Molecular Characterization of a Heterothallic Mating System in Pseudogymnoascus destructans, the Fungus Causing White-Nose Syndrome of Bats


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Molecular Characterization of a Heterothallic Mating System in Pseudogymnoascus destructans, the Fungus Causing White-Nose Syndrome of Bats

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Title:
Molecular Characterization of a Heterothallic Mating System in Pseudogymnoascus destructans, the Fungus Causing White-Nose Syndrome of Bats
Series Title:
Genes, Genomes, Genetics
Creator:
Palmer, Jonathan M.
Kubatova, Alena
Novakova, Alena
Minnis, Andrew M.
Kolarik, Miroslav
Lindner, Daniel L.
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English

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Geomyces ( local )
Sexual Reproduction ( local )
Mating Type ( local )
White-Nose Syndrome ( local )
Genetics Of Sex ( local )
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serial ( sobekcm )

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Abstract:
White-nose syndrome (WNS) of bats has devastated bat populations in eastern North America since its discovery in 2006. WNS, caused by the fungus Pseudogymnoascus destructans, has spread quickly in North America and has become one of the most severe wildlife epidemics of our time. While P. destructans is spreading rapidly in North America, nothing is known about the sexual capacity of this fungus. To gain insight into the genes involved in sexual reproduction, we characterized the mating-type locus (MAT) of two Pseudogymnoascus spp. that are closely related to P. destructans and homothallic (self-fertile). As with other homothallic Ascomycota, the MAT locus of these two species encodes a conserved α-box protein (MAT1-1-1) as well as two high-mobility group (HMG) box proteins (MAT1-1-3 and MAT1-2-1). Comparisons with the MAT locus of the North American isolate of P. destructans (the ex-type isolate) revealed that this isolate of P. destructans was missing a clear homolog of the conserved HMG box protein (MAT1-2-1). These data prompted the discovery and molecular characterization of a heterothallic mating system in isolates of P. destructans from the Czech Republic. Both mating types of P. destructans were found to coexist within hibernacula, suggesting the presence of mating populations in Europe. Although populations of P. destructans in North America are thought to be clonal and of one mating type, the potential for sexual recombination indicates that continued vigilance is needed regarding introductions of additional isolates of this pathogen.
Original Version:
Genes, Genomes, Genetics, Vol. 8, no. 8 (2014-09-01).

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GENETICSOFSEXMolecularCharacterizationofaHeterothallic MatingSystemin Pseudogymnoascusdestructans , theFungusCausingWhite-NoseSyndromeofBatsJonathanM.Palmer,*AlenaKubatova,†AlenaNovakova,‡,§AndrewM.Minnis,*MiroslavKolarik,†,§andDanielL.Lindner*,1*CenterforForestMycologyResearch,NorthernResearchStation,USForestService,Madison,Wisconsin53726,†DepartmentofBotany,FacultyofScience,CharlesUniversityinPrague,Benátská2,CZ-12801Praha2,CzechRepublic,‡InstituteofSoilBiology,BiologyCentreCzechAcademyofSciences,NaSadkach7,CZ-37005 CeskeBud ejovice,Czech Republic,and § LaboratoryofFungalGeneticsandMetabolism,InstituteofMicrobiologyoftheASCR,v.v.i,Víde nská 1083,CZ-14220Praha4,CzechRepublic ORCIDIDs:0000-0003-0929-3658(J.M.P.);0000-0003-4016-0335(M.K.);0000-0002-2951-4481(D.L.L.)ABSTRACT White-nosesyndrome(WNS)ofbatshasdevastatedbatpopulationsineasternNorthAmerica sinceitsdiscoveryin2006.WNS,causedbythefungus Pseudogymnoascusdestructans ,hasspreadquickly inNorthAmericaandhasbecomeoneofthemostseverewildlifeepidemicsofourtime.While P.destructans isspreadingrapidlyinNorthAmerica,nothingisknownaboutthesexualcapacityofthisfungus.To gaininsightintothegenesinvolvedinsexualreproduction,wecharacterizedthemating-typelocus( MAT ) oftwo Pseudogymnoascus spp.thatarecloselyrelatedto P.destructans andhomothallic(self-fertile).As withotherhomothallicAscomycota,the MAT locusofthesetwospeciesencodesaconserved a -boxprotein ( MAT1-1-1 )aswellastwohigh-mobilitygroup(HMG)boxproteins( MAT1-1-3 and MAT1-2-1 ).Comparisons withthe MAT locusoftheNorthAmericanisolateof P.destructans (theex-typeisolate)revealedthatthis isolateof P.destructans wasmissingaclearhomologoftheconservedHMGboxprotein( MAT1-2-1 ).These datapromptedthediscoveryandmolecularcharacterizationofaheterothallicmatingsysteminisolatesof P.destructans fromtheCzechRepublic.Bothmatingtypesof P.destructans werefoundtocoexistwithin hibernacula,suggestingthepresenceofmatingpopulationsinEurope.Althoughpopulationsof P.destructans inNorthAmericaarethoughttobeclonalandofonematingtype,thepotentialforsexualrecombinationindicatesthatcontinuedvigilanceisneededregardingintroductionsofadditionalisolatesofthis pathogen.KEYWORDSgeomyces sexual reproduction matingtype