatsarelocatedbetweenresidues290and315,323and350,357and384,and415and441(19).PedleyandWaltonNovember20,2001vol.98no.24PLANTBIOLOGY dilutedwith700lofZ-bufferandmixedwith160lof4mg-nitrophenyl--galactopyranoside(ONPG)dissolvedinZ-buffer.Afterincubationat30Cfor10min,400lof1Mwasaddedandthemeasured.Oneunitofgalactosidaseisdefinedastheamountwhichhydrolyzes1ofONPGperminpercell(30).BeforeanalysisoftheinteractionbetweenTOXEandthepromoters,itwasessentialtoknowthetranscriptionalstartsitesofthegenes.areclusteredanddiver-gentlytranscribed.Their5-UTRsare162and145bp,respec-tively(13).The5-UTRofwasdeterminedbyrandomamplificationofcDNAends(RACE)tobe17bp(Y.Cheng,K.F.P.,andJ.D.W.,unpublishedresults).alsoclusteredanddivergentlytranscribed,andhave5-UTRsof59and46bp,respectively(17).The5-UTRofdeterminedbyRACEtobe167bp(datanotshown).TOXEBindstoaSpecificSequenceinthePromotersoftheItwasnotpossibletoexpressTOXEinsolubleforminE.colitherebyprecludingtheanalysisofDNAbindingbygelmobilityshiftassays.TOXEwasinsteadexpressedinE.coliandanalyzedbysout

3 hwesternblotting(i.e.,separationofprotei
hwesternblotting(i.e.,separationofproteinsbySDSPAGEfollowedbytransfertonitrocellulose,renaturation,andprobingwithdouble-strandedradiolabeledDNAfragments).Foreachgenepromotertested,oneormoreDNAfragmentswerefoundtobindstronglytoaproteinofthecorrectsizeinTOXE-expressingE.coliextracts(Fig.2).AnalysisofthesequencesoftheDNAfragmentsthatboundtoTOXErevealedthepresenceofaconservednucleotidesequence,whichwasthereforeacandidatefortheTOXErecognitionsite.This10-bpmotif,whichwerefertoasthetox-box,ishighlyconservedateightpositions.Nucleotidesatfiveofthepositionsareabsolutelyconserved,includingthefirstfour,ATCT(Fig.3).ExaminationoftheDNAsequencesupstreamanddownstreamofthiselementgavenoindicationthatthetox-boxsequencehasdyadsymmetry.Thetox-boxwaspresentinallDNAfragmentsthatboundTOXEandwasabsentfromallfragmentsthatdidnotbindTOXE(Fig.2).Thepromotersof,andcontaintwocopiesofthetox-boxandcontainsonecopy(Fig.2).Noevidencewasfoundforatox-boxinthepromoterof,suggestingthatdoesnotregulateitself.Asfurtherevidencefortheimportanceofthetox-boxforTOXEbinding,changingthefirstfiveoligonucleotidesofthetox-boxeliminatedbindingofTOXE(Fig.4).TOXEFunctionsasaTranscriptionFactorinYeast.TotesttheabilityofTOXEtobindspecificallytothetox-boxinvivoaswellas,ayeastexpressionsystemwasused.Fourcopiesofwild-typeormutantversionsofthetox-boxsequence,inbothforwardandreverseorientations,wereclonedupstreamofapromoterdrivingexpression.Thispromoterisinactivebyitselfbecauseofalackofupstreamactivationsequences(ref.27;Fig.5).Yeaststrainscontainingthevarioustox-boxconstructswerethentransformedwithanexpressionplasmidcontainingdrivenbytheconstitutiveyeastpromoter.Onlystrainscontainingboththewild-typetox-boxandexpressinghaddetectablelevelsof-galactosidaseactivity(Fig.5Bothorientationsofthewild-typetox-boxwereactive(Fig.5IdentificationofRegionsofTOXEImportantforDNABinding.InthebZIPfamilyofproteinsthebasicregiondeterminesDNAbinding-sitespecificitythroughdirectcontactswithDNA(20).TotestwhetherthiswasalsotrueforthebasicregionofTOXE,weanalyzedthisregionbyalaninescanning(31).Sevenbasicaminoacidsbetweenresidues19and34werechangedtoalaninesandexpressedinyeaststrainKP50.1(seeFig.5).Onlystrainsexpressingwild-typeTOXEhaddetectablelevelsof-galactosidaseactivity(Fig.6).Becausefailuretoactinvivocouldhavebeenduetopoorexpression,reducedproteinsta-bility,orfailuretolocalizetothenucleus,andnotnecessarilyduetoreducedDNAbinding,allofthemutantconstructswerealsotestedfortheirabilitytobindtothetox-boxbysouthwesternblotting.AllofthemutantproteinswereexpressedinE.colilevelscomparabletothewild-type(Fig.6),butallhadreducedabilitytobindtothetox-box(Fig.6).MutationoffourofthehighlyconservedbasicresiduescompletelyeliminatedDNAbinding(Fig.6TheresultsofthemutationalanalysisoftheTOXEbasicregionindicatedthatthebasicregionneartheNterminuswasessentialforDNAbindingbutnotnecessarilysufficient.Totestthis,twocarboxy-terminaldeletionmutants,whicheliminatedthreeorfourofthefourankyrinrepeats,respectively,weretestedforinvitroDNAbinding.Bothofthetruncatedversions Fig.2.TOXEbindstothepromotersofthegenes.TotalproteinextractsfromE.coliexpressing()ornotexpressing()TOXEwereseparatedbySDSPAGEandtransferredtonitrocellulosemembranes.Afterrenaturingtheboundproteins,themembraneswereprobedwiththeindicatedlabeledDNAfragments.Hatchedboxesindicatetox-boxsequences(seeFig.3)relativetothetranscriptionalstartsitesandtranscriptionaldirectionsofthegenes,indicatedbythearrows.Notethatarenotdrawntoscale.PedleyandWalton ofTOXEwereexpressedinE.coliatlevelscomparabletowild-typeTOXE(Fig.7),butneitherwasabletobindDNA(Fig.7IdentificationoftheTranscriptionalActivationRegionofTOXE.fusedtotheGAL4andexpressedinyeaststrainY190,TOXEbyitself(i.e.,withoutcoexpressingtheactivationdomainofGAL4)inducedexpressionof,indicat

4 ingthatnativeTOXEhasthecapacitynotonlyto
ingthatnativeTOXEhasthecapacitynotonlytobindDNAbutalsotoactivatetranscription.TocharacterizetheregionorregionsofTOXEresponsibleforactivation,aseriesofamino-andcarboxy-terminaldeletionsofTOXEwereexpressedasfusionproteinstoGAL4andtestedfortheirabilitytoinduceexpressionof.OftheTOXEfragmentstested,thesmallestonethatcouldactasanactivationdomainencompassedaminoacids254(Fig.8).Allactivefragmentscontainedthisregion,whichextendsfrom35aainfrontofthefirstankyrinrepeattooneaminoacidafter.TheseresultsindicatethatTOXEhasadiscreteactivationdomainandthat,whereasactivationpossiblyinvolvesatleastoneoftheankyrinrepeats,itdoesnotrequireallfourTheactivationdomaincomposedofaminoacids254317wasfurtheranalyzedbyalaninescanning.Thisregionisnotpartic-ularlyrichinanyparticularsingleaminoacidorclassofaminoacid,although30%arehydrophobic(Fig.9).Hydrophobicresidueshavebeenimplicatedascriticalinvarioustranscriptionactivationdomains,eveninthosethatareclassifiedonthebasisoftheirpredominantaminoacids(3234).Allmutationsofhydrophobicresiduesinthisregion,exceptone,stronglyreducedactivationfunctioninyeast(Fig.9).Theexceptionwasamutantdomainwithtwochanges,I298AandL300A,whichactuallyledtoincreasedactivationactivity(Fig.9TOXEwasearliershowntoberequiredfortheexpressionofthegenesinvolvedinthebiosynthesisofHC-toxin,butthemecha-nismofregulationwasnotclearbecauseoftheunusualstructureofTOXE(19).HereweshowthatTOXEisaDNAbindingproteinthatrecognizesaspecificsequence,thetox-box,thatispresentinoneortwocopiesinthepromotersofalloftheknowngenesinvolvedinHC-toxinbiosynthesis,exceptTOXEitself.Thetox-boxisfunctionalinyeastinbothorientations,whichisbiologicallysignificantbecausetwopairsofthegenes)aretightlyanddivergentlyclusteredandthereforeshareoverlappingpromoters(13,18).TheN-terminalbZIP-typebasicregionisrequiredforDNAbinding.ThesouthwesternblottingresultsindicatethatTOXEbindsasamonomerorhomomultimer.Becausethetox-boxdoesnotcontaindyadsymmetry,TOXEprobablybindsasamono-mer.Inthisregard,TOXEmorecloselyresemblestranscriptionfactorsthatareknowntobindasmonomerse.g.,Skn-1fromCaenorhabditiselegansratherthanmosttruebZIPproteins,whichtypicallybindashomo-orheterodimerstoDNAsiteswithdyadsymmetry.Skn-1lacksaleucinezipperbutbindstoanasymmetricalDNAbindingsiteasamonomerviaitsbZIP-likebasicregion(35).However,Skn-1differssignificantlyfrom Fig.3.SequencesoftheTOXE-bindingsitesfromthepromotersofthegenes,asdeducedfromtheresultsshowninFig.2andfromcomparativesequenceanalysis.Sequencelocationsarerelativetothetranscriptionalstartsites.Fortheandthepromoters,thelocationsarerelativeto,respectively(seeFig.2).NotethattheÞrsttox-boxisintheoppositeorientationtotheothers. Fig.4.Mutationoftheconservedresiduesinthetox-boxeliminateTOXEbinding.()Representationofthepromoter.Hatchedboxesindicatethetwotox-boxes.()Double-strandedoligonucleotidescontainingthewild-type(Wt)tox-boxsequence(underlined)andsurroundingnucleo-tides,andamutantversion(Mut)inwhichallÞveofthehighlyconservednucleotideswerechanged(indicatedinboldlettering),wereusedaslabeledprobesagainstsouthwesternblots.ÔÔÕÕIndicatestotalproteinex-tractsfromE.colinotexpressing(control),andÔÔÕÕindicatesE.coliexpressingTOXE. Fig.5.TOXEactsasasequence-speciÞcDNAbindingproteinandtranscrip-tionalactivatorinyeast.()Fourtandemcopiesofadouble-strandedoligo-nucleotidecontainingeitherthewild-typeorthemutanttox-box(seeFig.4)werefusedinbothorientationsupstreamofwithoutupstreamactivat-ingsequences(UAS)fusedto(27).TOXEdrivenbytheconstitutiveyeastGPDpromoterwasexpressedfromaplasmid.()Resultingactivitiesofthevariousyeaststrains.ÔÔTOXEexpressionÕÕindicateswhethertheyeastcellscontainedtheplasmidexpressingTOXE.PedleyandWaltonNovember20,2001vol.98no.24PLANTBIOLOGY TOXEincontaininghomeodomainelementsandlackingankyrinrepeats(35,36).Twovers

5 ionsofTOXEthatweretruncatedatthecarboxyt
ionsofTOXEthatweretruncatedatthecarboxyterminusfailedtobindtothetox-box,suggestingthattheC-terminalankyrinrepeatscontributeeitherdirectlyorindi-rectlytoDNAbinding.Thefour-and-a-halfN-terminalankyrinrepeatsofthesubunitofthetranscriptionalregulatorGABPstabilizetheinteractionbetweentheGABPcomplexandDNA(37).ThereforeitispossiblethattheankyrinrepeatsofTOXEcontributetothestabilityofTOXEbindingtoDNAorarecriticalforthecorrectpositioningofthebasicregionovertheInadditiontoDNAbindingactivity,TOXEalsohasthecapacitytoactivatetranscriptioninaheterologoussystem,indicatingthatTOXEcontainsanactivationdomain.ThesixankyrinrepeatsoftheC.elegansGLP-1receptorarealonesufficienttoactivatetranscriptioninyeast(38).However,theactivationdomainofTOXEdefinedinourexperimentsresidesina63-aaportionoftheproteinjustbeforeandincludingthefirstankyrinrepeat.AllfourankyrinrepeatsarenotnecessaryforTOXEtofunctionasatranscriptionalactivator,althoughitcannotbeexcludedfromourexperimentsthatthefourankyrindomainsmightconstituteasecondactivationdomaininTOXE. Fig.6.MutationsintheTOXEbasicregionreduceinvivoactivityandinvitroDNAbinding.