Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum

36 Pages·2016·3.78 MB·English

RESEARCHARTICLE
Small RNA-Based Antiviral Defense in the
Phytopathogenic Fungus Colletotrichum
higginsianum
SoniaCampo1,2☯,KerriganB.Gilbert1☯,JamesC.Carrington1*
1 DonaldDanforthPlantScienceCenter,St.Louis,Missouri,UnitedStatesofAmerica,2 Centerfor
ResearchinAgriculturalGenomics,CSIC-IRTA-UAB-UB,CampusUAB,Bellaterra(CerdanyoladelVallès),
Barcelona,Spain
a11111 ☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
Eventhoughthefungalkingdomcontainsmorethan3millionspecies,littleisknownabout
OPENACCESS
thebiologicalrolesofRNAsilencinginfungi.TheColletotrichumgenuscomprisesfungal
Citation:CampoS,GilbertKB,CarringtonJC(2016) speciesthatarepathogenicforawiderangeofcropspeciesworldwide.Toinvestigatethe
SmallRNA-BasedAntiviralDefenseinthe
roleofRNAsilencingintheascomycetefungusColletotrichumhigginsianum,knock-out
PhytopathogenicFungusColletotrichum
mutantsaffectinggenesforthreeRNA-dependentRNApolymerase(RDR),twoDicer-like
higginsianum.PLoSPathog12(6):e1005640.
doi:10.1371/journal.ppat.1005640 (DCL),andtwoArgonaute(AGO)proteinsweregeneratedbytargetedgenereplacement.
Noeffectswereobservedonvegetativegrowthforanymutantstrainwhengrownoncom-
Editor:Hui-ShanGuo,ChineseAcademyof
Sciences,CHINA plexorminimalmedia.However,Δdcl1,Δdcl1Δdcl2doublemutant,andΔago1strains
showedseveredefectsinconidiationandconidiamorphology.TotalRNAtranscriptsand
Received:January6,2016
smallRNApopulationswereanalyzedinparentalandmutantstrains.Thegreatesteffects
Accepted:April26,2016
onbothRNApopulationswasobservedintheΔdcl1,Δdcl1Δdcl2,andΔago1strains,in
Published:June2,2016
whichapreviouslyuncharacterizeddsRNAmycovirus[termedColletotrichumhigginsia-
Copyright:©2016Campoetal.Thisisanopen numnon-segmenteddsRNAvirus1(ChNRV1)]wasderepressed.Phylogeneticanalyses
accessarticledistributedunderthetermsofthe
clearlyshowedacloserelationshipbetweenChNRV1andmembersofthesegmentedPar-
CreativeCommonsAttributionLicense,whichpermits
titiviridaefamily,despitethenon-segmentednatureofthegenome.Immunoprecipitationof
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare smallRNAsassociatedwithAGO1showedabundantloadingof5’U-containingviralsiRNA.
credited. C.higginsianumparentalandΔdcl1mutantstrainscuredofChNRV1revealedthattheconi-
DataAvailabilityStatement:Therawdata diationandsporemorphologydefectswereprimarilycausedbyChNRV1.Basedonthese
presentedinthisarticleisavailableon-line.Sequence results,RNAsilencinginvolvingChDCL1andChAGO1inC.higginsianumisproposedto
dataisavailableatGeneExpressionOmnibus
functionasanantiviralmechanism.
(GEO),accessionnumber:GSE62708.Thecomplete
genomesequenceofChNRV1isavailableat
GenBank,accessionnumber:KM923925.Remaining
primaryandintermediatefiles,includingPerlandR
scripts,areavailablethroughtheFigShareFileSet
AuthorSummary
doi:10.6084/m9.figshare.3124735.
Colletotrichumsp.comprisesadiversegroupoffungalpathogensthatattackover3000
Funding:SCwassupportedbyaMarieCurie
fellowship(FP7-PEOPLE-2010-IOF-273512).This plantspeciesworldwide.Understandingtheunderlyingmechanismsthatgovernfungal
workwassupportedbygrantsfromtheNational developmentandpathogenicitymayenablemoreeffectiveandsustainableapproachesto
ScienceFoundation(1330562;http://www.nsf.gov/)
PLOSPathogens|DOI:10.1371/journal.ppat.1005640 June2,2016 1/36
AntiviralRoleofRNASilencinginFungi
andNationalInstitutesofHealth(AI043288)toJCC.