white-nose syndrome GeneticsofSex Sinceitsdiscoveryin2006inNewYork,white-nosesyndrome(WNS) ofhibernatingbatshasspreadtomorethan25statesand5Canadian provinces,killingmorethan5.5millionbats(FroschauerandColeman 2012; www.whitenosesyndrome.org ).ThecausativeagentofWNS isapsychrophilicfungusnamed Pseudogymnoascusdestructans ( ¼ Geomycesdestructans )(Gargas etal. 2009;Lorch etal. 2011;Warnecke etal. 2012;MinnisandLindner2013)thathasbeenhypothesized tobeanintroducedpathogen,possiblyfromEurope,wherethe pathogenhasbeenconsistentlydetected(Martínková etal. 2010; Puechmaille etal. 2010,2011;Wibbelt etal. 2010)andcancause WNS(Pikula etal. 2012),althoughnomassmortalityhasbeenobservedinEurope.ThefungusisspreadingrapidlyinNorthAmerica (Lorch etal. 2013);isolatescollectedthusfarappeartohavebeen derivedfromasingleclonalintroductioninthenortheasternUnited States(Rajkumar etal. 2011;Ren etal. 2012).Thishasrecentlybeen supportedbywholegenomesequencingof26NorthAmericanisolatesof P.destructans (Chibucos etal. 2013;K.DreesandJ.Foster, Copyright©2014Palmer etal. doi:10.1534/g3.114.012641 ManuscriptreceivedJune11,2014;acceptedforpublicationJuly14,2014; publishedEarlyOnlineJuly21,2014. Thisisanopen-accessarticledistributedunderthetermsoftheCreative CommonsAttributionUnportedLicense( http://creativecommons.org/licenses/ by/3.0/ ),whichpermitsunrestricteduse,distribution,andreproductioninany medium,providedtheoriginalworkisproperlycited. Supportinginformationisavailableonlineat http://www.g3journal.org/lookup/ suppl/doi:10.1534/g3.114.012641/-/DC11Correspondingauthor:OneGiffordPinchotDr.,Madison,WI53726-2398. E-mail:dlindner@wisc.edu Volume4|September2014|1755

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unpublisheddata).Takentogether,thesedatasuggestthat P.destructans isspreadinginNorthAmericaexclusi velythroughasexualreproduction, giventhatconidiaarecommonlyobs ervedinclinicalspecimensand inculture(Meteyer etal. 2009;Chaturvedi etal. 2010).Althoughitis knownthat P.destructans reproducesasexually,itscapacityforsexual reproductionisunknown. Becausefungiandhumansarerelatedmembersoftheopisthokonts,sexualreproductioninfungihasbeenatopicofintenseresearchinterest(Heitman etal. 2013;DyerandO ’ Gorman2012;Ni etal. 2011;NielsenandHeitman2007).Althoughsexinvolvingtwo matingpartners,maleandfemale,isobligatoryinsomeeukaryotes ( e.g. ,humans),matinginfungicaninvolvemultiplematingtypes,but therearenomaleandfemalegendersand,thus,nosexchromosomes ( e.g. ,humanX&Y).Fungalmatingtypesaredeterminedbyasingle geneticlocustermedthemating-typelocus( MAT locus),which consistsofhighlydivergentnonhomologousgenesthataretermed idiomorphs(Heitman etal. 2013).Generally,the MAT idiomorphs encodefortwokeytranscriptionalregulators:wherethe MAT1-1 matingtypeiscontrolledbythe MAT1-1-1 a -boxtranscriptionfactor andwherethe MAT1-2 matingtypeiscontrolledbythe MAT1-2-1 high-mobilitygroup(HMG)transcriptionfactor(Ni etal. 2011). Whereassomefungalspecieshaveaheterothallic(outcrossing)matingsystem[ e.g. , Neurosporacrassa (MetzenbergandGlass1990)and Aspergillusfumigatus (O ’ Gorman etal. 2009)]involvingeachindividualhavingeitherthe MAT1-1-1 orthe MAT1-2-1 idiomorph,others canreproducehomothallically;theindividualcarriesbothidiomorphs,andthusasinglestrainiscapableofmatingwithitself, i.e. , itisself-fertile[ e.g. , Aspergillusnidulans (Paoletti etal. 2007), Sclerotiniasclerotiorum (Amselem etal. 2011), Sordariamacrospora (Klix etal. 2010)].Thespeci cgeneorganizationofthe MAT locuscanbe variableamongfungalspecies,althoughthecanonical MAT1-1-1 and MAT1-2-1 arealwayspresent(Figure1).Inseveralspecies,additional proteinsareencodedinthe MAT locus,forexample,Neurospora crassa (Ferreira etal. 1996)and Sordariamacrospora (Klix etal. 2010)containanadditionalHMG-boxgene( MAT1-1-3 )(Figure1). Althoughtheinfectiousparticlesofmanyfungalpathogensare asexualspores,sexualsporescanbeinfectious,asinthehumanpathogenicfungus Cryptococcusneoformans (Giles etal. 2009;Velagapudi etal. 2009).Thisisalsotrueofmanyfungalpathogensofplants, suchas Venturiainaequalis (applescab)(Machardy etal. 2001)and S.sclerotiorum (whitemold)(Amselem etal. 2011).Inmanypathogenicfungi,sexualsporesalsofunctionasimportantoverwintering orsurvivalstructures,allowingthefungustopersistforlongperiods oftimeintheabsenceofahost.Moreover,sexualreproductionin pathogenicfungiisofinterestbecause itisthebasisforgeneticvariability thathasthepotentialtocreateadditionalvirulentgenotypes.Mating populationsof P.destructans inNorthAmericacouldpotentially exacerbateWNS,soinformationregardingthesexualcapabilitiesof thisfungusisneededtohelpinformmanagementandtodevelopeffectivemitigationstrategies,especia llyinrelationtolong-distance(intercontinental)movementof P.destructans . Basedonarecentphylogeneticstudyofthegenus Pseudogymnoascus (MinnisandLindner2013),weselectedtwounnamedhomothallic (self-fertile)speciesthatproducesexualstructures(gymnothecia)in cultureandarerelativelycloselyrelatedto P.destructans