重寄生过程中哈茨木霉转化菌株大量表达胞外碱性蛋白酶.docx

重寄生过程中哈茨木霉转化菌株大量表达胞外碱性蛋白酶.docx

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重寄生过程中哈茨木霉转化菌株大量表达胞外碱性蛋白酶

Trichodermaharzianumtransformanthashighextracellularalkalineproteinaseexpressionduringspecificmycoparasiticinteractions

 GeneticsandMolecularBiology

Print ISSN 1415-4757

Genet.Mol.Biol. vol.21 n.3 SãoPaulo Sept. 1998

MariaHelenaS.Goldman1andGustavoH.Goldman2

1DepartamentodeBiologia,FaculdadedeFilosofia,CiênciaseLetrasdeRibeirãoPretoand2DepartamentodeCiênciasFarmacêuticas,FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,USP,Av.doCafés/n,14040-903RibeirãoPreto,SP,Brasil.Fax:

55-016-6331092.E-mail:

ggoldman@usp.br SendcorrespondencetoG.H.G.

ABSTRACT

ThemycoparasiteTrichodermaharzianumproducesanalkalineproteinasethatmaybespecificallyinvolvedinmycoparasitism.Wehaveconstructedtransformantstrainsofthisfungusthatoverexpressthisalkalineproteinase.Someofthetransformantswereassessedforalkalineproteinaseactivity,andthosewithhigheractivitythanthewildtypewereselectedforfurtherstudies.Oneofthesetransformantstrainsproducedanelevatedandconstitutivepbr1mRNAlevelduringmycoparasiticinteractionswithRhizoctoniasolani.

INTRODUCTION

ThemycoparasiteTrichodermaharzianummayprovetobeaneffectivecontrolagentformanyphytopathogenicfungi.Ithasbeenproposedthatitsmycoparasiticinteractionproceedsinthreemajorsteps（Goldmanetal.,1994a,b;Haranetal.,1996）.Initially,themycoparasitehyphaegrowtowardthehosthyphae（Chetetal.,1981）.Thentheparasiteattachestothetargethyphae,presumablymediatedbyahostlectin,andappressoria-likestructurescoilaroundtheattackedcells（Eladetal.,1983a,b;Baraketal.,1985）.Concurrently,degradationofβ（1,3）-glucansandchitinfromthehostcellwallhasbeenobserved（Eladetal.,1983b）.Probablybothmechanicalpressureandcellwalldegradationbyhydrolyticenzymesareinvolved.Finally,themycoparasitepenetratesand/orlysesthehosthyphae（Chetetal.,1981）andreleasescellularcontents,whichprovidesnutrientstosustaingrowth.Extracellularenzymescorrespondingtothemainchemicalconstituentsofthefungalcellwall,i.e.,chitin,glucans,andproteins,havebeendetectedwhenT.harzianumisgrownonRhizoctoniasolanimyceliaorcellwallsasthesolecarbonsource（Ridoutetal.,1988;Geremiaetal.,1993）.Theenzymesappearedsequentially;analkalineproteinasewasproducedfirst,followedbyglucanasesandchitinases（Geremiaetal.,1991,1993）.Recently,wepurifiedandclonedthisalkalineproteasegene（pbr1）specificallyinducedbyRhizoctoniasolanicellwallsandchitin（Geremiaetal.,1993）.Althoughtheresultssupportthehypothesisthatthisgeneisspecificallyinvolvedinmycoparasitism,thereisnodirectevidenceofitsroleinthisinteraction.

TheavailabilityofoverproducingalkalineproteasestrainsofT.harzianumwouldbeofhelptoestablishthisenzyme'sroleinmycoparasitism.Additionally,antagonisticactivityinthesoilcouldbeimprovedbyincreasedactivityofoneormoreenzymesinvolvedincellwalldegradationofthepathogens.Thisstudydemonstratedthefeasibilityofoverexpressingpbr1geneinT.harzianumbyincreasingitscopynumberthroughtransformation.Oneofthetransformantsshowedelevatedandconstitutivepbr1mRNAexpressionduringmycoparasiticinteractions.

MATERIALANDMETHODS

Strainsandgrowthconditions

T.harzianumstrainIMI206040wasusedthroughoutthiswork.ThisstrainwasgrownasdescribedbyGeremiaetal.（1993）.R.solanicellwallsandcolloidalchitinwerepreparedaccordingtoGeremiaetal.（1993）.BasicproteaseactivitywasdeterminedbyincubationwithSucc-Ala-Ala-Pro-Phe-pNA（SigmaChemicalCo.,St.Louis,MO）（Geremiaetal.,1993）.ProteinconcentrationwasdeterminedusingaBio-Radassay（Bio-Rad,USA）.Intheantagonisticdualinteractions,simplemycelialdiscs（1.0cmindiameterfromfive-day-oldculturesofR.solaniinpotato-dextrose-agar（PDA）plates）wereinoculatedoncellophanediscs,neartheedgeofPetridishescontainingminimalmedium（Geremiaetal.,1991）plus0.5%glucose.Aftertwodays,amycelialdisc（1.0cm）ofafour-day-oldcultureofT.harzianumfromaPDAplatewasplacedoppositetothegrowingR.solanicolony,andincubatedat28oCforfourto10days.