()UnderlinedaminoacidsindicatethechangesmadeinTOXEineachmutant.-Galactosidaseactivitiesweremea-suredinyeaststrainYKP50.1(seeFig.5)expressingmutantTOXEconstructs.)Southwesternblotting.MutantTOXEconstructswereexpressedinE.coli)TheSDSPAGEgelstainedwithCoomassieblue;()theautora-diographoftheblotprobedwithaP-labeledDNAfragmentfromthepromotercontainingasingletox-box. Fig.7.TOXElackingthecarboxy-terminusdoesnotbindDNA.()SDS(stainedwithCoomassieblue)oftotalextractsofE.coliexpressingdifferentTOXEconstructs.Lane1,emptyplasmid(negativecontrol);lane2,full-lengthTOXE(aminoacids1441)(positivecontrol);lane3,TOXEaminoacids1lane4,TOXEaminoacids1254.Thesizesoftheexpressedproteinsaregivenontheleft.()SouthwesternblottingofthegelshowninprobedwithaDNAfragmentfromthepromotercontainingasingletox-box. Fig.8.MappingoftheTOXEactivationregion.TheindicatedfragmentsofTOXEwerefusedtotheGAL4,expressedinyeaststrainY190,andtheyeasttransformantsassayedfor-galactosidaseactivity. Fig.9.MutationalanalysisoftheactivationdomainofTOXE.()Sequenceoftheactivationdomain.Hydrophobicresiduesthatweremutatedareboxed,andrstankyrinrepeatisunderlined.()Full-lengthTOXEproteinscontainingtheindicatedmutationswerefusedtotheGAL4,expressedinyeaststrainY190,andtheyeasttransformantsassayedfor-galactosidaseactivity.PedleyandWalton TheonlyknownphenotypeofmutantsisinhibitionofmRNAexpressionofthegenesdedicatedtoHC-toxinbiosynthesis,withconcomitantlossofspecificpathogenicityofC.carbonumonmaizethatishomozygousrecessiveattheloci(19).LiketheothergenesofpresentonlyinTox2isolatesofC.carbonumisolates,andatleastoneofitstwocopiesisclusteredwiththeothergenes(ref.19;J.-H.AhnandJ.D.W.,unpublisheddata).Therefore,appearstobeapathway-specifictranscriptionfactorwhosesolefunctionistocoordinatetheexpressionofthegenesinvolvedinHC-toxinbiosynthesis.AgenewhoseproducthasthesamestructureasTOXE,namelyabZIPbasicregion,ankyrinrepeats,andnoleucinezipper,wasrecentlyreportedinCladosporiumfulvum,afungalpathogenoftomato(39).Thus,TOXEmightbetheprototypeofanewclassoftranscriptionfactors,sofarfoundonlyinplantpathogenicfilamentousfungi.Bussinketal.(39)haveproposedthenamebANKproteinsforthisclassoftranscriptionfactor.Pathway-specifictranscriptionfactorshavealsobeenfoundtoregulatesecondarymetabolitebiosynthesisinotherfilamentousfungi.Forexample,TRI6,azinc-fingertranscriptionfactor,regulatestheexpressionofthetrichothecenebiosyntheticgenesFusariumsporotrichiodes(40,41).InspeciesofAspergillus,theaflatoxinbiosyntheticgenesareregulatedbyAFLR,azincclustertranscriptionfactor(4245).Anotherzincclusterpro-tein,ORFR,regulatestheACR-toxingeneclusterofAlternariaalternata(46).Inallofthesecases,thetranscrip

6 tionalregulatorsareclusteredwiththebiosy
tionalregulatorsareclusteredwiththebiosyntheticgenes,asisthecaseinBecausetrans-actingtranscriptionfactors,suchasTOXE,TRI6,AFLR,andORFR,shouldbecapableofactivatinggenesanywhereintheirrespectivegenomes,thebiologicalrationaleforthephysicallinkageofregulatoryandregulatedgenesinfungalsecondarymetabolitegeneclustersisnotclear.Itmaybearesultoftheirevolutionaryoriginsandmechanismsoftrans-mission(47WethankJohnPitkinforadviceintheearlystagesofthisworkandSteveTriezenbergforvaluablesuggestionsthroughout.Thisworkwassup-portedbytheU.S.DepartmentofEnergy,DivisionofEnergyBiosciences.1.Hammond-Kosack,K.