Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Industrial

Microbiology

Isolation

and

expression

of

two

polyketide

synthase

genes

from

Trichoderma

harzianum

88

during

mycoparasitism

Lin

Yao

_,

_Chong

_Tan

_,

_Jinzhu

_Song

_,

_Qian

_Yang

_,

_Lijie

_Yu

_,

_Xinling

_Li

a_{KeyLaboratoryofMolecularandCytogeneticsandGeneticBreedingofHeilongjiangProvince,HarbinNormalUniversity,Harbin,}

PRChina

b_{DepartmentofLifeScienceandEngineering,HarbinInstituteofTechnology,Harbin,PRChina}

c_{ResearchCenteronLifeSciencesandEnvironmentalSciences,HarbinUniversityofCommerce,Harbin,PRChina}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10August2013 Accepted5September2014 Availableonline2March2016 AssociateEditor:EleniGomes

Keywords:

Polyketidesynthese RealtimequantitativePCR Genedisruption

Fungalplantpathogens

a

b

s

t

r

a

c

t

MetabolitesofmycoparasiticfungalspeciessuchasTrichodermaharzianum88haveimportant biologicalroles.Inthisstudy,twonewketoacylsynthase(KS)fragmentswereisolatedfrom culturedTrichodermaharzianum88myceliausingdegenerateprimersandanalysedusinga phylogenetictree.Thegenefragmentsweredeterminedtobepresentassinglecopiesin

Trichodermaharzianum88throughsouthernblotanalysisusingdigoxigenin-labelledKSgene

fragmentsasprobes.ThecompletesequenceanalysisinformationofpksT-1(5669bp)and

pksT-2(7901bp)suggeststhatpksT-1exhibitedfeaturesofanon-reducingtypeIfungalPKS,

whereaspksT-2exhibitedfeaturesofahighlyreducingtypeIfungalPKS.Reverse transcrip-tionpolymerasechainreactionindicatedthattheisolatedgenesaredifferentiallyregulated

inTrichodermaharzianum88during challengewith threefungal plantpathogens,which

suggeststhattheyparticipateintheresponseofTrichodermaharzianum88tofungalplant pathogens.Furthermore,disruptionofthepksT-2encodingketosynthase–acyltransferase domainsthroughAgrobacterium-mediatedgenetransformationindicatedthatpksT-2isa keyfactorforconidialpigmentationinTrichodermaharzianum88.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Biologicalcontrolisanefficientandenvironmentallyfriendly waytopreventdampingoff.Trichodermaspp.are opportunis-ticbiologicalcontrolagentsand avirulentplantsymbionts.

∗ _{Correspondingauthor.}

E-mail:qianyangcn@yeah.net(X.Li).

T.harzianumbelongingtoTrichodermaspp.iswidelyusedasa

biopesticideandbiofertilizertocontroldiseasesandpromote positive physiologicresponsesinplants.Itcombatsseveral plantpathogenic fungisuchasFusariumoxysporum inbean and melon,1,2 _{Botrytis cinerea in tobacco,}3 _{Rhizoctonia solani}

incucumber,4_{sheathblightinrice,}5_{creepingbentgrassand}

http://dx.doi.org/10.1016/j.bjm.2016.01.004

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

tomatoandSclerotiniasclerotioruminsoybean.6–8_Thesereports

suggest that T. harzianumstrains parasitize a large variety ofphytopathogenicfungi. Itcan alsobeusedto controlR.

solani.9_{Therefore,themolecularbiocontrolmechanismofT.}

harzianumdeservesfurtherstudy.T.harzianumisreportedly

effectiveincontrollingfungithroughdifferentmechanisms, suchasbyinducingsystemicresistanceinplants, parasitiz-ingotherfungibyrecognizingsignalsfromthehosttoprovoke the transcriptionofmycoparasitism-related genes, enzyme productionandsecretion,competingfornutrientsandlight andbyproducingantifungalmetabolites.10 _{Harmanet al.}11

reportedthatT.harzianumstrainssignificantlyenhancethe systemicresistanceandseedgrowthofmaize,andinducesthe expressionofproteinsrelatedtosystemicinducedresistance and increases growth.12 _{Furthermore, although the}

under-lying mechanism is much more complex than previously considered,thesymbiosis-likeinteractioncanbegenetically improvedtobenefitplantsmore.13_{ProteinssuchasG-proteins}

and mitogen-activated protein kinases affect biocontrol-relevantprocessesinthemycoparasiticinteractionsbetween

Trichodermaandplantpathogens,suchastheproductionof

hydrolyticenzymesandantifungalmetabolitesandthe for-mation ofinfection structures.14 Thetranscriptional factor Pac1regulatessomeproteinsinvolvedinantagonism,such as chit42chitinase, papA protease, gtt1 glucose permease, andqid74cellwallprotein.15_{Competitionfornutrientsand}

space is alsoconsidered antagonism. T. harzianumshowed dominance over Alternaria alternata under different envi-ronmental conditions (water activity and temperature).16

However,aninvitroantagonismtestindicatesthatthe pro-duction of antifungal compounds under nutrient-limiting conditionsalsocontributestofungistasis.17 _Ingeneral,

elu-cidating the pathogen-induced biocontrolmechanism ofT.

harzianumenablesitsapplicationincontrollingplantdiseases.

T.harzianumiscurrentlyusedasabiofungicidetocontrolF.

oxysporumandSclerotiumrolfsiiefficiently.18

Antifungal metabolites isolated from Trichoderma spp., including non-ribosomal peptides, polyketides, terpenoids andpyrones,playtheimportantroleinbiologicalcontrol.10

Andstudyingbiologicalactivities,especiallytheirbiosynthetic pathwayofthosecompoundsisnecessaryforuncoveringtheir biologicalcontrolmechanisms.

Polyketidesynthases(PKSs)aremajorenzymesinplants, fungi, and bacteria that are responsible for polyketide biosynthesis.19_{FungalPKSsaregenerallylargemultidomain}

systemsthatcompriseaminimalsetofketosynthase, acyl-transferase and acyl carrier protein domains. Type I PKSs elongate their polyketide products iteratively. In addition, ketoreductase,dehydrataseandenoylreductasedomainsmay catalyseoptional␤-ketoprocessing reactions, and optional accessorydomainsprovidecyclaseactivityandmethyl trans-ferase activity.20 _{Type Ifungal PKSs are grouped into three}

classes based on their optional processing domains: non-reducing (e.g., melanin, aflatoxin), partially reducing (e.g., 6msas,calicheamicin),andhighlyreducingPKSs(e.g.,T-toxin, fumonisin).8,21

Avarietyoffungalpolyketidesynthasegeneshavebeen identified in recent ten years. However, there were no relatedresearchreportsthatpolyketidesynthasegenesofT.

harzianumwereclonedandidentified.Inthisstudy,thegenes

encoding type I PKSs from T. harzianum mycelia were iso-latedusinggenewalkingtechnology,andpredictiveanalysis ofitsstructureandencodingproduct.AndPKSgenes expres-sion from different growth stages of antagonism between

T. harzianum and fungal pathogens was detected by

real-time quantitative RT-PCR (RT-qPCR) method. Furthermore, PKSgenefunctionwasstudiedbygeneknockouttechnology. ThesestudieswerefoundationforfurtherrevealedPKSinthe roleofbiologicalmetabolicprocessanditsbiologicalactivity, andalsoprovidenewresearchideasforfuturestudyofother fungiPKSgene.

Materials

and

methods

Fungalstrains

ThebiocontrolagentT.harzianumstrain88(CGMCC5403; Gen-Bank accessionno. of ITS: JN579713) and the fungal plant pathogensRhizoctoniasolani(ACCC36246),S.sclerotiorum(ACCC 36462) and F. oxysporum (ACCC 36966) were stored at the MicrobialGeneticEngineeringLaboratory,HarbinInstituteof Technology(Harbin,China).

IsolationofPKSgenesviagenewalkingusingdegenerate primers

TotalDNAwasisolatedfromT.harzianum88usingaDNeasy Plant Minikit (Invitrogen,Carlsbad,CA,USA). PKSketoacyl synthase(KS)andacyltransferase(AT)sequenceswere ampli-fiedusingtheketosynthase–acyltransferase(KA)degenerate primersKAF1andKAR1previouslydesignedandreportedby Amnuaykanjanasinetal.22_{ThePCRcyclingconditionswere}

as follows:94◦C for5min;30 cycles of94◦C for30s, 58◦C for30sand72◦Cfor30s;and72◦Cfor7min.Theresulting 700bpproductwasgelisolatedandinsertedintothe pMD18-TEasyvector(Promega,Madison,WI,USA).Therecombinant vector wasthentransformedintocompetent Escherichiacoli

DH5␣(TaKaRa,Osaka,Japan)usingtheheatshockmethod.23

The transformants were spread onto LB agar medium (1% tryptone, 0.5% yeastextract, 1%NaCl and 1.5%agar atpH 7.0)(TaKaRa,Osaka,Japan)containingampicillin(100␮g/mL) (TaKaRa,Osaka,Japan),X-Gal(0.2mM)(TaKaRa,Osaka,Japan) and isopropylthio-␤-galactoside (40␮g/mL) (TaKaRa, Osaka, Japan)andincubatedovernightat37◦C.Whitecolonieswere selectedforPCRidentification.

The forward primer xKAF1 was designed based on the amino acid (aa)sequences inthe conservedKS domainof reducing fungal PKS.ThexKAF1forwardand KAR2reverse primerswereusedtoisolatean800bpfragment,whichwas clonedintothepMD18-TEasyvector(Promega,Madison,WI, USA)andsequenced.

Asetofsenseprimersfor3 endamplificationand anti-sense primers for 5 end amplification were designed for gene walking toobtain morecomplete genomic sequences of the two PKS genes. The reverse primers AP1, AP2, AP3

andAP4forthetwoPKSgenesweresynthesizedbyTaKaRa (Osaka, Japan). The sequences of all primers used in this study are listed in the Online Resource 1. The fragment sequenceswereassembledbyPhrap(http://www.phrap.org/)

and were then analysed using the BLASTx algorithm (http://www.ncbi.nlm.nih.gov/BLASTx/) and ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The putative PKSsweredesignatedaspksT.

Southernblotanalysis

TwospecificprimerpairsweredesignedaccordingtotheKS domainfragmentsequences,asshownintheschematic pro-videdintheOnlineResource1.TheDIG-labelledDNAprobes

pksT-a-S and pksT-b-S were generated from the cloned KS

domainPCRproductsusingaDIG-HighPrimeLabellingMix (RocheAppliedSciences,Basel,Switzerland).Approximately 180␮gofgenomicDNAwasdivided intosixequalportions anddigestedovernightat37◦Cwiththerestrictionenzymes

BglII(usedtodigesttwoportions),HindIII,SalIorSacI(Promega,

Madison,WI,USA)toensurecompletedigestion.Thedigested genomicDNAwasthenelectrophoresison0.7%(w/v)agarose gel(30V,12h)andtransferredontopositivelychargednylon membranes (GE Healthcare, London, UK). Themembranes werehybridizedat42◦CusingtheDIG-labelledprobes

pksT-a-SandpksT-b-S(sequencesprovidedinOnlineResource2)and

weredetectedwiththeDIGLuminescentDetectionSystem (RocheAppliedSciences,Basel,Switzerland).

Reversetranscription-polymerasechainreaction(RT-PCR) ofPKSgenes

T. harzianum88 mycelia (100mg) were groundinto powder

after freezing in liquid nitrogen. RNA was then extracted usinganRNeasyPlantMinikit(Invitrogen,Carlsbad,CA,USA) andusedforfirst-strandcDNAsynthesiswithSuperScriptII reversetranscriptase(Invitrogen,Carlsbad,CA,USA).Thefull cDNAsequenceofT.harzianum88pksTwasamplifiedusing RT-PCRprimersspecificforpksT-1andpksT-2(OnlineResource 1).Theamplifiedfragmentswerethenassembledusingthe programPhrap(http://www.phrap.org/).

Thepotentialopenreadingframes(ORFs)ofthetwoPKSs were scanned using FGENESH as the matrix (http://linux1. softberry.com) and compared with T. virens gene sequences. The conserved PKS domains were verified usingCDDSearch/PRS-BLAST(http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi).ThepksTKSdomainswerealigned with 55 fungal PKS protein sequences (reducing and non-reducing)retrieved from GenBank,andthe twoalignments analysedusingCLUSTALWwereembeddedintheMEGA4.0 program(http://www.megasoftware.net/).Bootstrapanalysis wasconductedusingtheMEGA4.0programwith1000 replica-tionstoobtaintheconfidencevalueforthealignedsequence dataset.Aphylogenetictreewasconstructedviamaximum parsimony. The resultant phylogenetic tree was rooted in animalfattyacid synthase(GallusgallusFAS),aspreviously described.21 _{The candidate secondary metabolite product}

waspredictedusingNaturalProductDomainSeeker(NaPDoS) (http://npdomainseeker.sdsc.edu//runanalysis.html). DifferentialexpressionofPKSgenes

RT-qPCRwasusedtoevaluatetherelativechanges ingene expression in T. harzianum 88 during challenge with the

fungalpathogensR.solani,S.sclerotiorumandF.oxysporum.T.

harzianum88waschallengedwithR.solani,S.sclerotiorumandF.

oxysporumonplatesusingtheproceduredescribedbyCarsolio

etal.24_{Agarwithcutfungalcolonieswereplacedonopposite}

sidesof9cmplatescontainingminimummedium(MM)with 2%agarand2%glucose(Promega,Madison,WI,USA)andwere covered with sterilecellophane sheets(Promega, Madison, WI,USA).T.harzianum88myceliawerecollectedwhenthey touchedthefungalpathogenat3,6,12,24,and36h.The pro-cedurewasrepeatedthreetimes;however,onlyTrichoderma

mycelia without spores and 0.5cm away from the original inocula were collected.The controlplate wasT. harzianum 88challengedwithT.harzianum88mycelia,whichwere col-lectedwhenthecoloniestouchedeachother(3,6,12,24,and 36h).Thefungiwere allowedtogrowat28◦Cinthe dark. Twoprimerpairswereselectedforreal-timeRT-PCRanalysis (OnlineResource1).