asexemplars forunderstandingthemating-typelocusin Pseudogymnoascus .We clonedandsequencedthemating-type( MAT )locusfromthesetwo homothallicspeciesanddiscoveredthatthesespeciesshareanearly identicalgenestructureatthe MAT locus(see Results section).Comparisonofthehomothallic PseudogymnoascusMAT locustothe P.destructans genomereferencestrainsuggestedthatthebatpathogen waslikelyheterothallic.Wescreenedisolatesof P.destructans from centralEuropeanddiscoveredtheoppositematingtype( MAT1-2 ). Differentialexpressionof P.destructans genesinvolvedinmatingwas alsoexaminedinculturesofeachmatingtypeindividuallyaswellasin mixedculture. MATERIALSANDMETHODS FungalstrainsusedinthisstudyarelistedinTable1.Newstrainsof P.destructans wereisolatedfrommuzzlesandwingsofbatswithsuspectedWNS( Myotismyotis and Plecotusauritus )usingsterilecotton orplasticswabsandculturedonyeastextractglucosechloramphenicolagarorSabourauddextroseagarat10 .Forroutinelaboratory experiments,isolatesweremaintainedonacombinationofglucoseminimalmedium(GMM)(ShimizuandKeller2001)andChampe ’ s medium(ChampeandEl-Zayat1989).AllisolateshavebeenpreservedintheculturecollectionoftheCenterforForestMycology Research(CFMR).TheCultureCollectionofFungi(CCF)andthe CollectionofMicroscopicFungi(CMF)CzechRepublicalsomaintain culturesasindicatedbytheiracronymsinTable1.Allprimersare listedinSupportingInformation,TableS1.PfuUltraIIpolymerase (Stratagene)wasusedforallPCRreactionsaccordingtothemanufacturer ’ srecommendations.Standardmolecularbiologytechniques wereusedaspreviouslydescribed(SambrookandRussell2001).BLASTsearcheswereconductedusingthedraftgenomesequence oftheNorthAmericanisolate20631-21of P.destructans ( Geomyces destructans SequencingProject,BroadInstituteofHarvardandMIT; http://www.broadinstitute.org/ ). DNAextractionfromfungi Fungalculturesweregrowninliquidstationaryculturefor3wkin Champe ’ smedium(ChampeandEl-Zayat1989),myceliumwascollected,lyophilizedovernight,groundtoa nepowder,mixedwith700 ulofLETSBuffer(100mMlithiumchloride,20mMEDTA,10mM Tris-HCL,pH8.0,and0.5%SDS),andextractedwithanequalvolumeofphenol:chloroform:isoamylalcohol(Ambion);theaqueous phasewascollectedaftercentrifugationfor10minat12,000 g at4 . DNAwasprecipitatedbyadding1.0mlof95%ethanolandcentrifugedfor10min(12,000 g at4 ).TheDNApelletwaswashedwith 70%ethanolandsubsequentlyresuspendedin10mMTris-HCl(pH 8.0)containing20unitsofRNAseA(5 9 ). Cloningof MAT locusinhomothallic Pseudogymnoascus species Primersdesignedatconservedinternallocationsof P.destructans MAT1-1-1 ( a -box)wereusedtoamplifyaPCRfragmentofthe MAT1-1-1 genefromthehomothallic Pseudogymnoascus species (WSF3629and23342-1-I1); 900-bpfragmentwasobtainedfor isolateWSF3629and 400-bpfragmentwasobtainedfor23342-1I1.ThePCRfragmentsweresubsequentlyclonedusingpCR-Blunt II-TOPO(LifeTechnologies)andsequenced.Sequencingoftheregion ankingthe MAT1-1-1 genewasachievedbyusingamodi edversion ofthermalasymmetricalinterlacedPCR(TAIL-PCR)calledfusion primerandnestedintegratedPCR(FPNI-PCR)(Wang etal. 2011). Brie y,degeneratefusionprimers(FP1 – FP9)werepooledinbatches ofthreeandusedincombinationwithgene-speci cprimers(GSP) followedbytwoconsecutivenestedPCRreactions.ThelargestPCR productfromthe nalnestedreactionwasgel-puri ed,clonedinto pCR-BluntII-TOPO,andsubsequentlysequenced.Fivesuccessive roundsofFPNI-PCRwereconductedforisolate23342-1-I1andfour roundswereconductedforWSF3629,whichwassuf cienttoidentify theconserved ankinggene sla2 .Theremainingportionofthe MAT1756 |J.M.Palmer etal.

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locusforeachisolatewasPCR-ampli edbyusingaprimeranchored intheconserved ankinggene apn2 andaGSPprimerfromthe FPNI-PCRwalking,clonedintopCR-BluntII-TOPO,andsequenced. GenepredictionwasperformedusingacombinationofFGENESH (Solovyev etal. 2006)andAUGUSTUS2.7(Stanke etal. 2004)using thepre-trainedhidden-Markovmodelsfor Botrytiscinerea . Identi cationof P.destructansMAT1-2 locus Twenty-threeisolatesof P.destructans fromcentralEuropewere screenedviaSouthernblotusinga900-bpPCRfragmentof MAT1-1-1 asaradio-labeled32Pprobeaccordingtostandardprocedures (SambrookandRussell2001).Isolatesmissingthisfragmentwere suspectedofhavingtheothermatingtype.The MAT1-2 locus of P.destructans wasclonedandsequencedfromisolatesCCF3942and CCF4124byPCRamplifyingtheentireregionbetween apn2 and sla2 .ThepreviousSouthernblotwasstrippedandre-probedwith a1.1-kbradio-labeled32Pprobecorrespondingtothe MAT1-2-1 sequence. RNAextractionandsemi-quantitativeRT-PCR Conidiawereharvestedinsterilewatersupplementedwith0.01% Tween-80from8-week-oldculturesof P.destructans grownonGMM mediumat15 .Conidiafroma MAT1-1 isolateanda MAT1-2 isolate wereenumeratedwithahemocytometerandusedtoinoculate50-ml liquidculturesofChampe ’ smediumataconcentrationof1 · 105conidiaperml.Cultureswereincubatedinashakerat15 and200 rpmfor14d.Myceliumwascollectedfromeachstrainbysterile ltrationoverMiracloth(CalBiochem)andsubsequentlytransferred tothesurfaceofsolidGMMmediumagarplates:oneplateforeach