Novel Mitoviruses and a Unique Tymo-Like Virus in Hypovirulent and Virulent Strains of the

20 Pages·2017·12.88 MB·English

viruses
Article
Novel Mitoviruses and a Unique Tymo-Like Virus in
Hypovirulent and Virulent Strains of the Fusarium
Head Blight Fungus, Fusarium boothii
YukiyoshiMizutani1,AdaneAbraham2,†,KazumaUesaka3,HidekiKondo2,HaruhisaSuga4,
NobuhiroSuzuki2 andSotaroChiba1,5,*
1 GraduateSchoolofBioagriculturalSciences,NagoyaUniversity,Nagoya464-8601,Japan;
[email protected]
2 InstituteofPlantScienceandResources,OkayamaUniversity,Kurashiki710-0046,Japan;
[email protected](A.A.);[email protected](H.K.);[email protected](N.S.)
3 CenterforGeneResearch,NagoyaUniversity,Nagoya464-8601,Japan;[email protected]
4 LifeScienceResearchCenter,GifuUniversity,Gifu501-1193,Japan;[email protected]
5 AsianSatelliteCampusesInstitute,NagoyaUniversity,Nagoya464-8601,Japan
* Correspondence:[email protected];Tel.:[phone]
† Presentaddress:DepartmentofBiotechnology,AddisAbabaScienceandTechnologyUniversity,P.O.Box
16417,AddisAbaba,Ethiopia.
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:2October2018;Accepted:23October2018;Published:26October2018
Abstract: Hypovirulenceofphytopathogenicfungiareoftenconferredbymycovirus(es)infections
andforthisreasonmanymycoviruseshavebeencharacterized,contributingtoabetterunderstanding
ofvirusdiversity. Inthisstudy,threestrainsofFusariumheadblightfungus(Fusariumboothii)were
isolatedfromEthiopianwheatsasdsRNA-carryingstrains: hypovirulentEp-BL13(>10,3and2.5kbp
dsRNAs),andvirulentEp-BL14andEp-N28(3kbpdsRNAeach)strains. The3kbp-dsRNAsshared
98%nucleotideidentityandhavesingleORFsencodingareplicasewhenappliedtomitochondrial
codonusage. PhylogeneticanalysisrevealedthesewerestrainsofanewspeciestermedFusarium
boothiimitovirus1inthegenusMitovirus. ThelargestandsmallestdsRNAsinEp-BL13appearedto
possesssingleORFsandthesmallerwasoriginatedfromthelargerbyremovalofitsmostmiddle
part. ThelargedsRNAencodedareplicasesharingthehighestaminoacididentity(35%)withthat
ofBotrytisvirusF,thesolememberofthefamilyGammaflexiviridae. Giventhatthephylogenetic
placement, largegenomesize, simplegenomicandunusual3(cid:48)-terminalRNAstructureswerefar
differentfrommembersintheorderTymovirales,thevirustermedFusariumboothiilargeflexivirus
1mayformanovelgenusandfamilyundertheorder.
Keywords: Fusariumheadblight;mycovirus;RNAgenome;mitovirus;Tymovirales;Ethiopia
1. Introduction
Thenumberofextantfungalspeciesiscurrentlyestimatedatupto1.5million. Virusesthatinfect
fungiareknownasmycovirusesorfungalvirusesandareubiquitousacrossthekingdomFungi[1].
Partitiviruses,andmitovirusesinparticular,areomnipresent. Interestingly,mycoviruseswereoften
found to have unique features such as genomic structures, virion structures, anti-viral defence
strategies,etc. thatdifferfrommajorplantandanimalviruses[2–4]. Mostmycovirusesinfectfungal
hostsasymptomaticallyandtheremainderrarelyexertapositiveornegativeimpactonfungalhost
traitssuchasvirulence,asexual/sexualsporeproduction,pigmentationandgrowth[5–7]. Virocontrol,
amethodofbiologicalcontrolusingmycovirusesthatattenuatethevirulenceofphytopathogenicfungi
(conferringhypovirulence),hasattractedtheattentionofresearchersforthelastfewdecades[8–11],
Viruses2018,10,584;doi:10.3390/v10110584 www.mdpi.com/journal/viruses
Viruses2018,10,584 2of20
andinformationonmycovirusdiversityhasexpandedanddeepeneddramaticallythroughintensive
mycovirushuntingaimingatscreeningforusefulvirocontrolagents[1]. However,whilemycovirus
huntinghasbeenextensiveinAsian,European,AmericanandOceaniancountries,itsexploration
in the African continent has been limited: Diaporthe ambigua RNA virus (an unclassified RNA
virus), Diplodia scrobiculata RNA virus 1 (a fusagravirus) and Sphaeropsis sapinea RNA virus 1
and2(victoriviruses)arereportedinSouthAfrica[12–16]withtheremainderbeingmycovirus-like
sequencesobtainedbymetagenomicviromeapproaches[17].