Total RNAwasextractedfrom the myceliaanddigested withDNaseI(TaKaRa,Osaka,Japan).25_{TotalRNA(4␮g)from}

eachpooledsamplewasreversetranscribedintocDNAinthe presenceofoligo (dT) inatotal volumeof25␮L.The syn-thesizedcDNAwasdilutedwith25␮LofRNase-freeddH2O

andusedasthetemplateforRT-qPCR.Threepairsofprimers weredesignedaccordingtotheITSDNAfromR.solani,S.

scle-rotiorum,andF.oxysporumtoavoidtheamplificationofhost

fragments.Consequently,nofragmentswereamplifiedfrom cDNAtemplates.ˇ-Tubulin(DY762540)transcriptswereused asendogenousreferencesforRT-qPCR.Theprimersetfor ␤-tubulinwasdesigned(additionaldataaregivenintheOnline Resource1)andRT-qPCRwasperformedusingtheSYBR®

Pre-mixExTaqTM_{II(TaKaRa,Osaka,Japan)onanABIModel7500}

HT sequence detector(ABI, Harbin Institute ofTechnology, China).Reagentmixeswerepreparedbycombining12.5mLof SYBR®_PremixExTaqTM_{II(2×)with2.5␮LofcDNA,0.4pmol/␮L}

forward primers,0.4pmol/␮Lreverse primers,and 8␮L dis-tilledwaterperreaction.The25␮Lreactionswerecarriedout usingprogrammedthermalcyclingconditionsconsistingof 30s at95◦C,followed by 40 cycles of 5s at 95◦C and 40s attheappropriateannealingtemperatureforeachreal-time RT-PCRprimerset.Threetechnicalreplicatesofeach ofthe threebiological replicateswere usedtonormalizethe rela-tivequantificationanalysis.Data fromthe thresholdcycles wereanalysedaccordingtothe2(C(T))methodsinPKSgene expression.26

AStudent’st-testwasusedtodeterminewhetherthepksT

genessignificantlyaffectedthesurvivalofT.harzianum88with andwithoutchallengefromthethreefungalplantpathogens. ThelevelofsignificanceofStudent’st-testwassettop<0.05 andwasnotedatp<0.01.

DeterminationofoptimalhygromycinBconcentration The wild type isolate (T) was assayed for growth on hygromycin B (hyg) (Promega, Madison, WI, USA) to opti-mizethenegativeselectionforsubsequenttransformations. Agar pieces from plate cultures of the isolate were used to inoculate M-100 plates amended with varying hyg concentrations. Individual wells were photographed after 3days.8

Table1–Theprimersusedinthisstudy. Primer Orientation Sequence(5–3) T2-P1 Forward CCGCTCGAGTGCTCTCCCTATGCCACTTC T2-P2 Reverse CCCAAGCTTAGATACACACCTCCCTCTTCAC T2-P3 Forward CGCGGATCCGCTTGATTCAGATTCCACAGG T2-P4 Reverse CTAGTCTAGAAAGGCTCGGGAGAGACCA

ConstructionofpksT-2disruptantstrain

Standard molecular techniques were used to construct all plasmidsusingrestrictionenzymes(Promega,Madison,WI, USA),alkalinephosphataseandPCRreagents(TaKaRaBioInc., Otsu,Japan).TheplasmidswerepurifiedusingtheMiniBEST PlasmidPurificationKitVer.3.0(QIAgen,USA).Thebinary vec-torpPZPtk8.10wasfromGardinerandHowlett.27

The5-flankingDNA(1.4kb,5and3)and3-flankingDNA (1.114kb, 5 and 3) ofpksT-2 were amplified byPCR using

T.harzianum88genomicDNA asthetemplate.Theprimers

usedtoamplifythe 5-flankingDNAwere T2-P1andT2-P2, whereasthoseforthe3-flankingDNAwereT2-P3andT2-P4 (Table1).The3-flankingDNAwasdigestedusingBamHIand

XbaI(Promega,Madison,WI,USA),andthedigestedfragment wasligatedintotheBamHIandXbaIsiteofthepPZPtk8.10.The generatedplasmidwasdesignatedaspPZPtk8.10-LR.The5 -flankingDNAwasdigestedwithXhoIand HindIII(Promega, Madison,WI, USA),and the digestedfragment wasligated

into theXhoI and HindIII(Promega,Madison,WI, USA)site

ofthepPZPtk8.10.Thegeneratedplasmidwasdesignatedas pPZPtk8.10-RR-LR. The hygromycin resistancegene Hyg

was amplified via PCR using the plasmid pCSN43 (Fungal GeneticsStockCenter,Kansas,USA)asatemplateandprimers hpgcassetteFand hpgcassetteR(OnlineResource1).The DNAfragmentandpPZPtk8.10-RR-LRweredigestedusing

HpaI (Promega, Madison, WI, USA), and the digested frag-mentwasligatedintotheblunt-endrestrictionHpaIsiteof thepPZPtk8.10-RR-LRvector,whichwastreatedwith alka-linephosphatase.Thegeneratedplasmidwasdesignatedas pPZPtk8.10-pksT-2.

Fungaltransformation

A. tumefaciens-mediated transformation of T. harzianum 88

was performed as described for other fungi with minor modifications. Briefly, A. tumefaciens carrying the plasmid DNA was grown on solid LB mediumcontaining 50␮g/mL kanamycinand50␮g/mLstreptomycin(LB-ka-str)(Promega, Madison, WI, USA) at 28◦C for 24h. Single colonies were inoculatedintoliquid (LB-ka-str)andincubated at28◦Cfor 12h.Thebacterialculturewasliquidinductionmedium(IM) (10mMK2HPO4,10mMKH2PO4,2.5mMNaCl,2mMMgSO4,

0.7mM CaCl2, 9nM FeSO4, 4mM NH4SO4, 10mM glucose,

40mM2-[N-morpholino]ethanesulfonicacid,pH5.3,and0.5% glycerol)containing200␮Macetosyringone(4-hydroxy-3,5 -dimethoxyacetophenone)AS(Promega,Madison,WI,USA)to anopticaldensityof0.15at660nm.8_{Aftercultivationof}

bacte-rialcellsat28◦Cfor6h,1×106_{fungalconidia/mLwasmixed}

withthebacterialsuspensionandspreadontofiltersplaced onIMagarcontaining200␮MAS.After48hofincubationat 28◦C,thefilterswere transferredtoMM-hygagar selection

mediumcontaining300␮g/mLcefotaxime(WakoChemicals) toinhibitthegrowth ofA.tumefacienscell.Thesamplewas frozeninliquidnitrogenandlyophilized.DNAwasextracted basedonthecetyltrimethylammoniumbromide(CTAB)mini extractionmethodwithslightmodifications.Thefrozen sam-plesweregroundinafrozenpestlewithliquidnitrogen.The finelygroundmyceliaweretransferredintosterilecentrifuge tubesandpre-warmedat65◦C.DNAextractionbuffer[100mM TrisHCl (pH8.0),1.4MNaCl,50mMEDTA(pH8.0),and2% CTAB](WakoChemicalsUSA,Inc.)wasaddedtothemixture, mixed well and incubatedwith gentle shaking for1h ina 65◦Cwaterbath.Afterincubation,anequalvolumeof chloro-form:isoamylalcohol(24:1,v/v)(WakoChemicalsUSA,Inc.) was addedgentlytodenature proteinsandcentrifuged for 10minat15,670×gand4◦C.Theaqueousphasewas trans-ferredintoasteriletubeandDNAwasprecipitatedfor20min with0.6volumeofcoldisopropanol(WakoChemicals USA, Inc.)at16,100×g and4◦C.Thesupernatantwaspouredoff andthepelletwaswashedtwicewithcold70%ethanol(Wako ChemicalsUSA,Inc.).Then,thepelletwasair-driedatroom temperatureandsuspendedinTEbuffer[10mMTrisHCl(pH 8.0),1mMEDTA(pH8.0)].ThesamplewastreatedwithRNase A (20mg/mL) (TaKaRa,Osaka, Japan)at40ng/mL DNA and incubatedfor1hat37◦C.Then,1␮LofgenomicDNAfromthe knockoutcandidatewasusedasthetemplateforPCR amplifi-cationusingspecificprimers(pksT-2checkFandpksT-2 check R(Online Resource1) and the Taq enzyme(TaKaRa, Osaka,Japan).