matingtypeaswellasonethatwasa1:1mixtureofmyceliumfrom MAT1-1 and MAT1-2 strains.TheplateswerewrappedinPara lm-M (Bemis)andaluminumfoilandincubatedat15 foranadditional 14d.Myceliumwasthenscrappedoffthesurfaceoftheplatesusing asterileglassslide,immediatelyfrozeninliquidnitrogen,andlyophilizedovernight.TotalRNAwasextractedfromthelyophilizedtissue usingIsol-RNALysisReagent(5Prime)followingmanufacturer ’ s recommendations,treatedwithDNaseI(NEB)accordingtothemanufacturer ’ sprotocol,andsubsequentlyusedtomakecDNAusingthe iScriptcDNASynthesisKit(Biorad).Genesinvolvedinsexualreproductioninother lamentousfungiwereidenti edthroughBLASTp searchingofthe P.destructans referencegenomeandprimerswere designedforthemating-typegenes( MAT1-1-1 – GMDG_01209.1, MAT1-1-3 – GMDG_01208.1,and MAT1-2-1 – KJ938434),thepheromonepathway( ppg1 – GMDG_06142.1, pre1 – GMDG_00660.1,and pre2 – GMDG_08410.1),theG-proteinsignalingpathway( fad1 – GMDG_04604.1, sfa4 – GMDG_08182.1, gpg1 – GMDG_01954.1, mpk2 – GMDG_04404.1,and ste1 – GMDG_05416.1),andthevelvetcomplex ( vel1 – GMDG_00043.1, vel2 – GMDG_08054.1,and lae1 – GMDG_07817.1); actin( act1 – GMDG_01001.1)wasusedasaloadingcontrol.Between 32and42ampli cationcycleswereusedtodetecttranscriptionof genesputativelyinvolvedinsexualreproduction. RESULTS Identi cationofthemating-typelocus The MAT locusof P.destructans wasidenti edbyaBLASTp(Altschul etal. 1997)searchofthe P.destructans draftgenomeassemblywiththe MAT a -box( MAT1-1-1 )proteinsequencefrom Aspergillus nidulans AN2755(Paoletti etal. 2007).Thisresultedinidenti cation ofasinglehitonSupercontig14,correspondingtoGMDG_01209.1. Inother lamentousfungi,conservedprimarymetabolismgenes apn2 and sla2 ankthe MAT locus(Figure1);thus,welookedat anking genesonSupercontig14andidenti edGMDG_01207.1as apn2 and GMDG_01210.1as sla2 .UsingtheConservedDomainDatabase Figure1 The MAT locusof lamentousfungiconsistsoftheconservedregulators MAT1-1-1 and MAT1-2-1 .Sexinmembersofthe lamentousAscomycotacanbeeitherhomothallic(self-fertile)or heterothallic(requiret heoppositematingtype). Themechanismofsexualreproductionrequires theactionsoftheconservedtranscriptionfactors, MAT1-1-1 and MAT1-2-1 ,whicharephysically linkedtothe ankinggenes apn2 and sla2 ofthe MAT locus. MAT1-1-1 containsan a -boxdomain, whereas MAT1-2-1 containsarelatedHMG-boxdomain.SomeAscomycotaalsohaveanadditional HMG-boxtranscriptionfactor( MAT1-1-3 )thathas alsobeenimplicatedinsexualreproduction. Volume4September2014| MAT Locusof P.destructans | 1757

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n Table1Isolatesof Pseudogymnoascus usedinthisstudy CollectionInformationSequenceAccession# IsolateSpeciesMATTypeLocationDateSubstrateCitationITSLSUTEFMAT 20631-21 P.destructansMAT1-1 USA,NewYork2008 M.lucifugus (Gargas etal. 2009)EU884921KF017865KF017806KJ938437 WSF3629 P .sp.HomothallicUSA,Wisconsin1960Amorphouspeat(ChristensenandWhittingham 1965) KF039897KF017870KF017811KJ938436 23342-1-I1 P .sp.HomothallicUSA,Wisconsin2008 P.sub avus (Muller etal. 2012)JX415266KF017868KF017809KJ938435 CCF4801 P.destructansMAT1-1 CR,SWBohemia2013 M.myotis Thisstudy CMF2498 P.destructansMAT1-1 Slovakia,HarmaneckaCave2013 M.myotis Thisstudy CMF2583 P.destructansMAT1-1 CR,Moravia,NaPomezíCaves2013 M.myotis Thisstudy CMF2584 P.destructansMAT1-2 CR,Moravia,NaPomezíCaves2013 M.myotis ThisstudyKJ938418KJ938423KJ938428 CCF3937 P.destructansMAT1-1 CR,BohemianKarst,MalaAmerika2010 M.myotis (Kubátová etal. 2011) CCF3938 P.destructansMAT1-1 CR,Solenice2010 M.myotis (Kubátová etal. 2011) CCF3941 P.destructansMAT1-1 CR,BohemianKarst,MalaAmerika2010 M.myotis (Kubátová etal. 2011) CCF3942 P.destructansMAT1-2 CR,BohemianKarst,MalaAmerika2010 M.myotis (Kubátová etal. 2011)KJ938422KJ938427KJ938432KJ938434 CCF3944 P.destructansMAT1-1 CR,NovyKnin2010 M.myotis (Kubátová etal. 2011) CCF4103 P.destructansMAT1-1 CR,Herlikovice2011 P.auritus Thisstudy CCF4124 P.destructansMAT1-2 CR,HorniAlberice2011 M.myotis ThisstudyKJ938421KJ938426KJ938431KJ938433 CCF4125 P.destructansMAT1-1 CR,HorniAlberice2011 M.myotis Thisstudy CCF4127 P.destructansMAT1-1 CR,Herlikovice2011 M.myotisThisstudy CCF4128 P.destructansMAT1-1 CR,Herlikovice2011 M.myotis Thisstudy CCF4129 P.destructansMAT1-1 CR,Pistov2011 M.myotis Thisstudy CCF4130 P.destructansMAT1-1 CR,Fucna-Otov2011 M.myotis Thisstudy CCF4131 P.destructansMAT1-2 CR,Vyskov2011 M.myotis ThisstudyKJ938420KJ938425KJ938430 CCF4132 P.destructansMAT1-1 CR,Pernink2011 M.myotis Thisstudy CCF4247 P.destructansMAT1-1 CR,Morina2012 M.myotis Thisstudy CCF4350 P.destructansMAT1-1 CR,BohemianKarst,MalaAmerika2012 M.myotis Thisstudy CCF4351 P.destructansMAT1-2 CR,BohemianKarst,MalaAmerika2012 M.myotis ThisstudyKJ938419KJ938424KJ938429 CCF4471 P.destructansMAT1-1 CR,BohemianKarst,VelkaAmerika2013 M.myotis ThisstudyITS,internaltranscribedspacerregion;LSU,nuclearlargesubunitregion;TEF,translationelongationfactorEF-1 a ;MAT,mating-typelocus;CR,CzechRepublic.1758 |J.M.Palmer etal.