&Jones,J.D.G.(2000)inBiochemistryandMolecularBiologyofPlants,eds.Buchanan,B.B.,Gruissem,W.&Jones,R.L.(Am.Soc.PlantBiologists,Rockville,MD),pp.11022.Kohmoto,K.&Otani,H.(1991)Experientia3.Walton,J.D.(1996)PlantCell4.Meeley,R.M.,Johal,G.,Briggs,S.P.&Walton,J.D.(1992)PlantCell5.Johal,G.S.&Briggs,S.P.(1992)6.Multani,D.S.,Meeley,R.B.,Paterson,A.H.,Gray,J.,Briggs,S.P.&Johal,G.S.(1998)Proc.Natl.Acad.Sci.USA7.Taunton,J.,Hassig,C.A.&Schreiber,S.L.(1996)8.Brosch,G.,Ransom,R.,Lechner,T.,Walton,J.D.&Loidl,P.(1995)PlantCell9.Darkin-Rattray,S.J.,Gurnett,A.M.,Myers,R.M.,Dulski,P.M.,Crumley,T.M.,Alloco,J.,Cannova,C.,Meinke,P.T.,Colletti,S.L.,Bednarek,M.A.,etal.Proc.Natl.Acad.Sci.USA10.Ransom,R.F.&Walton,J.D.(1997)PlantPhysiol.11.Panaccione,D.G.,Scott-Craig,J.S.,Pocard,J.-A.&Walton,J.D.(1992)Proc.Natl.Acad.Sci.USA12.Scott-Craig,J.S.,Panaccione,D.G.,Pocard,J.-A.&Walton,J.D.(1992)J.Biol.Chem.13.Pitkin,J.W.,Panaccione,D.G.&Walton,J.D.(1996)Microbiology14.Ahn,J.-H.&Walton,J.D.(1997)Mol.PlantMicrobeInteract.15.Kennedy,J.,Auclair,K.,Kendrew,S.G.,Park,C.,Vederas,J.C.&Hutchinson,C.R.(1999)16.Cheng,Y.-Q.,Ahn,J.-H.&Walton,J.D.(1999)Microbiology17.Ahn,J.-H.&Walton,J.D.(1996)PlantCell18.Cheng,Y.-Q.&Walton,J.D.(2000)J.Biol.Chem.19.Ahn,J.-H.&Walton,J.D.(1998)Mol.Gen.Genet.20.Ellenberger,T.E.(1994)Curr.Opin.Struct.Biol.21.Bork,P.(1991)Proteins22.Sedgwick,S.G.&Smerdon,S.J.(1999)TrendsBiochem.Sci.23.Frohmann,M.A.,Dush,M.K.&Martin,G.R.(1988)Proc.Natl.Acad.Sci.24.Sambrook,J.,Fritsch,E.F.&Maniatis,T.A.(1989)MolecularCloning:ALaboratoryManual(ColdSpringHarborLab.Press,Plainview,NY),2ndEd.25.Guthrie,C.&Fink,G.R.,eds.(1991)MethodsEnzymol.26.Myers,L.C.,Gustafsson,C.M.,Hayashibara,K.C.,Brown,P.O.&Kornberg,R.D.(1999)Proc.Natl.Acad.Sci.USA27.Li,J.J.&Herskowitz,I.(1993)28.Schena,M.,Picard,D.&Yamamoto,K.R.(1991)MethodsEnzymol.29.Sarkar,G.&Sommer,S.S.(1990)30.Miller,J.H.(1972)ExperimentsinMolecularGenetics(ColdSpringHarborLab.Press,Plainview,NY).31.Cunningham,B.C.&Wells,J.A.(1989)32.Tjian,R.&Maniatis,T.(1994)33.Triezenberg,S.J.(1995)Curr.Opin.Genet.Dev.34.Sullivan,S.M.,Horn,P.J.,Olson,V.A.,Koop,A.H.,Niu,W.,Ebright,R.H.&Triezenberg,S.J.(1998)NucleicAcidsRes.35.Blackwell,T.K.,Bowerman,B.,Priess,J.R.&Weintraub,H.(1994)36.Kophengnavong,T.,Carroll,A.S.&Blackwell,T.K.(1999)Mol.Cell.Biol.37.Batchelor,A.H.,Piper,D.E.,delaBrousse,F.C.,McKnight,S.L.&Wolberger,C.(1998)38.Roehl,H.,Bosenberg,M.,Blelloch,R.&Kimble,J.(1996)EMBOJ.39.Bussink,H.-J.,Clark,A.&Oliver,R.(2001)Eur.J.PlantPathol40.Proctor,R.H.,Hohn,T.M.,McCormick,S.P.&Desjardins,A.E.(1995)Appl.Environ.Microbiol.41.Hohn,T.M.,Krishna,R.&Proctor.R.H.(1999)FungalGenet.Biol.42.Payne,G.A.,Nystrom,G.J.,Bhatnagar,D.,Cleveland,T.E.&Woloshuk,C.P.Appl.Environ.Microbiol.43.Yu,J.H.,Butchko,R.A.E.,Fernandes,M.,Keller,N.P.,Leonard,T.J.&Adams,T.H.(1996)Curr.Genet.44.Fernandes,M.,Keller,N.P.&Adams,T.H.(1998)Mol.Microbiol.45.Todd,R.B.&Andrianopoulos,A.(1997)FungalGenet.Biol.46.Tanaka,A.&Tsuge,T.(2000)Mol.Plant-MicrobeInteract.47.Keller,N.P.&Hohn,T.M.(1997)FungalGenet.Biol.48.Walton,J.D.(2000)FungalGenet.Biol.49.Rosewich,U.L.&Kistler,H.C.(2000)Annu.Rev.Phytopathol.PedleyandWaltonNovember20,2001vol.98no.24PLANTBIOLOG