Single correct homologous integration through replace-ment of the resident pksT-2 with pksT-2:: hyg was confirmed via Southern blot analysis. The primers for the pksT-2-Sprobe intheOnlineResource1andits sequence areprovidedintheOnlineResource2.

Results

and

discussion

IsolationofPKSgenesviagenewalkingusingdegenerate primers

ThePCRoftheT.harzianum88genomicDNAusing

degener-ateprimersbasedontheKS-ATregionyieldedonefragment. The KA-seriesprimers are described in OnlineResource 1.

The700bppksT-a-1fragmentwasclonedinto thepMD18-T

Easyvector(Promega,Madison,WI,USA).Thesequencewas homologous toPenicilliummarneffeiPKSs(58%identity, Gen-Bankaccessionno.XP002151003)basedonBLASTxsearches againsttheNCBI/GenBankdatabase.ThepksT-a-1fragment, amplifiedusingtheKAF1/KAR1primers,wasaputative non-reducingPKSfragment.However,PCRusingotherKAprimer pairsforHP-reducingPKSsandPR-reducingPKSsdidnot pro-duceanyDNAfragment.WedesignedxKAF1andxKAF2as forwardprimersfromtheconservedregionsofKSdomains toobtain highlyreducing PKSs.PCRusing thexKAF1/KAR2 primersrevealedoneproductwiththeexpectedsize(800bp),

pksT-b-1,whichwashighlysimilartoHP-reducingPKSsgenes

fromTalaromycesstipitatus(57%similarity,GenBankaccession

no.XP002478229.1)underBLASTxanalysis.xKAF1primerwas derivedfromtheaasequenceEM(S)HGTGTQintheKSdomain ofHR-type PKSs,whereas xKAF2 primerwas derivedfrom

EAEQMDPQ,whichisconservedamongPRPKSs,but ampli-ficationusingxKAF2/KAR1andKAR2primersdidnotproduce anyviableDNAfragment.Bakeretal.28_{usedaphylogenomic}

approachtostudythePKSrepertoireencodedinthegenomes

ofTrichodermareesei,T.atrovirideandT.virens.Theyfoundonly

onePRPKSgeneinT.atroviride.Therefore,T.harzianummay

notexpressthisclassofPKS.

Asidefromthetwoaforementionedsequences,noother sequenceswereobtainedthroughgenewalkingeventhough differentapproacheswereusedtoisolatefurthersequences. Therefore,asetofsenseprimersfor5endand3end ampli-ficationwasdesignedforgenewalking(OnlineResource1). Thesequenceswereobtainedbyoverlappingthefragments isolatedthroughdegeneratePCR,andotherfragmentswere generatedusingthegenewalkingprimersets.Finally,mostof the5and3endregionsoftheputativePKSsweredetermined viagenewalkingamplification, andtheputative␤-ketoacyl synthasegeneswere identifiedthroughBlastsearches.Two finalPKS sequences,7187bp(pksT-a)and 10,976bp(pksT-b), wereobtained.

DetectionofPKSgenecopynumbers

T.harzianum88genomicDNAwasisolatedusingDNeasyPlant

Maxicolumns.SouthernblotanalysiswiththeDIG-labelled pksT-aproberevealedasingle2.08kbbandafterBglII cleav-ageanda3.36kbbandafterHindIIIcleavage(Fig.1).Similarly, Southernblot analysis usingthe DIG-labelled pksT-bprobe yieldeda6.0kbbandafterPstIcleavageanda4.2kbbandafter

SacIcleavage(Fig.1).Thesefindingsdemonstratethat

pksT-aandpksT-bwerepresentassinglecopiesinT.harzianum88.

TheiterativetypeofPKS(iPKS)onlyposesasinglecopyofeach

Bgl II Lane 1 2 3 4 — 2.08kb — 3.36kb — 6.0 kb — 4.2 kb Hind lll Pst I Sac I

Fig.1–SouthernblotanalysisofgenomicDNAfromT. harzianum88digestedwithBglII,HindIII,PstIorSacIas indicated.BandsweredetectedwithpksT-a(Lane1,2)and pksT-b(Lane3,4)digoxigenin-labelledprobes.

catalyticdomain,howeverthesecanbedeployedrepeatedly duringsynthesisofasinglepolyketidemolecule.Andsingle copiesofthegenomearemoststructuralgenesthatcarryouta varietyvariousfunctions.Therefore,identificationofspecific genecopynumberisimportantforin-depthunderstandingof itsbiologicalfunction.

DomainorganizationofPKSgenes

AdditionaldomainanalysiswasperformedonpksT-aand pksT-bsequencesafterusingdegenerateprimersandgenewalking toobtain thetwoPKSgenefragments, whichincludedtwo complete PKSfragments(pksT-1 andpksT-2,respectively).A

Pyridine nucleotide-disulphide oxidoreductase

protein sequence

protein sequence

Putative short-chain dehydrogenase

90 202 ₃₆₀ KS AT KS DH ER KR ACP AT ACP 791 889 1174 1199 2371 2319 2157 1977 1956 1639 1103 919 836 533 432 417 370 282 163 5 1698 7194 bp 1719 aa 10 976 bp 2382 aa 1650 1188 pksT-a pksT-1 pksT-b pksT-2 pksT-2 pksT-1

A

B

Fig.2–StructureanddomainorganizationofT.harzianum88geneclusters.(A)pksT-1geneclusterstructureinT.

harzianum88:twoORFsencodingaputativepyridinenucleotide-disulphideoxidoreductase,andpksT-1,respectively;pksT-1 genestructure:thefourintronsarerepresentedbylinesandexonsarerepresentedbyblocks.Schematicorganizationofthe domains:KS,␤-ketoacylsynthase(360–791),AT,acyltransferase(889–1174),ACP,acylcarrierprotein(1650–1698).(B)pksT-2 geneclusterstructureinT.harzianum88:twoORFsencodingaputativeshort-chaindehydrogenase,andpksT-2,

respectively;pksT-2genestructure:thefourintronsarerepresentedbylinesandtheexonsarerepresentedbyblocks. Schematicorganizationofthedomains:KS(5–417),AT(533–836),DH(919–1103),ER(1639–1956),KR(1977–2157)andACP (2319–2371).TheapproximatedomainboundariesweredeterminedusingtheiterativePKSdomainsearchprogram (http://www.nii.ac.in/∼pksdb/sbspks/master.html).