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(CDD)search(Marchler-Bauer etal. 2011)withGMDG_01209.1,we identi edthe MAT a -boxdomain(pfam04769).Interestingly, GMDG_01208.1isalsolocatedinthe MAT locusandhasapredicted HMG-boxdomain(cd01389).ABLASTpsearchusingGMDG_01208.1 ofthenonredundantproteindatabase(nr)atNCBIrevealedthetophits tobe MAT1-2-1 proteins(ACA51904.1,AFY11134.2,AGH03115.1, CBY44653.1).Therefore,weinitiallythoughtthat P.destructans could behomothallicbecausethe MAT locusharboredboth MAT1-1-1 ( a -box)and MAT1-2-1 (HMG-box)genes.However,becausewehave neverobservedfruitingbodiesfrom P.destructans 20631-21inculture andthe MAT locusofsomefungicontains twoHMG-boxdomaingenes, wecouldnotruleoutthat P.destructans 20631-21wasa MAT1-1 genotype. Cloningandsequencingof Pseudogymnoascus homothallic MAT loci Severalspeciesof Pseudogymnoascus areknowntobehomothallic, andthusproducesexualfruitingbodiesinculture(RiceandCurrah 2006;Tsuneda1982).Becausehomothallicasocmycetestypicallyhave both MAT idiomorphsatthe MAT locus,wereasonedthatcomparisonofthe MAT locusfromacloselyrelatedhomothallicspecies wouldaidincharacterizationofthe P.destructans matingsystem. Weselectedtwounnamedhomothallicisolatesfromarecentstudy: Pseudogymnoascus sp.WSF3629(cladeG – P.roseus complex)and Pseudogymnoascus sp.23342-1-I1(cladeD)(MinnisandLindner 2013). Pseudogymnoascus sp.WSF3629doesnotproduceconidia inculture;however,itproducesvisiblegymnothecia(Figure2A), whicharecomposedoflooselywoven,pigmentedperidialhyphae (Figure2B),asci(Figure2C),andascospores(Figure2D).Wehave observedasimilarsexualstatefor Pseudogymnoascus sp.23342-1-I1; formalidenti cationand/ordescriptionofthesespeciesarepresented elsewhere. Aftercon rmationofhomothallismintwo Pseudogymnoascus species,PCRprimersforthe P.destructansMAT1-1-1 wereused toamplify,clone,andsequenceaportionofthe MAT1-1-1 gene fromboth Pseudogymnoascus species(WSF3629and23342-1-I1). Figure2 Homothallicspeciesof Pseudogymnoascus producedgymnotheciaandcontaina MAT locusconsistingoftheconservedregulators MAT1-2-1 , MAT1-1-3 ,and MAT1-1-1 .(A)Gymnotheciaof Pseudogymnoascus WSF3629grownat25 for4wkonsolidoatmealmediuminthe dark.Scalebarsaredrawnoneachoftheimages.(B)GymnotheciaofWSF3629arecomposedoflooselywoven,pigmentedperidialhyphae,and amongtheperidialhyphaethereareasci.(CandD)Highermagni cationofascicontainingascosporesandascosporesliberatedfromasci.(E) Schematicofthemating-typelocus( MAT )forthehomothallicspecies Pseudogymnoascus sp.WSF3629and Pseudogymnoascus sp.23342-1-I1. TheNorthAmericangenomereferencestrainof P.destructans (20631-21)isthe MAT1-1 matingtype,whereasthe MAT locusof MAT1-2 strainsis depictedbytheCzechstrainof P.destructans CCF3942. Volume4September2014| MAT Locusof P.destructans | 1759

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SubsequentroundsoffusionprimerandnestedintegratedPCR (FPNI-PCR)(Wang etal. 2011)wereusedtoobtainsequence ofthe ankingregionsineachdirect ion,yieldingtheentiresequencebetweenthe apn2 and sla2 genesfromWSF3629and 23342-1-I1(13.2kBand12.4kB,respectively)(Figure2).Using the ab-initio genepredictionprogramsAUGUSTUS2.7(Stanke etal. 2004)andFGENESH( www.softberry.com ),wededucedthat thehomothallic MAT locusfrombothWSF3629and23342-1-I1 containanearlyidenticalg enestructureconsistingof vepredictedopenreadingframes(ORFs).AcombinationofBLAST (Altschul etal. 1997),CDD(Marchler-Bauer etal. 2011),and InterProScan(Quevillon etal. 2005)searchesidenti edaclear MAT a -boxprotein( MAT1-1-1 )andtwohigh-mobilitygroup (HMG)domain-containingproteins( MAT1-2-1 and MAT1-1-3 ) (Figure2).Thisanalysisalsoidenti edtwoadditionalORFs ( MAT1-1-6 and MAT1-2-5 );however,BLASTsearchdidnotrevealanysigni canthomologywithotherknownproteins,suggestingthatthesepredictedORFsareeithernovel MAT genes uniquetothePseudeurotiaceaeo rperhapspseudogenes.Pairwise comparisonofthe MAT locusfromthehomothallic Pseudogymnoascus speciestothe P.destructans genomesequencedreference strain(20631-21)indicatedthatt hegenomereferencestrainwas missingthe MAT1-2-1 HMGboxdomaincontaininggene,aswell asthehypothetical MAT1-2-5 gene,indicatingthatitwasa MAT1-1 ( a -box)matingtype. Identi cationofthe P.destructansMAT idiomorph The P.destructans genomereferencestrain(20631-21)isaNorth Americanisolatethathasbeenhypothesizedtobespreadingclonally (Ren etal. 2012;Rajkumar etal. 2011),whichhasrecentlybeensubstantiatedbecauseanalysisofwholegenomesequencingdataof26 NorthAmericanisolatesof P.destructansrevealedthattheyareallthe MAT1-1 genotype(Chibucos etal. 2013;K.DreesandJ.Foster,unpublisheddata).Althoughdiversitystudiesof P.destructans isolates collectedfromEuropehavenotbeenconducted,ithasbeenhypothesizedthatthefungusmayhaveoriginatedfromEurope(Warnecke etal. 2012);therefore,welookedfor alternativematingtypesin P.destructans isolatesfromcentralEurope(CzechRepublicand Slovakia).Wescreened23isolatesof P.destructans forthepresenceof the MAT1-1-1 geneviaSouthernblottingandfoundthat veofthe isolates(CMF2584,CCF3942,CCF4124,CCF4131,andCCF4351)were missing MAT1-1-1 (Figure3A).Theseisolateswerecon rmedtobe P.destructans bymorphologyaswellassequencingoftheITS,LSU,and TEFregions(Table1).Wenextc lonedandsequencedtheentire MAT locusfromCCF3942aswellasthegenomereferencestrain20631-21 asacontrol(Table1).ConsistentwithitsSouthernblot,CCF3942did Figure3 CentralEuropeanisolatesof P.destructans havetwomatingtypes( MAT1-1 or MAT1-2 ).(A)Southernblotofthe MAT locusoftheNorth Americanisolate(20631-21)and23isolatesfromcentralEurope.Expectedbandingpatternsforan EcoRI digestionof MAT1-1 strainsisasingle bandof3.183kb.Expectedbandingpatternfor EcoRI digestionusing MAT1-2 asaprobeisthreebandsof2.6kb,2.063kb,and0.699kb. Europeanisolatesof P.destructans collectedfromthesamehibernaculumanddate,differentindividualbats,butoppositematingtypesare demarcatedwitharedlineabovetheisolatename.(B)Schematicofthetwo MAT idiomorphsin P.destructans illustratingthegeneprediction structureandrestrictionenzymecutsites.Radio-labeledprobesusedintheSouthernblotareindicatedbyablackline. 1760 |J.M.Palmer etal.