Thismaterialisbaseduponworksupportedbythe cropdiseasemanagementandcontrol.Inmostorganisms,RNAsilencingisanimportant
NationalScienceFoundationunderGrantNo.DBI- mechanismtocontrolendogenousandexogenousRNA.RNAsilencingutilizessmallreg-
0922879foracquisitionoftheLTQ-VelosProOrbitrap
ulatorymolecules(smallRNAs)producedbyproteinscalledDicer(DCL),andexercise
LC-MS/MS.Thefundershadnoroleinstudydesign,
theirfunctionthougheffectorproteinsnamedArgonaute(AGO).Here,weinvestigated
datacollectionandanalysis,decisiontopublish,or
theroleofRNAsilencingmachineryinthefungusColletotrichumhigginsianum,bygener-
preparationofthemanuscript.
atingdeletionsingenesencodingRNAsilencingcomponents.Severedefectswere
CompetingInterests:Theauthorshavedeclared
observedinbothconidiationandconidiamorphologyintheΔdcl1,Δdcl1Δdcl2,andΔago1
thatnocompetinginterestsexist.
strains.AnalysisoftranscriptsandsmallRNAsrevealedanuncharacterizeddsRNAvirus
persistentlyinfectingC.higginsianum.Theviruswasshown(1)tobede-repressedinthe
Δdcl1,Δdcl1Δdcl2andΔago1strains,and(2)tocausetheconidiationandsporemutant
phenotypes.OurresultsindicatethatC.higginsianumemploysRNAsilencingasanantivi-
ralmechanismtosuppressvirusesandtheirdebilitatingeffects.
Introduction
RNA-mediatedsilencingmechanismsregulategeneexpressionatthetranscriptionalandpost-
transcriptionallevel[1].Althoughpathwayshaveproliferatedandspecializedinvariousline-
ages,acoreRNAsilencingmechanismisconservedamongplants,animals,fungiandother
eukaryotes[2].CanonicalRNAsilencinginvolveshighlybase-pairedordouble-strandedRNA
(dsRNA)thatisprocessedinto21–30-nucleotidesmallRNAsbytheactivityofoneormore
ribonucleaseIII-likeenzymescalledDicerorDicer-like(DCL)[3].SmallRNAsareincorpo-
ratedintoanRNA-inducedsilencingcomplex(RISC)thatcontainsamemberoftheArgo-
naute(AGO)proteinfamily[4].ThesmallRNAprogramsthecomplextorecognizetarget
RNA(s)throughbasepaircomplementarity,whiletheAGOproteinfunctionsasaneffectorto
modulatetheabundanceoractivityofthetarget[5,6].
Thefungalkingdomcomprisesanenormous,diversegroupoforganisms.Twomajorfungal
RNAsilencingpathwayshavebeendescribed:thequellingandthemeioticsilencingby
unpairedDNA(MSUD)pathways[7,8].Bothappeartobeeffectivegenomedefensemecha-
nismsthatoperateduringasexual(quelling)[9,10]andsexualdevelopment(MSUD)[11].
FungalRNAsilencingpathwaysfunctioningenomeprotectionandhavebeenproposedtobe
involvedinpathogenicity[12],development[13]andantiviraldefense[14].
Fungalviruses,ormycoviruses,arewidespread.Mostcharacterizedmycoviruseshave
dsRNAgenomespackagedinsphericalparticles(Totiviridae,Partititiviridae,Chrysoviridae)or
a(+)singlestranded(ssRNA)genomewithouttheabilitytoformparticles(Hypoviridae,
Endornaviridae)[15].Both(-)ssRNA[16]andssDNAmycoviruses[17]havealsobeen
described.Mycovirusesdonothaveanextracellularphase,butratheraretransmittedvertically
bysporesorhorizontallybyhyphalanastomosis[15].Althoughmycoviralinfectionsaregener-
allyassociatedwithcryptic(non-symptomatic)andlatent(expressedunderspecificcondi-
tions)infections,somemycovirusescausedebilitatingphenotypesintheirhost,making
mycovirusesapotentialtoolforthecontroloffungalplantpathogens[18–20].Inothercases,
mycoviruseshavecoevolvedmutualisticallywiththeirhosts[21,22].Cryphonectriaparasitica
isamodelfilamentousfungusforthestudyofvirus-hostinteractions[23,24],andprovidedthe
firstexamplethatRNAsilencingfunctionsasanantiviraldefencemechanisminfungi[14].C.
parasiticahasfourRNA-dependentRNAPolymerases,twoDicer,andfourArgonautegenes,
butonlydcl-2andagl-2havebeenshowntohaverolesinantiviraldefense[14,25,26].Genetic
studieshaverevealedthatC.parasiticaantiviraldefenseisactiveagainstmembersfromthe
Hypoviridae,Reoviridae[14,25],Partitiviridae[27],Totiviridae[28]andMegabirnaviridae
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AntiviralRoleofRNASilencinginFungi
[29]families.IndirectevidenceofanRNAsilencing-mycovirusinteractionhasalsobeen
describedinfungiwithkillerviruses.InSaccharomycescerevisiaetheMsatellitesofthedsRNA
mycovirusL-AfromtheTotiviridaefamilyproduceatoxinthatkillsuninfectedneighbour
cellsbutrendersthehostimmunetothetoxin[30].StrainswithactiveRNAsilencingsuppress
thevirusandlosetheadvantageprovidedbythevirus[31].Theincompatibilitybetweenthe
killervirusandtheRNAsilencingmachinerymightexplaintheexistenceofseveralRNA
silencing-deficientfungi[31,32].