Fig.3–AlignmentofT.harzianum88predictedPKSactive regionswithcloselyrelatedfungalPKSs.(A)Alignmentof putativecatalyticmotifsfoundinthededucedaminoacid sequenceofT.harzianum88pksT-1withthoseofT.virens PKS(EHK16308.1);B.fuckelianaPKS15(BAE80697.1);Xylaria sp.BCC1067PKS(ABB90283.1);C.chiversiiRADS2

(ACM42403.1);M.chlamydosporia(ACD39770.1).

putativepyridinenucleotide–disulphideoxidoreductasegene clustered with pksT-1 on the sequenced pksT-a fragment (Fig.2A).AnothergenewasclusteredwithpksT-2onthe pksT-bsequence,whichwasputativelyidentifiedasashort-chain dehydrogenase(Fig.2B).ThepksT-1sequencewasdeposited intheNCBIGenBankdatabaseundertheaccessionnumber JN556039.1.

pksT-1,whichcontains5669bpandencodesa1719aa

pro-tein with three exons(positions 31–299,354–639, 699–5306 from the transcript inicial point), was confirmed via RT-PCR. The protein sequence of pksT-1 contained the entire ancestral domain structure of type I non-reducing PKSs based on thepresence ofconservedmotifs: KS(␤-ketoacyl synthase)/AT/ACP. Comparison between the pksT-1 protein sequence and the corresponding domains of other fungal PKSs showedconservationoftheactivesiteresidues inKS (EMHGTGT), AT(GHSLGEYAAL)and ACP (GVDSLV)(Fig.3A).

ThepksT-1proteinsequencewaspredictedtoproduce

non-reducednon-cyclicPKS.20

AnotherPKSgene,pksT-2isa7901bpgenethatencodesa 2382aaprotein.Thepresenceoffiveexons(positions228–718, 766–1074,1127–1337,1399–1522and1590–7603fromthe tran-script inicialpoint)wasconfirmedvia RT-PCR.Sixcatalytic domainswereidentifiedinthepksT-2proteinsequencebased onthepresenceofconservedmotifs.Thesedomains,inorder from the N-terminus to the C-terminus were KS, AT, DH, ER,KRandACP.ComparisonofthepksT-2proteinsequence withthecorrespondingdomainsofotherfungalPKSsshowed conservation of the active site residues in KS (ECHGTGT), AT (GHSSGEIAAAY),DH (HELLGTR),ER (GRGVNVIVNSL), KR (YLLVGCLGGLGR), and ACP (FGVDSML) (Fig. 3B).ThepksT-2

sequencewasdepositedintheNCBIGenBankdatabaseunder accessionnumberJN556040.1.

PhylogeneticanalysisofthePKSfragments

TheKSdomaingenealogy wasusedtoinferthe genealogy oftype Ifungal PKSs.Reliable sequences alignment ofthe proteinswasimpossiblebecauseoftheinsufficientsimilarity betweenmembers.Thedomainsweregroupintopreviously established chemistry-based clades that represented non-reducing,partiallyreducingandhighlyreducingfungalPKSs based on a maximum parsimony phylogenetic analysis of the translated sequences. These three major clades were clusteredinto alargercladeoftypeIfungal PKSs,whichis asistercladeoftheFASsfoundinanimals.Inthis present study,wechoseseveralproteinsequencesfrom3Trichoderma

(B)ComparisonwiththehighlyconservedmotifsofT. harzianum88pksT-2,T.virens(EHK18438.1),T.reeseiQM6a PKS(EGR47100),T.atroviride(EHK47038.1),G.moniliformis PKS14(AAR92221.1)andG.moniliformisFUM1

(AAR92218.1).Aminoacidresiduesconservedamongall sequencesaremarkedwithanasterisk.Positionswith conservedsubstitutionsaremarkedwithtwodots,and thosewithsemi-conservedsubstitutionsaremarkedwith onedot.Highlightedregionsindicatethecatalyticamino acidresiduesintheactivesite.

spp.,15filamentousfungalspeciesandG.gallustoconstruct aphylogenetictree.

The full aa sequences of the KS domains from several reducing/non-reducingfungalPKSswereemployedto gener-ateaphylogenetictree(Fig.4).Phylogeneticanalysisrevealed

thatpksT-1wasmostcloselyrelatedtotheputativePKSfromT.

virens(80%similarity;GenBankaccessionno.EHK16308.1)and

BotryotiniafuckelianaPKS15(38%similarity;accessionnumber

AAR90251.1). pksT-1 generated a dependent subclade that wasclosetothefungalnon-reducingPKScladeIIandIIIwith strongbootstrapvalues(100%).Thesehomologous proteins alsocontainthesamedomainorganizationasKS-AT-ACPin typeIfungalPKSs.TheKS-AT-ACPdomainstructureis uni-versalinfungi,suchasinC.chiversiiRADS2andMetacordyceps

chlamydosporiaPKSs,whichareinvolvedinRALbiosynthesis.

ThephylogeneticanalysisdemonstratedthatpksT-1wasmore closelyrelatedtothePKSsinvolvedinRALbiosynthesisthan otherknownPKSs.RALbiosynthesisgenerallyrequiresa non-reducingRADS2,areducingPKS01,ahalogenaseandother clustergenes,buttheirfunctionsareunknown.29Alignment ofthe twoproteinsequences using the NCBI-Blast2p algo-rithmshowedthatonegenefromT.virens(accessionnumber EHK16311.1),whichencodesareducingPKS,ismostclosely relatedtoC.chiversiiPKS01.TheotherproteinfromT.virens

(accessionnumberEHK16308.1)ishighlysimilartoC.chiversii

RADS2basedonBlast2palignment.Therefore,T.virenslikely hasasimilargeneclusterstructureforRALbiosynthesis.

pksT-2generatedanothersubcladewithverystrong

boot-strapvalues(99%;Fig.4)closetothefungalreducingPKSclade IV.Thesehomologousclonesalsocontainthesamedomain organizationasKS-AT-DH-ER-KR-ACPinthetypeIPKSgenes. Phylogeneticanalysisusingtheaminoacidsequencesofthe KSdomainrevealedthatpksT-2ismostcloselyrelatedtothe putativePKSfromT.virens(86%sequenceidentity;accession numberEHK18438.1)andG.moniliformis(33%sequence iden-tity;accessionnumberAAR92218.1).TheKSdomainanalysis showedthatpksT-2maybeinvolvedinfumonisinproduction basedonananalysisoftheNaPDoSdatabase.ReducingPKS subcladeIV,and subcladesIand IImayor maynothavea conservedmethyltransferasedomain.Theonlycharacterized PKS inthis subcladewas G. moniliformisFUM1, which con-tains the KS-AT-DH-(C-MT)-ER-KR-ACP domains and forms thelinearPKprecursorofthetoxinfumonisin.30 _TheFUM1

expressionlevelswashighlycorrelatedwithfumonisinsbased onreal-time RT-PCR,which suggests thatreal-time RT-PCR is suitable for studying the influence of abiotic and biotic factors on the expression of these genes.31,32 Cox et al.33 foundthattheC-MeTdomaintransfersamethylgroupafter thefirstandsecondroundsofiteration,butitisinactivein the finalround along withthe ERdomain.However,many ofthe predicted PKSs in the PKS clade producing reduced PKshavehighdivergenceandpresumablynonfunctionalMT domains.21 _{pksT-2does notcontaintheC-MTdomain}

com-pared with domains of G. moniliformis FUM1, which may synthesizeacompoundsimilartothelinearPKprecursorof fumonisin.

TheNCBIENTREZaccessionnumbersofthe55fungalPKS sequencesandaFASsequencefrom G.gallusare shownin abbreviationstable.