Fusariumheadblight(FHB)isaneconomicallyimportantdiseaseofwheatcausedbymembers
of the Fusarium graminearum species complex (FGSC) that consists of at least 16 species including
F. boothii. The disease symptoms are reduced yields, shrivelled kernels, reduced seed quality,
anddiminishedseedweight. Inaddition,thepathogenleavesmycotoxinssuchasdeoxynivalenol
(DON)andnivalenol,typeBtrichothecenes,ongrainsurfacewhichcancausediarrhoeaandvomiting
inanimalsingestingthem. Anincreasingnumberofmycoviruseshavebeenreportedtoinfectvarious
Fusariumspecies,includingFGSCfromdifferentregionsoftheworld. Thesevirusesincludemembers
of Chrysoviridae, Hypoviridae, Partitiviridae, Narnaviridae, Totiviridae, and Mymonaviridae families;
proposedAlternaviridaeandFusariviridaefamilies; andotherunclassifiedvirusgroups[9,18–27].
Thosewiththegreatestpotentialasvirocontrolagentsare: Fusariumgraminearumvirus1strainDK21
(FgV1-DK21),FusariumgraminearumvirusstrainChina9,Fusariumgraminearumhypovirus2strain
JS16,andFusariumvirguliformedsRNAmycovirus1and2[18,21,23,28].
Asabovementioned,mitovirusesinthegenusMitovirusinthefamilyNarnaviridaeareomnipresent
infilamentousfungiandthemembershaveapproximately3kbpositivesensesinglestrandedRNA
((+)ssRNA)genomesthatpossessthesingleopenreadingframe(ORF)encodinganRNA-dependent
RNA polymerase (RdRp) [29]. The ORF accommodates “UGA” triplets that is an interrupting
stop codon when applied nuclear gene code but translated as tryptophan in the mitochondrial
gene code. Cryphonectria parasitica mitovirus 1 (CpMV1) is the first mitovirus described,
andco-presenceofvirus-deriveddoublestrandedRNA(dsRNA;consideredasareplicativeform)
withsubcellularlyfractionatedmitochondriasuggeststhelocalizationofCpMV1inmitochondria[30].
Khalifa and Pearson [31] observed the accumulation of filamentous structures in mitochondria
of Sclerotinia sclerotiorum under electron microscope, specifically when a mitovirus infected to
the host. Therefore, mitoviruses are expected to uniquely replicate in the host mitochondria.
Botrytiscinereamitovirus1,Thanatephoruscucumerismitovirus(TcMV,knownalsoasRhizoctonia
M2 virus), Sclerotinia sclerotiorum mitovirus 1/KL-1, Sclerotinia sclerotiorum mitovirus 2/KL-1,
Sclerotiniasclerotiorummitovirus3/NZ1,andSclerotiniasclerotiorummitovirus4/NZ1wereshown
tohaveahypovirulence-conferringabilitybuteffectsofmostmitovirusesonhostfungalphenotypes
have not been elucidated because mitoviruses are generally hard to cure and introduce [7,31–35].
SeveralmitoviruseshavealsobeenreportedfromFusariumspeciesincludingFGSCbutbiological
propertiesofthosearelargelyunknown[25,36,37].
The order Tymovirales contains viruses that have a non-segmented (+)ssRNA genome with
single or multiple ORFs and its members mostly infect plants. The order consists of five
families—Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Deltaflexiviridae and Tymoviridae in
International Committee on Taxonomy of Viruses (ICTV)—based on the genome organisation,
phylogeneticrelationshipsandvirionmorphology. Asofnow,Alpha-,Gamma-andDeltaflexiviridae
includemycovirusesand,toourknowledge,atleast10suchmycoviruseshavebeencharacterised
includingtheonesassociatedwithhoststhatexhibithypovirulentphenotypes[20,27,38–45].
Botrytis virus F (BVF, Gammaflexiviridae) was isolated from a hypovirulent strain of Botrytis
cinerea as the first mycovirus taxonomically classified in the order Tymovirales, and was followed
by Botrytis virus X (BVX, Alphaflexiviridae) [39,40]. S. sclerotiorum also hosts mycoviruses of this
order,namelySclerotiniasclerotiorumdebilitation-associatedRNAvirus(SsDRV,Alphaflexiviridae)
and Sclerotinia sclerotiorum deltaflexivirus 1 (SsDFV1, Deltaflexiviridae). The former is associated
with hypovirulence of its host and the latter is recognised as the first mycovirus in the order
Viruses2018,10,584 3of20
Tymoviralesthathasnocapsidprotein(CP)[41,42]. Recently,Rhizoctoniasolaniflexivirus1(RsFV-1,
unclassifieddeltaflexivirus-like)wasreportedtohavethelargestgenomeintheorderanditsunique
genomicorganisationbroadenedourperceptionofgenomeplasticitywithintheorder[38]. Inaddition,
twomycoviruses,Fusariumgraminearummycotymovirus1(FgMTV1,unclassifiedtymovirus-like)
andFusariumgraminearumdeltaflexivirus1(FgDFV1,Deltaflexiviridae),havebeenisolatedfromthe
FHBpathogen,F.graminearum[20,27]. BiologicalassaysdemonstratedthatFgMTV1infectiondoesnot
alterthehostvirulence,butdecreasesthegrowthrateandDONproduction. Finally,althoughoyster
mushroomsphericalvirus(OMSV)wasearlierreportedtobeatymo-likemycovirus[44],itstaxonomic
placementisinconclusivetilldate.
This study characterised a novel member of Tymovirales, potentially forming a new genus or
family,andmembersofanewspeciesinthegenusMitovirusinthefamilyNarnaviridae. Thesevirus
classeswereidentifiedforthefirsttimeinFusariumboothiistrainsfromtheAfricancontinent. Itis
noteworthy that the tymo-like virus possesses the largest genome size (over 12.5 kb) among the
currentlyreportedTymoviralesmembers. ThisstudycontributestoourunderstandingofTymovirales
virusandmitovirusdiversityandevolution.
2. MaterialsandMethods
2.1. FungalStrainsandCulture
F. boothii strains (Ep-BL13, Ep-BL14 and Ep-N28) were isolated from wheats with the FHB
symptomsinHadiyaregionofsouthernEthiopia. AJapaneseisolateofF.graminearumsensustricto
(s.str.) strainwasalsousedasareference. Allstrainsweremaintainedat28◦Conpotatodextroseagar
(PDA,BDDifcoLaboratories,Detroit,MI,USA)andsyntheticlownutrientagar(SNA)andstoredat
4◦Cinthedark. Myceliawereculturedinpotatodextrosebroth(PDB,BDDifcoLaboratories,Detroit,
MI,USA)liquidmediumatroomtemperaturefor4–7daysfornucleicacidextractions.
2.2. BiologicalAssessmentofFGSCStrains
Growthrateandcolonymorphology. MycelialplugsweretransplantedonPDAandSNAplates
(threereplicates)andincubatedat20◦Cfor1week. Thelongestandshortestdiametersofeachcolony
weremeasured3–7dayspost-transplantingandthemeandiameterwasusedforthecalculationof
colonyarea.
Pathogenicitytest. Conidialspores(1×105spores/mL)suspendedina1%(v/v)Tritonsolution
were used for inoculation, and the FHB-sensitive wheat cultivar Apogee was grown at 27 ◦C in a
chamber(KG-50HLA;KoitoElectricIndustries,Yokohama,Japan)toavoidpossiblereleaseofinocula
into the environment [46,47]. Briefly, 10 µL of spore suspension was injected into wheat spikelets,
andthenumberofdiseasedspikeletswascountedat15dayspost-inoculation. Alternatively,thespore
suspensionwassprayedthreetimes(500µL/push)toawheathead,anddiseaseseveritywasevaluated
accordingtoBanandSuenaga’scriteria(index,mildtosevere;0,5,10,20,30,50,60,80,100)at15days
post-inoculation[48]. Inoculatedplantswereplacedinatransparenthumidboxforoneday(injection
method) or two days (spray method) to promote fungal germination, prior to the maintenance in
thechamber.
2.3. GenomicPolymeraseChainReaction
Fungal genomic DNA was purified by conventional CTAB (cetyl trimethylammonium
bromide) extraction. The partial internal transcribed spacer (ITS) of rDNA and transcription
elongation factor 1-α gene (TEF1-α) sequences were PCR-amplified with the oligonucleotide
primers reported by O’Donnel et al. [49,50]. Primers used in this study were: primer 1
(5(cid:48)-TCAAAATGGGTAAGGA(A/G)GACAAGAC-3(cid:48)) and primer 2 (5(cid:48)-GCCTGGGA(G/A)GTAC
CAGT(G/C)ATCATGTT-3(cid:48))forITS;andprimer3(5(cid:48)-GTGGGGCATTTACCCCGCC-3(cid:48))andprimer4
Viruses2018,10,584 4of20
(5(cid:48)-GAGTGGCGGGGTAAATGCC-3(cid:48))forTEF1-α. Fungalspecieswereidentifiedbyhomologysearch
withandbyphylogeneticanalysiswithsequencesfrompublicdatabases.
2.4. dsRNAExtractionandcDNALibraryConstruction
dsRNA fraction was extracted from fungal mycelia by conventional cellulose column
chromatography. Filtrated mycelia were homogenised in the presence of liquid nitrogen,
and total RNA fractions were obtained by treatment with one round each of phenol,
phenol–chloroform–isoamylalcohol (25:24:1), and chloroform–isoamylalcohol (24:1) extraction.
dsRNAswerefurtherisolatedfromthetotalRNAfractionsbyusingCellulosePowderC(Advantec,
Tokyo, Japan). To eliminate fungal chromosomal DNA and ssRNA species, these fractions
were treated with RNase-free RQ1 DNase I (Promega, Madison, WI, USA) and S1 Nuclease
(Promega). ConventionalcDNAlibraryconstructionwasperformedforviralsequencedetermination.