ThegenusColletotrichumisconsideredoneofthemosteconomicallyimportantgroupsof
plantpathogens,causinganthracnosediseaseinover3,200speciesofmonocotanddicotplants
[33],withsomeinfectionsleadingtopost-harvestlossesofupto100%.Colletotrichumhiggin-
sianuminfectsplantsoftheBrassicaceaefamily,includingArabidopsisthaliana,andisemerg-
ingasamodelforthestudyofplant-pathogeninteractionsindicotyledonousspecies[34].C.
higginsianumisahemibiotrophicfungusthatformsanintracellularhyphaeduringtheinitial
symptomlessbiotrophicstagebeforeenteringadestructivenecrotrophiccolonizationphase.C.
higginsianumhasarelativelysmallhaploidgenomethatwasrecentlysequenced,theabilityto
beculturedaxenically,andstabletransformationmethodsthatallowfortheanalysisofgene
functionbytargeteddisruption[35].
TheprimarygoalofthisstudywastoidentifyandanalyzetheroleoftheRNAsilencing
machineryinthefungalpathogenC.higginsianum.Knock-outmutantsandhigh-throughput
sequencingwasusedtofunctionallycharacterizetranscriptomesandsmallRNApopulations
inC.higginsianummycelia.ChAGO1andChDCL1weredeterminedtobecriticalformain-
taininglowlevelsofaccumulationofanoveldsRNAvirus,designatedasChNRV1.Production
ofviralsmallRNAswasChDCL1-dependentandloadingintoChAGO1demonstratedastrong
preferencefor21-nt,5’Usequences.
ResultsandDiscussion
ColletotrichumhigginsianumRNAsilencingmachinery
GenesencodingthreehomologsofRNA-dependentRNAPolymerase(RDR1,RDR2and
RDR3),twohomologsofDicer(DCL1andDCL2)andtwohomologsofArgonaute(AGO1
andAGO2)wereidentifiedintheColletotrichumhigginsianumgenome(Fig1A).Allthree
RDRscontainedanRNA-dependentRNAPolymerasedomain(RdRP).BothDCLscontainan
RNAhelicasedomain,double-strandedRNA-bindingdomain(dsRBD)andtwoRNaseIIIcat-
alyticdomains.NeitherDCLcontainedaPAZ(Piwi/Argonaute/Zwille)domain,whichbinds
thetwo-nucleotide,3’overhangsoncanonicalDCLsubstrates[36,37].Dicer-likeproteinsin
someeukaryoteslackthePAZdomain[38–40]butretainthecapacitytogeneratesmallRNAs,
suggestingthepossibilityofothersubstraterecognitionmechanisms(S1AFig).BothC.higgin-
sianumAGOproteinspossessconservedPAZandPIWIdomains,andaconservedMID
domainrequiredfor5’anchoringoftheguideRNA.Thecatalytictriadresidues(Aspartic-
Aspartic-Aspartic),requiredforsliceractivity[41],arepresentinthePIWIdomainofboth
proteins(S1BFig).Additionally,AGO1containsanamino-terminalRGGbox,with12copies
oftheArginine-Glycine-Glycinemotif(Fig1A).TheRGGdomainfunctionsinnucleic-acid
bindingandprotein-proteininteractions,andispresentinsomeothereukaryoticAGOpro-
teins[39,42,43].
TheevolutionaryrelationshipsofC.higginsianumRDRs,DCLsandAGOswiththoseof
otherfungalspeciesfromtheAscomycotacladewasassessedbyphylogeneticanalysisofpro-
teinsequencesforeachfamily.MembersofC.higginsianumRDR,DCL,andAGOfamilies
groupedclearlywithknownmembersthatfunctioninquellingorMSUDpathways(Fig1B
and1D).RDR3,DCL1,andAGO1groupedwithquellingfactors,whileRDR1,DCL2,and
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Fig1.RNAsilencingintheascomycetefungusColletotrichumhigginsianum.(A)DomainorganizationofRNADependentRNApolymerases
(RDR),Dicer(DCL)andArgonaute(AGO)proteinsinC.higginsianum.RRM,RNARecognitionmotif.dsRBD,dsRNABindingDomain.Theconserved
asparticacidresiduesrequiredforAGOcatalyticactivityinthePIWIdomainareindicated(DDD).RGG,arginine-glycine-glycinerichdomain.(B-D)
PhylogeneticanalysisofRDR(B),DCL(C)andAGO(D)proteinsequences.Rootedmaximumlikelihoodneighborjoiningtreeswereconstructedby
alignmentoffull-lengthproteinsequencesfromrepresentativemembersoftheAscomycotaclade(Sordariomycetesinblue,Eurotiomycetesingreen,
Dothideomycetesinorange,Leotiomycetesinpink).C.higginsianumproteinsareindicatedwithreddots.Forthesakeofclarity,onlymaximum
likelihoodbootstrapsvalueshigherthan90%areshown.Twomaingroupsarelabeled,theQuellingpathway(shadedingreen)andtheMeiotic-
SilencingbyUnpairedDNA(MSUD)pathway(shadedinpurple).AccessionnumbersforproteinsequencesusedinthealignmentareinS1Table.