ExpressionpatternsofpksT-1andpksT-2genes

WestudiedtheeffectofT.harzianum88 confrontationwith differentfungalpathogens(S.sclerotiorum,R.solaniandF.

oxys-porum)inthemRNAlevelsofpksT-1andpksT-2toelucidatethe

pksTgenesexpression.Inthepresentstudy,fungalpathogens inductionoftwogeneswasnoticeablyhighinT.harzianum88

comparedwiththeuntreatedcontrols.

pksT-1 expression washighly upregulated by S.

sclerotio-rum,increasing1.88-foldwithin3handpeakingat4.74-fold within6h.ThepksT-1expressionwasdecreasedat12,24and 36hafterinduction.SignificantchangesinpksT-1mRNA lev-elswereobservedwhenT.harzianum88waschallengedwith

S.sclerotiorum(p≤0.01).R.solanichallengeinducedthe

high-estpksT-1mRNAlevelsat1.22-foldwithin3h.However,the

pksT-1mRNAlevelsdecreaseafter6,12,24and36h,andthe

lowestlevelwasobservedat12h.Thesesignificantchangesin

pksT-1mRNAlevelswerealsoobservedwhenT.harzianum88

waschallengedwithR.solani(p≤0.01).pksT-1wasalso upre-gulated1.07-foldbytheF.oxysporumchallengecomparedwith theuntreatedcontrolat3h,buttheincreasewasnot statis-ticallysignificant(p>0.05).TheincreasesinpksT-1expression at6handat12hweresignificant,reachingitspeakat 3.42-foldafter24h,andsignificantlydecreasingafter36h(p≤0.05;

Fig.5A).

TheS.sclerotiorum-inducedpksT-2expressionincreasedto

almost0.8-foldcomparedwiththeuntreatedsamplesat3h.

ThepksT-2expressiondecreasedafter6h,butincreasefrom

12hto36h(p<0.05;Fig.5B).Wealsoobservedthehighest increaseinpksT-2expression(1.33-fold)at6hafterR.solani

challenge (p<0.05;Fig.5B).Similarly,F.oxysporumincreased

pksT-2expressionby∼0.93-foldcomparedwiththeuntreated

samples(p<0.05at3,6,12,24and36h;Fig.5B).TheRT-qPCR analysisshowedthatthefungalpathogensinducepksT-2gene expression.Insummary,fungal pathogensinducethe tran-scriptionofpksTgenesinT.harzianum88.

Theseresultsweresimilartothatofnumerouspolyketide synthases, which are upregulated in creeping bentgrass infected with Sclerotinia homoeocarpa.34 _{By contrast, the}

PcPKS2 transcript levels in the leaves are highly upregu-lated by pathogens.35 _{6msas activates disease resistance}

pathways by enhancing the accumulation of PR proteins and resistance to tobacco mosaic virus.36

PKS/non-ribosomal peptide synthetase (NRPS) hybrid enzyme is also involved in Trichoderma–plant interactions that induce defence responses.37 _{In addition, T. harzianum is}

a mycoparasitic filamentous fungus that produces sev-eral antifungal compounds when challenged with fungal pathogens to contribute to fungistasis. These antifungal compoundsmayparticipateinthebiocontrolmechanismof

Trichoderma.

Inaddition,PKSexpressioninfungihasnotbeenstudiedin relationtopathogenesisandTrichoderma.Theresultsindicate thatwithatleastoneofthethreefungalpathogenscausesthe differentialregulationofthetwoPKSgenes.However,these genes generallydisplaydifferentexpressionpatterns when

Trichoderma encounters different fungal pathogens, which

suggests that T. harzianumgeneratesspecific response pat-ternstodifferentchallenges.

43 Tv_EHK20748.1 Tr_EGR44538 Ta_EHK46843.1 An_WA GF_PKS4 Xy_melanin At_at5 Ta_EHK46042.1 Tv_EHK20655.1 Tr_EGR44834.1 Cc_RADS2 Mc_PKS Bf_PKS15 Th_pksT-1 Tv_EHK16308.1 Tv_EHK23001.1 Ta_EHK42299.1 Tr_EGR49884.1 Tv_EHK17233.1 Tr_EGR52182.1 Ta_EHK44445.1 Xs_PKS1 pg_6msas Ta_EHK46840.1 At_6msas Tr_EGR46929 Ta_EHK49839.1 At_lovB Sm_PKS Ta_EHK50965.1 At_lovF Ta_EHK41169.1 Tv_EHK16311.1 Cc_PKS01 Tv_EHK16940.1 Tr_EG46571.1 Ta_EHK47389.1 Bf_PKS8 At_PKS Tr_EGR49751.1 Ta_EHK42694.1 Vd_PKS Tv_EHK21872.1 Bf_BcBOA9 Ag_PKS Gm_PKS14 Ta_EHK47038.1 Tv_EHK18438.1 Th_pksT-2 reducing PKS clade IV reducing PKS clade III reducing PKS clade I “6MSAS-type” PKS non-reducing PKS clade III non-reducing PKS clade II and III non-reducing PKS clade I and II non-reducing PKS clade I Tr_EGR47100 Tv_EHK21975.1 Ta_EHK42118.1 Gm_FUM1 Tv_EHK17164.1 Ta_EHK47613.1 Gg_FAS 37 87 81 55 52 89 35 94 52 67 25 80 68 54 99 95 94 96 100 78 92 92 97 62 43 51 99 99 87 98 39 100 100 100 100 100 100 91 87 100 100 100 100 99 80 54 82 98 96 91 34

Fig.4–RelationshipofT.harzianum88PKSstofungaltypeIiterativePKSs.ThetreewasconstructedwithtypeIiterative PKSproteinsequences.␤-KetoacylsynthaseofG.gallusFASwasusedastheout-group.Amultiplesequencealignment wasperformedusingtheCLUSTALW(1.83)program,andthetreewasgeneratedwiththeMEGA(4.0)program.The indicatedscalerepresents0.1aminoacidsubstitutionpersite.PKSsofT.harzianum88areindicatedinboldandframed withrectangles.Abbreviations,genusandspeciesnamesandtheNCBIENTREZaccessionnumbersofthe55fungalPKS sequencesandtheFASsequencefromG.gallusaregiveninabbreviationtable.

6

*

*

*

A

B

old induction relativ

e to β -tubin nor maliz ed) pksT -2 mRNA e xpression ( f old induction relativ e to β -tubin nor maliz ed) 5 4 3 2 1 0 3 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 3 6 12 24 36 6 12 24 36 Time (h) Time (h)

Fig.5–TimecourseexpressionanalysesofPKSgenesrespondingtoconfrontationwithR.solani,S.sclerotiorumorF. oxysporum.X-axis:timepointsofconfrontationwithphytopathogens;Y-axis:mRNAexpression(foldinductionrelativeto ␤-tubinnormalized).ThefoldchangeinPKSgeneexpressionwascalculatedusingtheformula2−CT.Whitebars: expressionrespondingtoS.selerotiorum;horizontalbars:expressionrespondingtoR.solani;Blackbars:expression respondingtoF.oxysporum.*Significantdifferenceat0.05level(p<0.05)bycomparingT.harzianum88whichwas

confrontationwiththreefungalplantpathogensandnon-confrontationwiththreefungalplantpathogens.Arrowsindicate thehighestexpressionofPKSgenesrespondingtodifferentphytopathogens.