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notcontainthe MAT1-1-1 sequence;instead,thisisolateharbors aHMGboxdomaincontainingprotein( MAT1-2-1 ),suggestingthat thisisolateistheoppositematingtype(Figure3B).Moreover,aSouthernblotusingaprobefor MAT1-2-1 identi edtheremainingfour isolatesasbeingidenticaltoCCF3942(Figure3A).Thereisalsoan additionalfaintbandintheSouthernblotof MAT1-1 isolateswhen probedwith MAT1-2-1 ,whichcouldbeduetohomologyinthe HMG-boxdomainof MAT1-1-3 .Ithasrecentlybeenrecognizedthat the MAT transcriptionfactorsshareanevolutionaryhistory,because eventhe MAT1-1-1 a -boxisderivedfromtheHMGgenefamily (Martin etal. 2010). Wealsoclonedandsequencedthe MAT locusfromCCF4124, whichwasa MAT1-2 isolatethatwascollectedonadifferentdate andlocation.Thesedatacorroboratethattherearetwo MAT idiomorphsfortheisolatesexamined: MAT1-1 and MAT1-2 .Interestingly,bothmatingtypeswereisolatedfromsamplestakenatdistinct timesfromdifferentindividualbatsfromthesamehibernaculum,even thoughonly23isolatesofEuropean P.destructans werescreenedinthis study(Figure3A). Analysisofgenesinvolvedinsexualreproduction Althoughthisisthe rstmolecularcharacterizationofsexualreproductionin Pseudogymnoascus ,muchisknownaboutthemolecular pathwaysinothermodelfungalsystemssuchas Saccharomycescerevisiae , Neurosporacrassa , Aspergillusnidulans ,andothers(Dyerand O ’ Gorman2012).Usingdatafromtheaforementionedmodelsystems, wesoughttoexamineexpressionofseveralconservedgenesinvolvedin sexualreproductionbysemi-quantitativereverse-transcriptasePCRof P.destructans mRNAfromtwomating-typeisolatesgrownaloneorin mixedculture(Figure4A).Thesedataareconsistentwithatypicalheterothallicmatingsysteminotherfungi,wherethe MAT1-1 locuscontrolstheexpressionoftheprecursorof a -pheromone( ppg1 ),whichis involvedinproductionofthe a -matingpheromone.The a -pheromone isrecognizedbytheG-protein-coupledreceptors( PRE1 and/or PRE2 ), whichourdatasuggestare,inturn,underthecontrolofthe MAT1-2 locusin P.destructans (Figure4A).In P.destructans ,itappearsthat co-cultivationof MAT1-1 and MAT1-2strainsresultsintheweakinductionofthe MAT1-1-3 HMGdomain – containinggene(Figure4A). Expressionofgenesinthesi gnaltransductionpathway( fad1 , sfa4 , gpg1 , mpk2 ,and ste1 )aswellasinthevelvetcomplex( vel1 , vel2 ,and lae1 )were notdrasticallyalteredineitherofthematingtypesorinmixedculture (Figure4A).Takentogether,thesedataindicatethat P.destructans has thenecessarygeneticsforsexualr eproductionandallowustopropose aheterothallicsexualrepro ductionpathway(Figure4B). DISCUSSION Togaininsightintothemolecularcomponentsofsexualreproduction inthePseudeurotiaceae,weselectedtwohomothallic(self-fertile) isolatesfromarecentstudycharacterizingspeciesrelatedto P.destructans (MinnisandLindner2013).Cloningandsequencingofthe MAT locusineachofthesespeciesrev ealeditwasnearlyidenticaland encodesforaconserved a -boxdomainprotein( MAT1-1-1 )andtwo conservedHMGboxdomainproteins( MAT1-1-3 and MAT1-2-1 ). Thisisconsistentwiththe MAT locusofotherwell-studiedhomothallic Ascomycotaspeciessuchas Sordariamacrospora (Klix etal. 2010), Fusariumgraminearum (Yun etal. 2000),and Sclerotiniasclerotiorum (Amselem etal. 2011),wherethematinggenesarelocatedinone conservedlocus ankedbytheprimarymetabolismgenes sla2 and apn2 .Comparisonofthehomothallic MAT locuswiththegenome referencestrainof P.destructans (20631-21)revealedthatitwasmissing the MAT1-2-1 HMGboxprotein,suggestingitwasheterothallic.Interestingly,therearetwomorepredictedORFsinthehomothallic MAT locus, MAT1-1-6 and MAT1-2-5 ,whichappeartohavenoknown functionaldomainsorhomologytootherknownproteinsandthus mayrepresentnovel MAT genesinthePseudeurotiaceae. PertinenttoWNSmanagement,wefoundisolatesofbothmating typesof P.destructans coexistinginEuropeanhibernacula,indicating thatincentralEuropethereisthepotentialformatingpopulations. Althoughthesedatasuggestthat,inourlimitedsampling,the MAT1-1 matingtypeisfoundmorefrequentlyon Myotismyotis(18outof23), moresamplingofEuropeanfungal isolatesisnecessarytounderstandtheprevalenceofmatingtypesin P.destructans .Preliminary Figure4 Putativegenesinvolvedinsexualreproductionareexpressedinlaboratoryculture. (A)Semi-quantitativereverse-transcriptasePCR wasusedtomeasuregeneexpressionofgenes predictedtobeinvolvedinsexualreproduction. AllPCRreactionswereconductedwith32 ampli cationcyclesexceptforthosemarked withanasterisk(),where42ampli cationcycles wereused.Matingtype MAT1-1 isrequiredfor expressionoftheprecursortoalpha-pheromone ( ppg1 ),whereas MAT1-2 isrequiredforexpressionoftheG-protein-coupledreceptors pre1 and pre2 .Expressionof MAT1-1-3 isonlyfound whenbothmatingtypeswereco-cultured.(B) Proposeddiagramofgenesinvolvedinsexual reproductionin P.destructans basedonhomologyandexpressioninlaboratoryculture. Volume4September2014| MAT Locusof P.destructans | 1761