PhylogenetictreesweregeneratedusingRAxMLunderthemodelLG+G+Fofaminoacidsubstitution.Scalebarineachpanelrepresents0.1amino
acidsubstitutionspersite.(E)ExpressionanalysisofRDR,DCLandAGOgenesinC.higginsianummycelium.Silencinggenesaregroupedintothe
Quellingpathway(leftpanel),MSUDpathway(middlepanel)andUnknownpathway(rightpanel).Threebiologicalreplicateswereusedforeachgene;
valueswerenormalizedtothemeanofACTINandTUBULINgenesandthemeanexpressionofeachRNAsilencinggeneisrepresentedasarelative
valuecomparedtoAGO1.
doi:10.1371/journal.ppat.1005640.g001
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AGO2groupedwithproteinsfromtheMSUDpathway.RDR2wasidentifiedinaseparate
groupwithRRP3fromNeurosporacrassaandRDR4fromC.parasitica(Fig1B),thefunction
ofwhicharenotclear[44].ExpressionofRRP3andRDR4iselevatedbytheintroductionof
dsRNA[45]orinresponsetoviralinfection[26],respectively.
ExpressionlevelsofC.higginsianumsilencinggenesweredeterminedusingquantitative
realtimePCR(qRT-PCR).Basedonthephylogeneticanalysis,wehypothesizedthatgenesin
thequellingandMSUDpathwayswouldbeexpressedduringasexualandsexualstagesoffun-
galdevelopment,respectively.Colletotrichumisgenerallyrecognizedasanasexualgenus,
althoughsomespeciesareabletoadoptasexualform,whichareclassifiedunderthegenus
nameGlomerella[33].Colletotrichumasexualmorphsaregenerallyassociatedwithdisease
symptoms,whilesexualstagestendtodevelopinmoribundordeadhosttissues[33].TheC.
higginsianumstrainIMI349063Ausedinthisstudyhasnotbeenobservedtoreproducesexu-
ally[35],thus,geneexpressionanalysiswaslimitedtomycelia,conidia,andgerminated
conidia.RDR3,DCL1andAGO1hadthehighestlevelsofexpressioninmycelia(Fig1E,left
panel),conidia,andgerminatedconidia(S2Fig,leftpanels),whichisconsistentwiththeirpre-
dictedroleinthequellingpathwayduringvegetativegrowthandasexualreproduction.Con-
versely,transcriptsforRDR1,DCL2andAGO2(MSUDpathway)wereexpressedatdetectable
butrelativelylowlevelsinmycelia,conidia,andgerminatedconidia(Figs1EandS2,middle
panels).Thus,whilearoleforRDR1,DCL2andAGO2duringsexualreproductionisantici-
pated,itispossiblethatthesegeneshaverolesinasexualdevelopmentalphases.Transcriptsfor
RDR2wereexpressedatintermediatelevelsinmycelia,butrelativelyhighinconidiaandger-
minatedconidia(Fig1EandS2Fig,rightpanels).
LossofDCL1orAGO1leadstoreducedconidiationandalteredconidia
morphology
ToidentifyfunctionsoftheRNAsilencingcomponentsinC.higginsianum,singledeletion
mutantswithlossofRDR(S3Fig),DCL(S4Fig)andAGO(S5Fig)genesweregeneratedby
targetedgenereplacement.AdoubleDCL1/DCL2deletionmutantwasalsogenerated(S4Fig).
Single-sitegenedisruptionswereconfirmedbySouthernblotandRT-PCRanalysis,andfour
independentdeletionmutantswereselectedforfurtheranalysis(S3andS5Figs).Previous
workinMucorcircinelloides[46],Magnaportheoryzae[47],Candidaalbicans[48],Botrytis
cinerea[12],andTrichodermaatroviride[13]demonstratedthatdisruptionsofDCL1ortho-
logsweresufficienttoslowvegetativegrowth.However,inotherfungi,suchasC.parasitica
[14]andSaccharomycescastellii[38],comparablemutationsdidnotaffectgrowth.Toevaluate
C.higginsianumsilencingmutants,mycelialgrowthandmorphologyonconidiation
(Mathur’s),complete(PDA),andminimal(CDA)mediaweremeasured.Forallsingleand
doublemutants,colonymorphologyandgrowthwereindistinguishablefromthatofthewild-
typestrain(Figs2AandS6).TheΔdcl1,Δdcl2,andΔdcl1Δdcl2mutantswerealsoexposedto
oxidativestress(H O andMethylViologen),cellwallstress(Calcofluorwhite),salt-related
2 2
stress(NaCl,andLiCl),osmoticstress(sorbitol,andsucrose)andnutrientdeprivation(carbon
andnitrogen).GrowthoftheΔdcl1,Δdcl2,andΔdcl1Δdcl2mutantstrainswasindistinguishable
fromthatofthewild-typeonallmedia(S7Fig),suggestingthatsmallRNA-mediatedprocesses
arenotnecessaryforvegetativegrowthunderseveralabioticstressconditions.Theseresults
areinagreementwiththosereportedforsilencingmutantsoftheyeastsS.castellii[38]andC.
neoformans[49].