ConstructionofthepksT-2disruptantstrain

The effect of the hygromycin B dose on the regeneration frequencyofconidiawasdeterminedbeforetransformation. HygromycinBat100␮g/mLcompletelyinhibitedthe regen-erationofuntransformed protoplasts. Therefore, growth at this concentration was used as the selection criterion for hygromycinB-toleranttransformants(Fig.6).

Weconstructeda pksT-2disruptant strainto examine thefunctionofpksT-2inT.harzianum.A.tumefaciens-mediated

transformation obtained six candidate colonies. Subse-quently,thesecolonieswerescreenedvia colonyPCRusing the primers pksT-2 checkRand pksT-2 checkF(Online Resource 1). We further confirmed the pksT-2 disrup-tant strain through Southernblot analysis.These analyses revealed the expected hybridization signal at7.8kband at 6.5kbinthedigestedgenomicDNAisolatedfrom the

wild-typeT.harzianum88andthepksT-2candidatetransformant

with BglII (Fig. 6). These results indicate successful gene

Bgl II Bgl II

–7.8 Kb –6.5 Kb

Lane 1 2

Fig.6–SouthernblotanalysisofgenomicDNAfrompksT-2 disruptantandT.harzianum88digestedwithBglIIas indicated.BandsweredetectedwithpksT-2-S(Lane1,2) digoxigenin-labelledprobes.

CharacterizationofthepksT-2disruptantstrain

Weexamined thepksT-2disruptantstraintodetermineits growthcharacteristics.Celldryweightofwild-typeandpksT-2 disruptantstrainsincreasedwithincubationtime,andwhich

ofwildtypeT.harzianum88reachedthehighestweightat84h.

However,based onthe dataobtainedfrom the cell growth curvesofT.harzianum88straininourlab(datanotshown), itscellhasenteredadeclinephaseat108h,Atthispointthe celldryweightisslightlylowerat108hthanat84h. Further-more,celldryweightofpksT-2disruptantstraingrowthslower

thanwildtypeT.harzianum88,andwhichreachedthehighest

weightat108h,butonly33%ofthedryweightofthe wild-typestraincell.Theresultshowedthatthegrowthrateofthe strainharbouringthepksT-2disruptantonsolidandliquid mediawassignificantlylowerthanthatofthewild-typestrain (Fig.7).

PhenotypeofthepksT-2disruptantstrain

WeobservedthephenotypeofthepksT-2disruptantstrainto confirmthefunctionofpksT-2andtoevaluatethegene target-ingefficiency.T.harzianum88producedgreenconidia(Fig.8A).

1.0 0.8 0.6 0.4 0.2 0.0 24 36 48 60 72 Time (h) T88 T5 Dr y w eight (g) 84 96 108 120

Figure7–Growthratesofthewild-typestrainandpksT-2 disruptant.Thewild-typestrainandpksT-2disruptant wereinoculatedinliquidMMmedium(1×106_conidia/mL) andgrownat28◦Cfor24,48hand72h,150rpm/min respectively.Thedatarepresentthemean±S.D(n=3). Blackbar:wild-type;whitebar:pksT-2disruptant.

UnderthesamegrowthconditionsT.harzianum88harbouring thepksT-2disruptantstraindisplayedapinkconidial pheno-type(Fig.8B),and theyellowconidiationspreadinto solid medium.ThisresultindicatesthatinactivationofthepksT-2

geneinfluencestheconidialpigmentationofT.harzianum88.

Polyketidesareinvolvedinthebiosynthesisofimportant nat-uralproducts,suchasfungalpigments,38 _{antibiotics,anda}

varietyoftherapeuticcompounds.39_{Thefunctionofa}

num-berofgenesinT.harzianum88genomicDNAthatareinvolved insecondarymetabolicpathways,suchasPKSandNRPSare unknown.Thus,T.harzianum88withthe pksT-2disruptant willbeusefulforgenedisruptionexperimentstoanalysegene function.

Fig.8–GenerationofapksT-2disruptant.PhenotypeofthepksT-2disruptantgrownonMMagarat28◦Cfor3days.The wildstraindisplayedthetypicalgreenconidia(A)andthepksT-2disruptantdisplayedpinkconidia(B).

Conclusions

In conclusion, pksT-1 and pksT-2 were isolated from T.

harzianum88.Althoughsecondarymetabolitesareunrelated

topksT-1 and pksT-2,analysisoftheir domainorganization

impliedthatpksT-1 providesasidechaininRAL

biosynthe-sisandpksT-2isinvolvedincompoundbiosynthesissimilar

tofumonisinbiosynthesis.TheexpressionlevelsofthepksT-1

andpksT-2geneswereevaluatedviareal-timeRT-PCRduring

challengewithdifferentfungalpathogens(S.sclerotiorum,R.

solaniandF.oxysporum).TheresultsindicatedthatpksT-1and

pksT-2aredifferentiallyregulatedwhenT.harzianum88is

chal-lengedwithatleastoneofthreefungalpathogens.ApksT-2 disruptantstrainwasobtainedthroughgenefunctional inac-tivation,anditsgrowthratewasslowerthanthatofthewild type.ThepksT-2disruptantstrainshowedthatthepksT-2gene encodesakeyenzymewhichmaybeinvolvedwith pigmen-tation.

Futurestudieswillfocusontheheterologousexpressionof

pksT-1andpksT-2toconfirmtheirfunctions.Thesestudieswill

provideabasisforthesustainableapplicationofT.harzianum 88secondary metabolitesinpharmaceuticaltreatmentand biocontrolofplantpathogens.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ProjectsupportedbytheNationalScienceFoundationfor Dis-tinguishedYoungScholarsofChina(GrantNo.31501839).

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.01.004.

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s

1. GilardiG,BaudinoM,GullinoML,GaribaldiA.Attemptsto controlFusariumrootrotofbeanbyseeddressing.Commun

AgricApplBiolSci.2008;73:75–80.

2. Martínez-MedinaA,PascualJA,Pérez-AlfoceaF,AlbaceteA, RoldánA.TrichodermaharzianumandGlomusintraradices modifythehormonedisruptioninducedbyFusarium

oxysporuminfectioninmelonplants.Phytopathology.

2010;100:682–688.

3. ChengCH,YangCA,PengKC.AntagonismofTrichoderma

harzianumETS323onBotrytiscinereamyceliuminculture

conditions.Phytopathology.2012;102:1054–1063.

4. HuangX,ChenL,RanW,ShenQ,YangX.Trichoderma

harzianumstrainSQR-T37anditsbio-organicfertilizercould

controlRhizoctoniasolanidamping-offdiseaseincucumber seedlingsmainlybythemycoparasitism.ApplMicrobiol

Biotechnol.2011;91:741–755.

5. NaeimiS,KocsubéS,AntalZ,etal.Strain-specificSCAR markersforthedetectionofTrichodermaharzianumAS12-2,a biologicalcontrolagentagainstRhizoctoniasolani,thecausal agentofricesheathblight.ActaBiolHung.2011;62:73–84.

6.LoCT,NelsonEB,HayesCK,HarmanGE.Ecologicalstudiesof transformedTrichodermaharzianumstrain1295-22inthe rhizosphereandonthephylloplaneofcreepingbentgrass.

Phytopathology.1998;88:129–136.

7.AmerMA,Abou-El-SeoudII.Mycorrhizalfungiand

Trichodermaharzianumasbiocontrolagentsforsuppression

ofRhizoctoniasolanidamping-offdiseaseoftomato.Commun

AgricApplBiolSci.2008;73:217–232.