The dsRNAs extracted from F. boothii Ep-BL13, Ep-BL14 and EP-N28 and the 3 kbp- and
2.5-kbp dsRNA bands were gel-purified using a Zymoclean™ Gel RNA Recovery kit (Zymo
Research, Irvine, CA, USA). cDNA libraries of each dsRNA fragment were constructed using a
non-PCR and a PCR-based method. After denaturation of dsRNA templates at 99 ◦C for 5 min,
cDNAwassynthesisedusingReverTraAce-α-(Toyobo,Osaka,Japan)withanadapter-taggedprimer
(5(cid:48)-CCTGAATTCGGATCCTCCNNNNNN-3(cid:48)). TheresultingcDNAfragmentswerepurifiedfrom
an agarose gel with a size exclusion (1.0–2.0 kbp) and amplified by PCR with an adapter primer
(5(cid:48)-CCTGAATTCGGATCCTCC-3(cid:48)). ThePCRconditionsareasfollows: 94◦C(2min),then1cycleat
94◦C(2min)/65◦C(1min)/72◦C(1min),followedby35cyclesat94◦C(40s)/52◦C(30s)/72◦C
(3min),andafinalextensionat72◦Cfor7min. DNAfragmentswereclonedintothepGEM-Teasy
(Promega) or pCR-Blunt (Thermo Fisher, Waltham, MA, USA) cloning vectors and used for the
transformationofEscherichiacolistrainDH5αforSangersequencinganalyses.
2.5. RNASeq,ReverseTranscriptionPCRandRapidAmplificationofcDNAEnds
RNA seq. The F. boothii Ep-BL13 virome was established on an Illumina-Hiseq 2500 system
(Illumina,SanDiego,CA,USA)byapair-endsequencingrun(2×100bp)using10ngofdsRNAasa
template. cDNAandlibraryconstructionwereoutsourcedtoBGIJapan. Fastqcversion0.11.5software
(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)wasusedtocheckthequalityofthe
readdata[51]. ReadswerefilteredusingSickleversion1.33software(https://github.com/najoshi/
sickle) by requiring an average Phred quality (Q score) of at least 30 and to only retain reads of
20nucleotides(nt)orgreaterinlength[52]. DenovoassemblywascarriedoutusingSPAdesversion
3.11.1software(http://cab.spbu.ru/software/spades/)atthedefaultsettings[53].
Gap-fillingRT-PCR.TargetcDNAswereproducedusingReverTraAceandagene-specificprimer
setfromgivendsRNAfragmentsandamplifiedwithDNApolymerasessuchasPrimeSTAR(Takara,
Shiga,Japan),KODFXNeo(Toyobo)orGO-Taq(Promega).
3(cid:48)-RLM-RACE (RNA ligase mediated RACE). The terminal sequences of dsRNAs were
determinedbythismethod. Pre-denatureddsRNAsindimethylsulphoxide(DMSO)(90%,65◦C)
were ligated at their 3(cid:48)-ends with a 5(cid:48)-phosphorylated oligodeoxynucleotide, 3(cid:48)-RACE-adaptor
(5(cid:48)-CAATACCTTCTGACCATGCAGTGACAGTCAGCATG-3(cid:48)),usingT4RNAligase(Takara)at16◦C
for16h. LigatedDNA–RNAstrandswereDMSO-denaturedinthepresenceoftheoligonucleotide
3(cid:48)-RACE-1st (5(cid:48)-CATGCTGACTGTCACTGCAT-3(cid:48)) and used as templates for cDNA synthesis.
TheresultingcDNAwasamplifiedbyPCRwith3(cid:48)-RNACE-2nd(5(cid:48)-TGCATGGTCAGAAGGTATTG-3(cid:48))
andgene-specificprimers. Plasmidclonesandgenomic-orRT-PCRampliconswerepurifiedwith
spin columns (GeneElute, Sigma, St. Louis, MO, USA; Nucleospin, Takara) and used for BigDye
(ABI,ThermoFisher)sequencingbyfollowingthemanufacturer’sinstructions. Sangersequencingwas
carriedoutona3100-Avantsequencer(ABI/HITACHI).Allvirus-specificoligonucleotideprimers
usedinthisstudyweredesignedbasedonsequencesobtainedfromcDNAlibrarysequencingand
Illuminasequencing.
Viruses2018,10,584 5of20
2.6. BioinformaticsAnalyses
SequenceassembliesweremanipulatedusingGeneStudio(http://genestudio.com/),SPAdesand
Genetyx (Genetyx, Tokyo, Japan) software. Sequence homology searches were performed with
the Basic Local Alignment Search Tool (BLAST) programme provided by the National Center for
BiotechnologyInformation(NCBI,http://www.ncbi.nlm.nih.gov/)andtheGenetyx. Motifsearching
was conducted using the online tool, InterPro: protein sequence analysis & classification (https:
//www.ebi.ac.uk/interpro/)[54]. ForsecondaryRNAstructureprediction,the3(cid:48)-and5(cid:48)-terminal
viralsequencesweresubmittedtomfold(http://unafold.rna.albany.edu/?q=mfold/rna-folding-form)
andpotentialstem-loopswerepredicted[55].