ConidiationwasanalyzedforallmutantstrainsaftergrowthinMathur’smediaforseven
days.Comparedtothecontrolstrains,conidiationwassignificantlyreducedintheΔdcl1,
Δdcl1Δdcl2,andΔago1strainsby8.8-fold,11.6-fold,and4-fold,respectively(Fig2B).There
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Fig2.PhenotypeanalysisofC.higginsianumRNAsilencing-mutantstrains.(A)ColonymorphologyandradialgrowthphenotypefortheC.
higginsianumRNAsilencingmachinerymutantsandControlstrainsonMathur’smedium.Arepresentativeexampleofcolonymorphologyafter6
daysofgrowth(leftpanel),andradialgrowthmeasurementsfromdays2to5(rightpanel)(mean+/-SE).Scalebar=1cm(B)Conidiaproductionin
theControlandRNAsilencingmutantstrains.Conidiawerecollectedafter7daysofgrowthinMathur’smediumandcountedwithahemocytomer.
Valuesplottedarefromthreebiologicalreplicatesforeachoffouruniquetransformants;redbarindicatesthemean.Strainswithsignificantly
differentconidiaproductionfromtheControlstrainareindicated(“a”:p=0.05).(C)Boxplotsrepresentinglength(upperpanel)andwidth(lower
panel)ofconidia.Conidiawerecollected,observedandmeasuredbylightmicroscopyusingaconfocalmicroscope.Atleast200conidiawere
measuredforeachstrain.Errorbarsrepresentthefirstandthirdquartile.Thehorizontallinewithintheboxrepresentsthemedianvalue(ie.50th
percentile).Blackdotsrepresentoutliers.(D)ConfocalimagesofconidiafromtheC.higginsianumControlandRNAsilencingmutantstrains.Scale
bar=2μm.
doi:10.1371/journal.ppat.1005640.g002
wasnosignificantdifferencebetweentheΔdcl1andΔdcl1Δdcl2strains,indicatingthatconidia-
tioneffectwasduetoDCL1withlittleornoredundancywithDCL2.DCLgeneshavebeen
implicatedinconidiationinotherfungi[13,50].InB.cinerea,atleastpartialredundancyofthe
twoDCLproteinswasobserved[12]wherethedoublemutantdcl1dcl2hadastronger,negative
effectonsporulationthandidthesinglemutantsdcl1anddcl2.Argonauteswerealsoshownto
affectsporeproductioninM.circinelloides[51],inwhichanago-1mutantstrainproduced
~50%fewersporesthanthecontrolstrain.
ConidiaproducedbytheΔdcl1,Δdcl1Δdcl2,andΔago1mutantswereshorterandwider
thanthoseproducedbythecontrolsstrain(Fig2C).MostconidiafromtheΔdcl1and
Δdcl1Δdcl2strainsexhibitedanabnormal,slightlyroundedmorphology(Fig2D),andwere
morevariableinlengthandwidththanthosecontrolstrains.DeletionmutantslackingRDR1,
RDR2,orRDR3geneshadnomeasurablegrowthorconidiationphenotypes(Fig2B).How-
ever,itisnotclearwhetherRDR1,RDR2andRDR3arenotfunctionalduringgrowthandconi-
diation,orarefunctionalbutredundant,ascombinatorialmutantswerenotanalyzed.
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Fig3.TranscriptandsmallRNAreadsunmappedtotheC.higginsianumgenome.Percentageofreads
notalignedtotheC.higginsianumgenomeintheControlandRNAsilencingmutantstrains,from(A)
transcriptand(B)smallRNAlibraries.(C)PercentageofsmallRNAsreadsunalignedtotheC.higginsianum
genomefromΔago1/6His-3FLAG-AGO1InputandIPlibraries.6H3F,6His-3FLAG.
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LocinotpresentintheC.higginsianumgenomeareamajorsourceof
smallRNAandtranscriptRNA
ThedeletionmutantanalysisrevealedthatDCL1andAGO1bothaffectconidiaproduction
andasexualsporemorphologyinC.higginsianum.ToidentifylocithatareaffectedbyRNA
silencingfactors,inparticularDCL1andAGO1,transcriptsandsmallRNAfromallmutant
strains(Fig3)wereanalyzed.Inaddition,smallRNAswereanalyzedafterimmunoprecipita-
tion(IP)of6His-3FLAG(6H3F)epitope-taggedversionsofAGO1(6H3F-AGO1)andAGO2
(6H3F-AGO2),whichwereexpressedinΔago1andΔago2strains,respectively(S8Fig).Trans-
formantscontainingandexpressing6H3F-AGO1hadsignificantlyincreasedconidiaproduc-
tionrelativetotheoriginalΔago1background,indicatingthetaggedversionofAGO1
complementedthelossofAGO1(S8BandS8DFig).