DNA/RNAmanipulations

Restrictionenzymedigestswereperformedaccordingtomanufacturer'srecommendations.IsolationofplasmidDNAfromEscherichiacoliandSouthernblotswasperformedusingstandardprocedures（Sambrooketal.,1989）.DNAprobesweremadeusingarandomprimersystem（Boehringer）.NorthernanalysiswasmadeasdescribedpreviouslybyGoldmanetal.（1992）.AcellophanearearepresentingdifferentstepsoftheinteractionwascuttoobtainRNAfromthedualantagonisticinteractions.ThemyceliawerecarefullywashedwithTESbuffer,disruptedandtotalRNAextracted（Goldmanetal.,1994b;Vasseuretal.,1995）.CotransformationofthebasicproteasegenewasmadeincombinationwithplasmidspHAT-aandpBP2.2（Herrera-Estrellaetal.,1990;Goldmanetal.,1990;Geremiaetal.,1993）.Amolarratioofapproximately1to4wasused;afterwards,selectionwasmadeinPDAplateswith1.2MSorbitoland100µg/mlhygromycin.PlasmidpBP2.2wasmadeofpT3T7.lac（Boehringer）witha5.5-kbEcoRIfragmentofthepbr1gene（Geremiaetal.,1993）.ThisinserthasthreeHindIIIandfourPstIsites.Furthermore,ithasafragmentofabout1.5kb,downstreamfromthestartcodon.DensitometryofthefungaltransformantswasperformedusinganLKB-Pharmacia2202UltrascanLaserDensitometer.

RESULTSANDDISCUSSION

HydrolyticenzymessecretedbyT.harzianumarebelievedtoplayanimportantroleintheparasitismofphytopathogenicfungi.Thealkalineprotease（PBR1）canbeinducedinsimulatedmycoparasiticconditions;furthermore,colloidalchitincanalsoinducethisenzyme（Geremiaetal.,1991）.Thegeneencodingthisalkalineproteinasehasbeencloned,andNorthernanalysisshowedthatinductionofthisenzymewasduetoanincreaseinmRNAlevel（Geremiaetal.,1993）.Todetermineifthepbr1genewasinducedduringdualinteractionassays,totalRNAwaspreparedfromdifferentstagesofantagonismbetweenT.harzianumandR.solani（Figure1A）.Asthemycoparasiticinteractionprogressedpbr1mRNAlevelsincreased（Figure1B）.Goldmanetal.（1992）reportedrepressionofthepgkgeneexpressionduringasimulatedmycoparasiticstate.Infact,expressiondroppedmarkedlyduringthedualinteractionassays（Figure1C）.Therewasconstantb-tubulinRNAexpressionunderalltestconditions（Figure1BandC）.Theseresultsindicatethatpbr1mRNAaccumulatesprogressivelyduringantagonisticinteractionsbetweenT.harzianumandR.solani.Furthermore,simulatedmycoparasiticconditions,wheregrowthtakesplaceinminimalmediumwiththecellwallsofaphytopathogenicfungusastheonlycarbonsource,mimicantagonisticdualinteractionassays,atleastinsofaraspbr1andpgkgeneexpressionareconcerned（Figure1BandC）.Thisisthefirstmoleculargeneticdemonstrationofsimulatedmycoparasiticconditionsthatreflectswhatoccursduringantagonisticdualinteractions.DuetothecomplexityoftheinvivointeractionbetweenT.harzianumandR.solaniontheplantrhizosphereorinthesoil,dualinteractionassaysprovidetheclosestandmostplausiblemethodologicalapproachforstudyingthemolecularbiologicalaspectsofmycoparasitism.

Figure1-NorthernblotofthemRNAobtainedduringspecificmycoparasiticinteractionsbetweenTrichodermaharzianumandRhizoctoniasolani.A:

Differentstagesoftheinteraction.B:

Northernanalysisofthepbr1mRNAobtainedduringdualinteractions.Theleftpanelshowshybridizationwithpbr1gene,whereastherightpanelshowshybridizationwiththetub1gene.C:

NorthernanalysisofthepgkmRNAobtainedduringdualinteractions.Theleftpanelshowshybridizationwithpgkgene,whereastherightpanelshowshybridizationwiththetub1gene.InpanelAII,T.harzianumhasgrownoverR.solanimycelia.R:

R.solani;T:

T.harzianum;G:

T.harzianumgrowninMMplateswith0.5%glucose;CW:

T.harzianumgrowninMMbrothwith0.1%cellwallsofR.solani.

Therearemanyexamplesofgeneoverexpressioninfunguscausedbyanincreaseincopynumber（FowlerandBerka,1991）.Consequently,wedecidedtooverexpresspbr1genebyco-transformingT.harzianumwithitsgenomicclone,plasmidpBP2.2,andaplasmidcontainingaselectedhygromycinBresistantmarker,pHAT-α（Herrera-Estrellaetal.,1990）.Protoplasts（107-108ml-1）weretransformedwithbothplasmidsandselectedforhygromycinBresistance.ElectroporationofT.harzianumprotoplastsinthepresenceofthesetwoplasmidsyieldedtheexpectedhightransformationfrequency（Goldmanetal.,1990）.Fifty-threetransformantswereselected,purified,andstabilizedthroughfourconsecutiveroundsoftransferinselectivemedium.Thesetransformantswereassessedforalkalineproteaseactivityandthosewithactivityhigherthanthewildtypewereselectedforfurtherstudies.

DNAfromfourtransformantsandthewild-typestrainwasisolated,digestedwithPstIorHindIII,andhybridizedwitha5.5-kbEcoRIfragmentcontainingthepbr1gene.DigestionwithHindIIIproducedhybridizingfragmentsofabout5.0and0.9kb,inthewildtypeandtransformants（Figure2A）.Thisdigestionalsoshowedanadditionalfragmentofabout3.0kbinalltransformants（Figure2A）.DigestionwithPstIproducedhybridizingfragmentsofabout2.3,1.7and0.6kbinthewildtypeandtransformants（Figure2B）.Additionalfragmentsofabout5.5,2.8,and1.5kbcouldalsobeseeninthetransformants（Figure2B）.Theadditionalfragmentspresentinbothdigestions