2.7. PhylogeneticAnalysis
MultipleaminoacidornucleotidesequencealignmentswereconstructedusingMUSCLE[56]
and with the MEGA X [57]. Phylogenetic analysis was conducted by using PhyML ver. 3.0 (http:
//www.atgc-ontpellier.fr/phyml/)withthebest-fitmodelssuggestedbySmartModelSelectionin
PhyML(http://www.atgc-montpellier.fr/sms/)[58],andtheresultswerevisualisedwithFigTreeand
furtherenhancedinAdobeIllustrator.
3. Results
3.1. DetectionofdsRNAsinF.boothiiStrains
ThefungalstrainsEp-BL13,Ep-BL14andEp-28wereisolatedfromwheatsexhibitingFHBin
EthiopiabyusingFusarium-selectingmedium,whichwerefoundtobedsRNA-positive. Agarosegel
electrophoresisofdsRNAfractionsextractedfromthesestrainsshoweda3kbpcommontoallthree
strainsandadditional>10and2.5kbpbandsonlyintheEp-BL13strain(Figure1A),therebysuggesting
infectionsbyunknownRNAmycoviruses. ThesefungalstrainswereidentifiedasF.boothiibasedon
comparisonofpartialTEF1-αandITSsequences(datanotshown)andbyphylogeneticanalysisof
partialTEF1-αsequences(FigureS1).
3.2. BiologicalCharacteristicsofF.boothiiStrains
Colony morphology of the F. boothii strains on nutrient-rich (PDA) and -poor (SNA) solid
media varied as shown in Figure 1B,C. The Ep-BL13 strain grew most slowly on PDA, followed
byEp-BL14andthenEp-28(Figure1B,D).ThestrainsalsoexhibiteddifferentphenotypesonSNA,
withEp-BL13andEp-N28growingfasterandslowerthanthoseonPDA,respectively(Figure1C).
ThepathogenicityofthethreeF.boothiiisolatesandofareferenceF.graminearums.str. strainonwheat
headwasinvestigatedwithinjectionandspraymethods. PlantsinoculatedwithF.boothiiEp-BL14
andEp-28exhibitedtypicalFHBsymptomssimilartothatoftheplantinfectedbyF.graminearum
s.str.,whereastheEp-BL13showedapparentlyweakvirulence(Figure1E,F).TheeffectsofthedsRNA
elementsonhostfungalgrowthandpathogenicitytowheatsareyettobedetermined.
3.3. AnalysisofViralSequences: ATymo-LikeVirus
The genome organisation of the coding strand of the largest dsRNA segment harboured in
Ep-BL13isshowninFigure2A.Thecompletegenomicsequenceoftheviruswas12,579ntinlength
andtheG+CcontentofthedsRNAis64.93%(accessionNo. LC425115). ABLASTXsearchofthe
wholenucleotidesequenceshowedthatthehighestaminoacid(aa)identity(35%)wastothereplicase
ofBVF,whichisthesolememberofthefamilyGammaflexiviridae(Table1).Basedonthisresult,thevirus
wastentativelynamedFusariumboothiilargeflexivirus1(FbLFV1). ThewholegenomeofFbLFV1
encompassesasinglelargeORFputativelyencodingapolypeptideof4070aa(162–12,374nt)witha
calculatedmolecularmassof443.6kDa. However,BVF,theclosestvirustoFbLFV1inaBLASTsearch,
possessestwoORFsencodingaputativereplicase(apotentialreadthroughproduct)andaputative
Viruses2018,10,584 6of20
CP.Methyltransferase(Met),helicase(Hel)andRNA-dependentRNApolymerase(RdRp)domains
of the FbLFV1 polypeptide showed low aa identities (22–36%, 20–38% and 25–37%, respectively)
to those of representative viruses in the order Tymovirales and related mycoviruses, as shown in
Table1. Threereplicasedomains,Met,HelandRdRp,arelocatedinthisorder,fromNtoCterminus,
andthisistypicalofthe“alphavirus-like”superfamilythatincludesmanyotherplant-infectingviruses
(Figure2A)[59]. ThereweretworegionsinthepolypeptidewithsimilaritytoaPHA03247domain
(N-terminal part, E-value: 3.94 × e−6; C-terminal part, E-value: 5.01 × e−3), which was found in
thelargetegumentproteinUL36ofherpesviruses(alargedsDNAvirus),althoughthefunctionof
PHA03247-likedomainsinFbLFV1isunknown.