TranscriptRNAreadswerede-multiplexedandalignedtotheC.higginsianumgenome
usingBowtie2(S2Table)[52].Strikingly,40±3%oftheRNA-seqreadsinΔago1,54±5%ofthe
readsinΔdcl1,and46±6%ofthereadsinΔdcl1Δdcl2didnotmaptotheC.higginsianum
genome(Fig3AandS2Table).SmallRNAlibrarieswerealsode-multiplexed,trimmedofthe
3’adaptorsequence,andalignedtotheC.higginsianumgenomeusingthealignerBowtie(S3
Table)[53].Here,63±3%ofthesmallRNAreadsinΔago1,and46±8%ofAGO1-boundsmall
RNAsdidnotmaptotheC.higginsianumgenome(Fig3Band3C,S3Table).
High-throughputsequencingusuallyyieldsaproportionofreadsthatdonotmaptothetar-
getgenomesuchassequencingerrors,sequencevariants,andchimericsequences[54].Draft
genomesequencespresentfurtherdifficulties;theavailableC.higginsianumIMI349063A
genomesequencecontainsapproximately48.2Mbpofapredicted53.4Mbpnucleargenome
across10,235contigs,where5.2%ofgenesaresplitacrossmultiplecontigs,4%ofgenesare
truncatedandtheassemblydoesnotincludethemitochondrialgenome[35].Further,exoge-
nousspecies(viralorbacterial)maybepresentinthetargetsample.Severalinfectiousagents
havebeenidentifiedfromhigh-throughputsequencingreadsthatdidnotmapreference
sequences[55–57].Sincetheincreaseinunmappedtranscriptandsmallreadswasspecificto
theΔdcl1,Δdcl1Δdcl2,Δago1strains,aswellasthesmallRNAreadsfromthe6F3H-AGO1IP,
wehypothesizedthattheC.higginsianumsilencingmachineryisimportantfortheregulation
ofoneormorelociofanundeterminedorigin.
C.higginsianumstrainIMI349063AisinfectedwithadsRNAmycovirus
IdentificationofunknownRNAmoleculesusingdenovotranscriptomeassemblyof
RNA-seqdata. Toidentifytranscript(s)missingfromthereferencegenomesequencewe
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Fig4.IdentificationofanoveldsRNAvirusinC.higginsianum.(A)Genomeorganizationandsizeofthe
ColletotrichumhigginsianumNon-segmenteddsRNAVirus1(ChNRV1)identifiedbydenovotranscriptome
assembly.ChNRV1containstwoORFthatareindifferentframes.ORF1encodesaputativecoatprotein
(CP)andORF2aputativeRNA-dependentRNApolymerase(RdRP).UTR,Untranslatedregion.(B)
AccumulationofChNRV1transcriptsintheC.higginsianumwild-typestrainIMI349063Aasdeterminedby
semi-quantitativeRT-PCRanalysis(centerlane,cDNA),usingprimersspanningbothORFs(S7Table).PCR
analysisfromgenomicDNA(gDNA)ofC.higginsianumIMI349063Astrainusingthesameprimerspairs
determinedthatChNRV1isnotintegratedintothefungalgenome.(C)Electrophoreticanalysisofviral
dsRNAin1%agarosegelwithouttreatment,treatedwithDNase,treatedwithRNaseinahigh-salt,ortreated
withRNaseinalowsaltbuffer.ResistancetodegradationbyRNaseAinbufferwithhighsaltconcentration
confirmedthenatureofthedsRNAmolecule.
doi:10.1371/journal.ppat.1005640.g004
createdadenovotranscriptassemblyusingacombinedsetofallreadsfromthefourΔdcl1rep-
licatesandtheprogramTrinity[58].Atotalof22,237contigswereassembledwithanaverage
lengthof509bppercontig.Fromthisassembly26non-redundantcontigswereidentifiedas
originatingfromtheC.higginsianummitochondrialgenome.Additionally,a4,077bpcontig
wasidentifiedasanadditionalcopyofthelargesubunitrRNAgene.Themitochondrialand
rRNAtranscriptsequenceswereaddedtotheexistingC.higginsianumgenomereference
sequenceforsubsequentwhole-genomeanalyses(S1Text).