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experimentsinducingsexualreproductioninthelaboratoryhavenot yieldedresultstodate;thisisnotsurprisingbecause P.destructans is slow-growingandsexualreproductionmaynotoccurforlongtime periods,asexempli edbyothermembersofthegenus(Riceand Currah2006).Moreover, ndingtheappropriateculturalconditions forfungiwithcrypticsexualcyclesistime-consuming.Forexample, althoughtheheterothallic MAT locus ofAspergillusfumigatus was characterizedin2005(Paoletti etal. 2005),ittookanother4yrto ndculturalconditionsconducivetosexualreproduction(O ’ Gorman etal. 2009).Molecularcharacterizationofthe MAT locusofisolates willhastentheprogressin ndingthesexualcycleof P.destructans . Intheabsenceofsexualstructuresof P.destructans ,wesoughtto furtherinvestigategeneticpathwaysinvolvedinsexthathavebeen well-studiedinotherfungi(DyerandO ’ Gorman2012).Consistent withotherAscomycota,ourexpressiondatasuggestthat MAT1-1 and MAT1-2 arelikelyresponsiblefordeterminationofmatingtype,becausetheprecursorto a -pheromone( ppg1 )wasonlyexpressedinthe MAT1-1 background.Moreover,bothoftheG-coupled-proteinreceptors( PRE1 and PRE2 )hypothesizedtorecognizethe a -pheromone areonlyexpressedinthe MAT1-2 background.Althoughwedidnot detectdifferencesinexpressionofgenesinvolvedinsexualdevelopmentinotherfungi,whichincludedthesignaltransductioncascade ( fad1 , sfa4 , gpg1 , mpk2 , ste1 )(DyerandO ’ Gorman2012)andthe velvetcomplexofproteins( lae1 , vel1 , vel2 )(BayramandBraus 2012;Bayram etal. 2008),thiswasnotsurprisinggiventhecentral importanceofthesegenesfornormalgrowthofthefungus.Interestingly, MAT1-1-3 ,theHMG-boxdomainproteinofthe MAT1-1 idiomorph,isonlyexpressedatlowlevelswhenbothmatingtypesare growninco-culture,suggestingthatitcouldbeinvolvedindownstreamtranscriptionalactivationofsexualreproduction. Giventheapparentclonalityof P.destructans inNorthAmerica, thisimportantdiscoveryofheterothallicmatingtypeshighlightsthe needforcontinuedvigilanceinpreventingadditionalintroductionsof thispathogeninNorthAmerica.Furtherworkisneededto ndand characterizethecrypticsexualcycleof P.destructans ,althoughdeterminationofthematingtypesofisol ateswillbecrucialtosuccessfully characterizingsexualreproductioninthisfungalpathogenunderlaboratoryconditions.Sexualrecombinationmayallow P.destructans to quicklyadapttoitsenvironmentan dhosts,despiteitsslowgrowth. Pertinenttopathogenicityof P.destructans ,matingtypesinotherfungi havebeencorrelatedtovirulence(CheemaandChristians2011;Nielsen etal. 2005;Kwon-Chung etal. 1992);therefore,thiswillbeanimportantconsiderationinelucidatingpathogenicityfactorsofWNS. ACKNOWLEDGMENTS WearegratefultoKevinDreesandJeffFosterforsharinggenomesequencingdatapriortopublication .WethankNancyKeller(University ofWisconsin)forsupport,aswellasJessieGlaeserandMarkBanik forcriticalreviewofthismanuscriptpriortosubmission.Thisstudy wasfundedbytheU.S.ForestService(NorthernResearchStation), theU.S.Fish&WildlifeService(FWS-R5-ES-12-001,Agreement #F11AP0078),andtheCzechScienceFoundation(ProjectNo. P506/12/1064). LITERATURECITEDAltschul,S.F.,T.L.Madden,A.A.Schaffer,J.Zhang,Z.Zhang etal. , 1997GappedBLASTandPSI-BLAST:anewgenerationofprotein databasesearchprograms.NucleicAcidsRes.25:3389 – 3402. Amselem,J.,C.A.Cuomo,J.A.vanKan,M.Viaud,E.P.Benito etal. , 2011Genomicanalysisofthenecrotrophicfungalpathogens Sclerotinia sclerotiorum and Botrytiscinerea .PLoSGenet.7:e1002230. Bayram,O.,andG.H.Braus,2012Coordinationofsecondarymetabolism anddevelopmentinfungi:thevelvetfamilyofregulatoryproteins.FEMS Microbiol.Rev.36:1 – 24. 