Viruses 2018, 10, x FOR PEER REVIEW 6 of 19

FigurFeig1u.reB 1io. lBoigoliocaglicpalr opproeprteiretsieso fofF .Fb. obootohthiiiis sttrraaiinnss aanndd ddssRRNNAA-b-banadnidnign gprpofriolefisl. e(sA.) (TAh)e TdhseRNdAsR-NA-
bandbinangdpinrgo fiplreosfiloefs eoaf cehacFh. bFo. obtohoitihisit rsatrianin( E(Epp-B-BLL1133,, EEpp--BBLL1144 oor rEEpp-2-82)8 a)nadn da raefreerfeenrceen pcaethpoagtehnoigc enic
strainst.raFi.ng. rFa. mgrianmeainreuamrums. sst.rs.tr.T Thhrreeee FF.. bbooootthhiiii ssttrraaininss alall clacrarrierdie ddsRdNsRAN fAragfmraegnmts eonf taspopfroaxpipmraotxeliym 3a tely
kbp. Aside from the 3-kbp dsRNA band, Ep-BL13 harboured two additional dsRNA bands of over 10
3kbp.Asidefromthe3-kbpdsRNAband,Ep-BL13harbouredtwoadditionaldsRNAbandsofover
kbp and about 2.5 kbp. (B,C) The colony morphologies of F. boothii strains and F. graminearum s.str.
10kbpandabout2.5kbp.(B,C)ThecolonymorphologiesofF.boothiistrainsandF.graminearums.str.
on PDA nutrition-rich media (B) and SNA nutrition-poor media (C) that were photographed at four
onPDAnutrition-richmedia(B)andSNAnutrition-poormedia(C)thatwerephotographedatfour
days after transplanting. The three F. boothii strains exhibited various colony morphologies in terms
daysaftertransplanting.ThethreeF.boothiistrainsexhibitedvariouscolonymorphologiesinterms
of colony size, the amount of aerial mycelium and pigmentation. (D) Growth rate of Fusarium strains
ofcolonysize,theamountofaerialmyceliumandpigmentation.(D)GrowthrateofFusariumstrains
on PDA media. Colony sizes were measured at 3–7 days post-transplantation. (E) Pathogenicity test
onPDAmedia.Colonysizesweremeasuredat3–7dayspost-transplantation.(E)Pathogenicitytest
I. A wheat spikelet was inoculated with Fusarium strains by injection and photographed at 15 days
I.Awpohset-aitnospcuiklaetlioent.w Tahse innuomcubelar toefd spwikiethletFsu esxahriibuimtinsgt rsayimnpstboymsin isje pcrteiosennatendd. (pF)h Poatothgorgaepnhiceidty atets1t 5II.d ays
post-Winhoecautl pathiootno.gTrahpehns utamkebne arso ifn s(pEi)k, beulet tasfteexrh sipbriatyin ignoscyumlaptitoonm. Dsiisseapsree isnednitceeds .(0(–F1)0P0)a aths opgeer nBiacnit ayntde stII.
WheaStupenhaogtao g(2r0a0p0h) s[4t8a]k aerne ashsoinwn(E. ),butaftersprayinoculation.Diseaseindices(0–100)asperBanand
Suenaga(2000)[48]areshown.
3.3. Analysis of Viral Sequences: A Tymo-Like Virus
The genome organisation of the coding strand of the largest dsRNA segment harboured in Ep-
BL13 is shown in Figure 2A. The complete genomic sequence of the virus was 12,579 nt in length and
the G + C content of the dsRNA is 64.93% (accession No. LC425115). A BLASTX search of the whole
nucleotide sequence showed that the highest amino acid (aa) identity (35%) was to the replicase of
BVF, which is the sole member of the family Gammaflexiviridae (Table 1). Based on this result, the virus
was tentatively named Fusarium boothii large flexivirus 1 (FbLFV1). The whole genome of FbLFV1
Viruses2018,10,584 7of20
Viruses 2018, 10, x FOR PEER REVIEW 8 of 19

FiFgiugurere2 2.. SScchheemmaatitcic rerpeprerseesnentattaiotino nofo tfheth geengoenmoem setrustcrtuucretu oref tohfe tlhaergela rFg. eboFo.thbioi omthyiicomviyrcuosv. i(rAu)s .
(AG)eGnoemnoem meampsa posf oFfbFLbFLVF1V. 1T.hTichkic bkabrsa rasraer enonno-nc-ocdoidnign gsesqeuqeunecnecse asnadn dopoepne nbboxoexse srerepprerseesnent tOORRFFs.s .