Next,theunmappedRNA-seqreadsfromoneΔdcl1replicatewerealignedtothedenovo
assembledtranscriptometoidentifyhighcoveragetranscriptsnotpresentinthegenome
sequence.Asinglesequence,assembledintotwocontigsrepresentingtheforwardandreverse
strandofthesamesequence,accountedfor79%ofthepreviouslyunalignedreads.ABLASTX
analysisagainstthenrdatabaseusingthelongerofthesetwosequences(2,835bpversus2,611
bp)identifiedtwoputativeopenreadingframes(ORF)(Fig4A).Thehighest-scoringalign-
mentforthe5’ORF(ORF1)wasORFA,aproteinofunknownfunctionfromBeauveriabassi-
anaRNAvirus1(e-value0.0).Thebestalignmentforthe3’ORF(ORF2)wastotheRNA-
dependentRNApolymeraseofPenicilliumjanczewskiiBeauveriabassiana-likevirus1(e-value
0.0).Theconserveddomaincd01699(RNA-dependentRNApolymerase)wasalsoidentified.
NovelRNAmoleculeisadouble-strandedRNAviruspresentinthewild-typestrain.
Bothbioinformaticandmolecularapproacheswereusedtoconfirmthepresenceofthevirusin
wild-typeC.higginsianumandwasthusancestraltothegeneratedRNAsilencingmutantlines.
RNA-seqreadsfromthecontrolsampleswerealignedtotheTrinitycontigs,resultingin9xcov-
erageofbothvirusgenomecontigs.Additionally,weanalyzedpreviouslypublishedRNA-seq
datafromfourdevelopmentalstagesofC.higginsianum[35]forthepresenceofthevirusand
foundsequencesthatuniquelymappedalong95%oftheviralgenome.Normalizedvirusread
counts(permillionmapped)fortheO’Connellandcolleagues[35]datasetwereapproximately
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AntiviralRoleofRNASilencinginFungi
300-timeslessabundantthanobservedinourcontroldataset,likelyduetodifferencesintotal
RNAextractionprotocols:O’Connellandcolleaguesusedpoly-ApurifiedtotalRNAasthetem-
plateforlibrarypreparation,whileweusedrRNA-depletedRNA.RT-PCRdemonstratedthe
presenceofaviraltranscriptinthewild-typestrainofC.higginsianum(IMI349063A)(Fig4B,
Lane2),andPCRanalysisoffungalgenomicDNAshowedthatthesequencewasnotintegrated
intotheC.higginsianumgenome(Fig4B,Lane3).Further,theRNAmoleculewasdetermined
tobedsRNAbasedonresistancetoDNase,resistancetoRNaseathighsaltconcentration(1M
NaCl),anddegradationbyRNaseatlowsaltconcentration(Fig4C).
RNA-ligase-mediatedRACE(RLM-RACE)[59]sequencingextendedthe5’and3’endsof
thecodingstrandby65ntand23nt,respectively,resultinginacompletedsRNAmoleculeof
2,923nt(Fig4A).Theadditional5’sequenceextendedORF1toincludeanATGstartcodon,
resultingina975ntlonggene,putativelyencodinga324aa(~35kDa)protein.ThedsRNA
sequenceincluded5’and3’untranslatedregions(UTR)of16and106ntrespectively.ORF1
andORF2areindifferentframesoftheplusstrand:ORF1inframe2(nucleotides17to991)
andORF2inframe1(nucleotides1081to2817),withan89-ntspacerinbetween.Aputative
slipperysiteheptamer,GGAUUUU,ispresentimmediatelyupstreamofthestopcodonof
ORF1,suggestingthatanORF1-ORF2fusionproteinmaybeproducedbya-1ribosomal
frameshift.
AsasearchthroughthedenovoTrinitytranscriptsdidnotrevealanyadditionalviral
RNAs,andthedsRNAsequencewasassembledasasinglefragment,wehavedesignatedthe
assembledcontigasColletotrichumhigginsianumnon-segmenteddsRNAvirus1(ChNRV1).
ThecompleteviralgenomesequenceisdepositedinGenBankundertheaccessionKM923925,
andGenBankproteinIDsforORF1andORF2areAIW81424andAIW81425,respectively.
TheidentificationofChNRV1promptedustoaskifitwaspresentinotherisolatesofC.
higginsianumandthecloselyrelatedspeciesC.destructivum,collectedfromvariousgeographi-
callocations(S4Table).RT-PCRwasusedtoscreenforthepresenceofChNRV1andonlyone
otherC.higginsianumstrain(IMI349061),whichwascollectedfromTrinidadandTobago
alongwiththestrainusedinthisstudy,haddetectablevirus(S4Table).Asthisassayspecifi-
callyqueriedforthepresenceofChNRV1sequences,theseC.destructivum/higginsianum
strainscouldbeinfectedwithotherviruses.ViraldsRNAelementshavebeendescribedprevi-
ouslyinthreeColletotrichumspecies,includingonefurthercharacterizedasaGammapartiti-
virus[60–62].Deepersurveysofmorestrainsfromadditionallocationswillbeneededtofully
definetherangeofChNRV1.