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Meteyer,C.U.,E.L.Buckles,D.S.Blehert,A.C.Hicks,D.E.Green etal. , 2009Histopathologiccriteriatocon rmwhite-nosesyndromeinbats. J.Vet.Diagn.Invest.21:411 – 414. Metzenberg,R.L.,andN.L.Glass,1990Matingtypeandmatingstrategies in Neurospora .BioEssays12:53 – 59. Minnis,A.M.,andD.L.Lindner,2013Phylogeneticevaluationof Geomyces andalliesrevealsnocloserelativesof Pseudogymnoascusdestructans ,comb.nov.,inbathibernaculaofeasternNorthAmerica.Fungal Biol.117:638 – 649. Muller,L.K.,J.M.Lorch,D.L.Lindner,M.O ’ Connor,A.Gargas etal. , 2012Batwhite-nosesyndrome:Areal-timeTaqManpolymerasechain reactiontesttargetingtheintergenicspacerregionof Geomycesdestructans .Mycologia105:253 – 259. Ni,M.,M.Feretzaki,S.Sun,X.Wang,andJ.Heitman,2011Sexinfungi. Annu.Rev.Genet.45:405 – 430. Nielsen,K.,andJ.Heitman,2007Sexandvirulenceofhumanpathogenic fungi.Adv.Genet.57:143 – 173. Nielsen,K.,R.E.Marra,F.Hagen,T.Boekhout,T.G.Mitchell etal. , 2005Interactionbetweengeneticbackgroundandthemating-typelocus in Cryptococcusneoformans virulencepotential.Genetics171:975 – 983. O ’ Gorman,C.M.,H.T.Fuller,andP.S.Dyer,2009Discoveryofasexual cycleintheopportunisticfungalpathogen Aspergillusfumigatus .Nature 457:471 – 474. Paoletti,M.,C.Rydholm,E.U.Schwier,M.J.Anderson,G.Szakacs etal. , 2005Evidenceforsexualityintheopportunisticfungalpathogen Aspergillusfumigatus .Curr.Biol.15:1242 – 1248. Paoletti,M.,F.A.Seymour,M.J.C.Alcocer,N.Kaur,A.M.Calvo etal. , 2007Matingtypeandthegeneticbasisofself-fertilityinthemodel fungus Aspergillusnidulans .Curr.Biol.17:1384 – 1389. Pikula,J.,H.Bandouchova,L.Novotny,C.U.Meteyer,J.Zukal etal. , 2012Histopathologycon rmswhite-nosesyndromeinbatsinEurope. J.Wildl.Dis.48:207 – 211. Puechmaille,S.J.,W.F.Frick,T.H.Kunz,P.A.Racey,C.C.Voigt etal. , 2011White-nosesyndrome:isthisemergingdiseaseathreattoEuropeanbats?TrendsEcol.Evol.26:570 – 576. Puechmaille,S.J.,P.Verdeyroux,H.Fuller,M.A.Gouilh,M.Bekaert etal. , 2010White-nosesyndromefungus( Geomycesdestructans )inbat,France.Emerg.Infect.Dis.16:290 – 293. Quevillon,E.,V.Silventoinen,S.Pillai,N.Harte,N.Mulder etal. , 2005InterProScan:proteindomainsidenti er.NucleicAcidsRes.33: W116 – 120. Rajkumar,S.S.,X.Li,R.J.Rudd,J.C.Okoniewski,J.Xu etal. ,2011Clonal genotypeof Geomycesdestructans amongbatswithWhiteNoseSyndrome,NewYork,USA.Emerg.Infect.Dis.17:1273 – 1276. Ren,P.,K.H.Haman,L.A.Last,S.S.Rajkumar,M.K.Keel etal. , 2012Clonalspreadof Geomycesdestructans amongbats,midwestern andsouthernUnitedStates.Emerg.Infect.Dis.18:883 – 885. Rice,A.V.,andR.S.Currah,2006Twonewspeciesof Pseudogymnoascus with Geomyces anamorphsandtheirphylogeneticrelationshipwith Gymnostellatospora .Mycologia98:307 – 318. Sambrook,J.,andD.W.Russell,2001 MolecularCloning:ALaboratory Manual ,ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY. Shimizu,K.,andN.P.Keller,2001GeneticinvolvementofacAMPdependentproteinkinaseinaGproteinsignalingpathwayregulating morphologicalandchemicaltransitionsin Aspergillusnidulans .Genetics 157:591 – 600. Solovyev,V.,P.Kosarev,I.Seledsov,andD.Vorobyev,2006Automatic annotationofeukaryoticgenes,pseudogenesandpromoters.Genome Biol.7:S10 – S12. Stanke,M.,R.Steinkamp,S.Waack,andB.Morgenstern,2004AUGUSTUS: awebserverforgene ndingineukaryotes.NucleicAcidsRes.32:W309 – 312. Tsuneda,A.,1982Scanningelectronmicroscopyof Pseudogymnoascus roseus .Mycologia74:844 – 847. Velagapudi,R.,Y.-P.Hsueh,S.Geunes-Boyer,J.R.Wright,andJ.Heitman, 2009Sporesasinfectiouspropagulesof Cryptococcusneoformans .Infect.Immun.77:4345 – 4355. Wang,Z.,S.Ye,J.Li,B.Zheng,M.Bao etal. ,2011Fusionprimerand nestedintegratedPCR(FPNI-PCR):anewhigh-ef ciencystrategyfor rapidchromosomewalkingor ankingsequencecloning.BMCBiotechnol.11:109. Warnecke,L.,J.M.Turner,T.K.Bollinger,J.M.Lorch,V.Misra etal. , 2012InoculationofbatswithEuropean Geomycesdestructans supports thenovelpathogenhypothesisfortheoriginofwhite-nosesyndrome. Proc.Natl.Acad.Sci.USA109:6999 – 7003. Wibbelt,G.,A.Kurth,D.Hellmann,M.Weishaar,A.Barlow etal. , 2010White-nosesyndromefungus( Geomycesdestructans )inbats, Europe.Emerg.Infect.Dis.16:1237– 1243. Yun,S.H.,T.Arie,I.Kaneko,O.C.Yoder,andB.G.Turgeon,2000Molecular organizationofmatingtypelociinheterothallic,homothallic,andasexual Gibberella/Fusarium species.FungalGenet.Biol.31:7 – 20.Communicatingeditor:D.Charlesworth Volume4September2014| MAT Locusof P.destructans | 1763


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