8.KimTG,KnudsenGR.Comparisonofreal-timePCRand microscopytoevaluatesclerotialcolonisationbya biocontrolfungus.FungalBiol.2011;115:317–325.

9.KrokenS,GlassNL,TaylorJW,YoderOC,TurgeonBG. PhylogenomicanalysisoftypeIpolyketidesynthasegenes inpathogenicandsaprobicascomycetes.ProcNatlAcadSciU SA.2003;100:15670–15675.

10.SchusterA,SchmollM.Biologyandbiotechnologyof

Trichoderma.ApplMicrobiolBiotechnol.2010;87:787–799.

11.HarmanGE,PetzoldtR,ComisA,ChenJ.Interactions betweenTrichodermaharzianumstrainT22andmaizeinbred lineMo17andeffectsoftheseinteractionsondiseases causedbyPythiumultimumandColletotrichumgraminicola.

Phytopathology.2004;94:147–153.

12.ShoreshM,HarmanGE.Therelationshipbetweenincreased growthandresistanceinducedinplantsbyrootcolonizing microbes.PlantSignalBehav.2008;3:737–739.

13.TucciM,RuoccoM,DeMasiL,DePalmaM,LoritoM.The beneficialeffectofTrichodermaspp.ontomatoismodulated bytheplantgenotype.MolPlantPathol.2011;12:341–354.

14.ZeilingerS,OmannM.Trichodermabiocontrol:signal transductionpathwaysinvolvedinhostsensingand mycoparasitism.GeneRegulSystBiol.2007;1:227–234.

15.Moreno-MateosMA,Delgado-JaranaJ,CodónAC,BenítezT. pHandPac1controldevelopmentandantifungalactivityin

Trichodermaharzianum.FungalGenetBiol.2007;44:1355–1367.

16.SempereF,SantamarinaMP.Invitrobiocontrolanalysisof

Alternariaalternata(Fr.)Keisslerunderdifferent

environmentalconditions.Mycopathologia.2007;163: 183–190.

17.VinaleF,GhisalbertiEL,SivasithamparamK,etal.Factors affectingtheproductionofTrichodermaharzianumsecondary metabolitesduringtheinteractionwithdifferentplant pathogens.LettApplMicrobiol.2009;48:705–711.

18.HannanMA,HasanMM,HossainI,RahmanSM,IsmailAM, OhDH.Integratedmanagementoffootrotoflentilusing biocontrolagentsunderfieldcondition.MicrobiolBiotechnol.

2012;22:883–888.

19.HertweckC.Thebiosyntheticlogicofpolyketidediversity.

AngewChemIntEdEngl.2009;48:4688–4716.

20.CoxRJ.Polyketides,proteinsandgenesinfungi:

programmednano-machinesbegintorevealtheirsecrets.

OrgBiomolChem.2007;5:2010–2026.

21.ChiangYM,OakleyBR,KellerNP,WangCC.Unraveling polyketidesynthesisinmembersofthegenusAspergillus.

ApplMicrobiolBiotechnol.2010;86:1719–1786.

22.AmnuaykanjanasinA,PunyaJ,PaungmoungP,etal. DiversityoftypeIpolyketidesynthasegenesinthe wood-decayfungusXylariasp.BCC1067.FEMSMicrobiolLett.

2005;251:125–136.

23.WeiQ,ed.Theguideofmolecularbiologyexperiment.Harbin:

HigherEducationPublishing;2007.

24.CarsolioC,GutiérrezA,JiménezB,VanMontaguM, Herrera-EstrellaA.Characterizationofech-42,aTrichoderma

harzianumendochitinasegeneexpressedduring

mycoparasitism.ProcNatlAcadSciUSA.

1994;91:10903–10907.

25.LiuZ,YangX,SunD,etal.Expressedsequencetags-based identificationofgenesinabiocontrolstrainTrichoderma

26.LivakKJ,SchmittgenTD.Analysisofrelativegeneexpression datausingreal-timequantitativePCRandthe2-deltadelta CTmethod.Methods.2001;25:405–408.

27.GardinerDM,HowlettBJ.Negativeselectionusingthymidine kinaseincreasestheefficiencyofrecoveryoftransformants withtargetedgenesinthefilamentousfungusLeptosphaeria

maculans.CurrGenet.2004;45:249–255.

28.BakerSE,PerroneG,RichardsonNM,GalloA,KubicekCP. Phylogenomicanalysisofpolyketidesynthase-encoding genesinTrichoderm.Microbiology.2012;158(pt1):147–154.

29.WangS,XuY,MaineEA,etal.Functionalcharacterizationof thebiosynthesisofradicicol,anHsp90inhibitorresorcylic acidlactonefromChaetomiumchiversii.ChemBiol.

2008;15:1328–1338.

30.SagaramUS,ShimWB.FusariumverticillioidesGBB1,agene encodingheterotrimericGproteinbetasubunit,isassociated withfumonisinBbiosynthesisandhyphaldevelopmentbut notwithfungalvirulence.MolPlantPathol.2007;8:375–384.

31.DeOliveiraRochaL,ReisGM,daSilvaVN,BraghiniR, TeixeiraMM,CorrêaB.Molecularcharacterizationand fumonisinproductionbyFusariumverticillioidesisolatedfrom corngrainsofdifferentgeographicoriginsinBrazil.IntJFood

Microbiol.2011;145:9–21.

32.MenendezAB,GodeasA.BiologicalcontrolofSclerotinia

sclerotiorumattackingsoybeanplants.Degradationofthecell

wallsofthispathogenbyTrichodermaharzianum(BAFC742). BiologicalcontrolofSclerotiniasclerotiorumbyTrichoderma

harzianum.Mycopathologia.1998;142:153–160.

33.CoxRJ,GlodF,HurleyD,etal.Rapidcloningandexpression ofafungalpolyketidesynthasegeneinvolvedinsqualestatin biosynthesis.ChemCommun(Camb).2004:2260–2261.

34.MitchellT,BoehmM,HuJN,VenuRC.Identificationof potentialpathogenicityfactorsandhostdefensegenesin

theSclerotiniahomoeocarpa–turfgrasspathosystemusing

transcriptomeanalysis.In:Proc.ASA,CSSAandSSSAAnnual

Meetings.2011.

35.MaLQ,PangXB,ShenHY,etal.AnoveltypeIIIpolyketide synthaseencodedbyathree-introngenefromPolygonum

cuspidatum.Planta.2009;229:457–469.

36.YalpaniN,AltierDJ,BarbourE,CiganAL,ScelongeCJ. Productionof6-methylsalicylicacidbyexpressionofa fungalpolyketidesynthaseactivatesdiseaseresistancein tobacco.PlantCell.2001;13:1401–1409.

37.MukherjeePK,BuensanteaiN,Moran-DiezME,Druzhinina IS,KenerleyCM.Functionalanalysisofnon-ribosomal peptidesynthetases(NRPSs)inTrichodermavirensrevealsa polyketidesynthase(PKS)/NRPShybridenzymeinvolvedin theinducedsystemicresistanceresponseinmaize.

Microbiology.2012;158(pt1):155–165.

38.RugbjergP,NaesbyM,MortensenUH,FrandsenRJ. Reconstructionofthebiosyntheticpathwayforthecore fungalpolyketidescaffoldrubrofusarininSaccharomyces

cerevisiae.MicrobCellFact.2013;12:31.

39.DasguptaA.Advancesinantibioticmeasurement.AdvClin