CCoololouur-rh-higighhlilgighhteteddb booxxeessi ninO ORRFFss rreepprreesseenntt tthhee pprreeddiicctteedd pprrootteeiinn ddoommaaiinnss aass iinnddiiccaatteedd.. TThhee lleennggtthh
ofoft htheew whhoollee ggeennoommee,, OORRFF aanndd UUTTRR aarere ggivivene.n T.hTeh deedleeteledt erdegrieogni oonf FobfLFFbVL1F iVn1 dienfedcteifveec tRivNeAR (NDA-
(DR-NRNA)A i)s idsedneontoetde dbyb ythteh edadsahsehde dlinliense sanadnd nnuuclceloetoitdide eppoosistiitoionns.s .MMete,t m,metehthyyltlrtarnansfsefrearsaes eddoommaainin; ;HHeel,l ,
hheleilciacsaesed doommaainin;;R RddRRpp,,R RNNAA-d-deeppeennddeenntt RRNNAA ppoollyymmeerraassee ddoommaaiinn,, FFSS,, ffrraammee--sshhiifftt.. ((BB)) CCoommppaarriissoonn
ofofn unuclceloeotitdideec oconntetexxtstss suurrrroouunnddiinngg ppootteennttiiaall iinniittiiaattiioonn ccooddoonnss ((rreedd bboolldd lleetttteerrss,, ppootteennttiiaall iinniittiiaattiioonn
cocdodoonn;r; erdedu unnddeerlrilnineeddl elettteterrss,,s suurrrroouunnddiinngg nnuucclleeoottiiddeess)) ooff FFbbLLFFVV11.. FFaavvoouurraabbllee ccoonntteexxttss ooff iinniittiiaattiioonn
cocoddoonni ninv veertretebbrraatete,,fi flialammeennttoouuss ffuunnggii aanndd yyeeaasstt aarree sshhoowwnn aass rreeffeerreenncceess.. ((CC)) SSttrruuccttuurree ooff tteerrmmininaal,l ,
nonno-nc-ocdodinigngR RNNAAr ergegioionnsso offF FbbLLFFVV11..5 5(cid:48)-′- aanndd 33(cid:48)′--tteerrmmiinnaall sseeqquueenncceess ooff tthhee ggeennoommeess ooff tthhee FFbbLLFFVV11 wweerree
ananalaylysesdedw witihths eseccoonnddaarryys strtruuccttuurreep prreeddiiccttiioonn ttoooollss.. RReepprreesseennttaattiivveess ooff ppuuttaattiivvee sstteemm--lloooopp ssttrruuccttuurreess
wwereered depepicitcetded..D Dootstsi nind dififffeerreenntt ccoolloorrss iinnddiiccaattee hhyyddrrooggeenn bboonnddss bbeettwweeeenn ddiiffffeerreenntt bbaassee ppaaiirrss: :bbluluee, ,
AA-U-Up paiarisr;sr; erded,G, G-C-Cp paairisr;s;g grereeenn,,G G-U-Up paairi.r.
3.6. TMhoeleicnuitlaiar tPiohnylcoogednoentica tA1n6a2ly–s1i6s 4ofn FtboLfFtVh1e largeORFwasinafavourablecontext,CACCAUGGa,
forfilamentousfungi,althoughanotherpotentialinitiationcodonat27–29ntappearedinframetothe
Amino acid sequences of whole FbLFV1 replicase and representative mycoviruses in the order
ORFwithaweakcontextuugCAUGcG(capitalandsmalllettersrepresentgoodandpoorresidues
Tymovirales, including taxonomically unassigned members, were aligned and the Met, Hel and RdRp
inthecontext,respectively;underlinedlettersaretheputativeinitiationcodon;Figure2B)[60–62].
domains were extracted (Table 1 and Figure 3). These three domains were found to be well conserved
FbLFV1hadshort5(cid:48)and3(cid:48)untranslatedregions(UTRs)thatare161and205nt,respectively(Figure2A).
in the deduced protein sequence of FbLFV1 replicase. The evolutionarily conserved relationship of
Althoughexperimentalevidencehasnotbeenobtained,thepresenceofaMetdomainsuggestedthe
FbLFV1 to the Tymovirales members was characterised by maximum likelihood (ML) phylogenetic
cappingof5(cid:48)-endofFbLFV1genomicRNA.TheMetdomainofthemembersoftheorderTymovirales
analyses based on the alignments of each of the three domains (Figure 4). As expected from BLAST
isgenerallylocatedattheextremeN-terminalregionofthereplicasebutthatofFbLFV1wasdistant
analyses, the FbLFV1 Met Hel and RdRp domains were distantly associated with those of viruses in
fromN-terminusofthepolypeptide(aapositions1261–1528)(Figure2A).
the order Tymovirales, with relatively close relationship to a gammaflexivirus (Figure 4A–C). The
concatenate sequence of these domains finally revealed that FbLFV1 was placed independently from
the clades of established families in the order Tymovirales, although the branching support value was
not sufficiently high in the ML tree (Figure 4D). Based on these phylogenetic analyses, FbLFV1 is
expected to belong to a novel virus species “Fusarium boothii large flexivirus 1” that may form an
independent genus, or even a new virus family, in the order Tymovirales.
Viruses2018,10,584 8of20
Table1.ListofvirusesintheorderTymoviralesandrelatedunassignedviruseswithsimilaritytoFbLFV1.