AnalysisofChNRV1proteinsandvirions. Double-strandedRNAvirusessharenumer-
ousstructuralandfunctionalsimilarities[63];manyareencapsidatedwiththenecessarycapsid
proteinsencodedforwithintheirgenome,alongwiththeRdRPusedforreplication.Therefore,
wehypothesizedthatORF1encodesacapsidprotein.Usingthepreviouslysolved3Dstructure
ofSaccharomycescerevisiaevirusL-A(ScV-L-A)(PDBID:1m1c)[64]topredicttheputative
3DstructureofORF1usingI-TASSER[65,66].Thetemplatemodelingscore(TM-score)is
reportedbetween0and1,whereaTM-scoregreaterthan0.5indicatesastrongsimilarityin
topology[67,68].TheTM-scoreforORF1alignedtoScV-L-Acapsidproteinwas0.872indi-
catingthatthetwoproteinshaveasimilartopology[67].Thenexthighestscoringstructural
analogwasthecapsidproteinfromPenicilliumchrysogenumvirus(PDBID:3j3iA)witha
TM-scoreof0.594.ThetopmodelpredictedbyI-TASSERwasdownloadedandvisualized
withChimera,wheretheORF1sequenceisinwhiteandtheScV-L-Acapsidsequenceincyan
(Fig5AandS1Movie).AsScV-L-Acapsidproteinsequenceis~2XlongerthanORF1(608aa
versus324aa),itwasexpectedthatalargepartoftheScV-L-Acapsidsequencewouldnothave
homologousregionsinORF1.Indeed,ORF1isstructurallysimilartoonlytheN-terminal414
aaofScV-L-A.ThisN-terminalregionisenrichedwithalphahelices(56%)comparedtofull
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AntiviralRoleofRNASilencinginFungi
Fig5.Characterizationofviralproteins.(A)PredictedsecondarystructureofChNRV1-ORF1,putative
coatprotein,(white)alignedtothemodelofScV-L-Acapsidprotein(cyan).The10aminoacidsfromtheN-
terminalandC-terminalendsofChNRV1-ORF1areinpurpleandorangerespectively.(B)Analysisofviral
proteinsbySDS-PAGE(upperpanel)anddsRNAbyagaroseelectrophoresis(lowerpanel)frompurified
virusfractions.Fourproteinsbands(p110,p36,p34andp25)andviraldsRNAaccumulateintheΔdcl1,
Δdcl1Δdcl2andΔago1strains.KDa,kilodaltons.(C)DistributionofChNRV1trypsin-digestedp110,p36,p34
andp26peptidesidentifiedbyMassSpectrometryalongthecapsidprotein(blueboxes)andtheRdRP
protein(orangeboxes).Valuesindicatethemeannormalizedspectralcountsandthepercentageof
sequencecoverageinparenthesis,p110:belowdiagram;p36,p34,p26:nexttodiagram.
doi:10.1371/journal.ppat.1005640.g005
lengthScV-L-A(38%)whiletheC-terminalend,whichisnotfoundinORF1,isenrichedin
betasheets(C-terminus:86%,entiresequence:63%)(S9Fig).ThusORF1appearstocontain
theconservedhelix-richcorefoundinScV-L-Aandothertotiviruses[69].
SDS-polyacrylamidegelelectrophoresis(SDS-PAGE)analysisdetecteddifferentialaccumula-
tionoffourproteinsofapproximately110,36,34,and26KDaintheΔdcl1,Δdcl1Δdcl2and
Δago1strainsthatwerevisiblyabsentinthewild-typestrain,thatwasaccompaniedbyspecific
accumulationofdsRNAmolecules(Fig5B).Eachproteinwasexcisedfromtwobiologicalrepli-
catesofΔago1andwild-typestrains,digestedbytrypsinandsubjectedtomassspectrometry
(MS).Proteinabundancewasestimatedonthebasisofspectralcountvalues[70](S5Table).In
allΔago1samples,proteinsencodedbyChNRV1werethemostabundantproteinsobserved.
Fromthep110sample,anaverageof70and66spectralcountsmatchedthecapsidandRdRP
proteins,respectively(Figs5CandS10A,S5Table).Theestimatedsizeofp110issimilartothe
predictedsizeofaCP-RdRPfusionprotein(933aa,103.9KDa),suggestingthattheseproteins
areexpressedasasinglepolyprotein,likelyduetoaribosomalframeshift.Howeverasthepre-
dictedm/zratioofthetrypsinproductsfromtheframeshiftregionexceededtheMSsurveyscan
m/zcut-off,thesefragmentswerenotobserved.Thecapsidproteinwasthemajorsourceofspec-
tralcountsintheΔago1mutantforthep36,p34,andp26samples(870,791,and970,counts
respectively)(Figs5CandS10A,S5Table).Bothp36andp34,butnotp26,hadtrypsin-derived
peptidesthatmatchedthecapsidN-terminus(Figs5CandS10C),suggestingthatp26isatrun-
catedformofthecapsidprotein.Althoughnotvisiblydetectableonthegel,thecapsidprotein
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