Taxon VirusName(Abbreviation) Accession Met* Hel* RdRp*
Alphaflexiviridae
Allexivirus ShallotvirusX(SVX) NC_003795 58/198(29%) 37/154(24%) 74/221(33%)
Botrexvirus BotrytisvirusX(BVX) NC_005132 60/196(30%) 45/178(25%) 76/221(34%)
Lolavirus Loliumlatentvirus(LLV) NC_010434 64/193(33%) 50/241(20%) 80/238(33%)
Mandarivirus Indiancitrusringspotvirus(ICRV) NC_003093 59/162(36%) 63/244(25%) 84/229(36%)
Platypuvirus Donkeyorchidsymptomlessvirus(DOSV) NC_022894 60/201(29%) 49/189(25%) 69/225(30%)
Potexvirus PotatovirusX(PVX) NC_011620 60/198(30%) 58/245(23%) 79/227(34%)
Sclerodarnavirus Sclerotiniasclerotiorumdebilitation-associatedRNAvirus(SsDARV) NC_007415 63/198(31%) 49/191(25%) 71/227(31%)
Betaflexiviridae
Quinvirinae
Carlavirus PotatovirusM(PVM) NC_001361 55/217(25%) 35/127(27%) 62/224(27%)
Foveavirus Applestempittingvirus(ASPV) NC_001749 60/222(27%) 17/45(37%) 63/222(28%)
Robigovirus Cherrynecroticrustymottlevirus(CNRMV) NC_002468 52/224(23%) 44/159(27%) 61/219(27%)
Trivirinae
Capillovirus Applestemgroovingvirus(ASGV) NC_003462 64/214(29%) 33/97(34%) 72/231(31%)
Chordovirus CarrotChvirus1(CChV) NC_025469 54/214(25%) 33/118(27%) 66/225(29%)
Citrivirus Citrusleafblotchvirus(CLBV) NC_003877 46/205(22%) 46/160(28%) 68/223(30%)
Divavirus DiurisvirusA(DVA) NC_019029 53/217(24%) 26/90(28%) 66/224(29%)
Prunevirus Apricotveinclearingassociatedvirus(AVCSV) NC_023295 49/183(26%) 24/62(38%) 66/224(29%)
Tepovirus PotatovirusT(PVT) NC_011062 56/186(30%) 34/115(29%) 67/219(30%)
Trichovirus Applechloroticleafspotvirus(ACLSV) NC_001409 62/195(31%) 43/172(25%) 64/218(29%)
Vitivirus GrapevinevirusA(GVA) NC_003604 50/201(24%) 22/57(38%) 65/218(29%)
Gammaflexiviridae
Mycoflexivirus BotrytisvirusF(BVF) NC_002604 63/193(32%) 71/252(28%) 82/224(36%)
Deltaflexiviridae
Deltaflexivirus Sclerotiniasclerotiorumdeltaflexivirus1(SsDFV1) NC_038977 54/202(26%) 61/253(24%) 45/174(25%)
Sclerotiniasclerotiorumdeltaflexivirus2(SsDFV2) MH299810 63/222(28%) 44/184(23%) 59/207(28%)
Fusariumgraminearumdeltaflexivirus1(FgDFV1) NC_030654 67/204(32%) 37/157(23%) 57/211(27%)
Soybeanleaf-associateddeltaflexivirus1(SlaDFV1) KT598226 54/203(26%) 63/260(24%) 46/174(26%)
Tymoviridae
Maculavirus Grapevinefleckvirus(GFV) NC_003347 57/203(28%) 43/144(29%) 54/143(37%)
Marafivirus Maizerayadofinovirus(MRFV) NC_002786 58/197(29%) 57/201(28%) 45/140(32%)
Tymovirus Turnipyellowmosaicvirus(TUMV) NC_004063 56/196(28%) 58/214(27%) 61/204(29%)
Unassigned
Mycotymovirus Fusariumgraminearummycotymovirus1(FgMTV1) KT360947 60/196(30%) 57/197(26%) 73/219(33%)
Unassigned Rhizoctoniasolaniflexivirus1(RnFV1) NC_030655 57/198(28%) 53/228(23%) 65/224(29%)
Unassigned Rhizoctoniasolaniflexivirus2(RnFV1) KX349069 N/A** N/A** 50/177(28%)
Unassigned Oystermuschroomsphericalvirus(OMSV) AY182001 48/189(25%) 61/237(25%) 73/219(33%)
Unassigned Fusariumboothiilargeflexivirus1(FbLFV1) LC425115
*ResultsofhomologysearchagainstFbLFV1foraminoacidcoverregionandidentityareshown.**N/Aindicates“notapplicable”duetotheunavailabilityofhomologoussequences.
Viruses2018,10,584 9of20
3.4. PresenceofaDefectiveSegmentofFbFV1intheEp-BL13
The cDNA-library-based sequencing of the approximately 2.5 kbp dsRNA revealed that the
segmentwasofadefectiveRNA(D-RNA)ofFbLFV1. ThetotallengthofthedsRNAwas2408bp,
andthepositive-senseRNAretainedanORFencodingpolypeptidewithapotentialmolecularmassof
76.8kDa(716aa;Figure2A)(accessionNo.LC425116).Thisoccurredbecauseofthelossofaa303–3693
oftheFbLFV1largepolypeptide(removalofFbLFV1genomepositions1066–11,235in-frame)andthe
additionof36aabecauseofaframe-shiftfromasinglenucleotidedeletionat−5ntpositionrelativeto
theoriginalterminationcodon,leadingtoanewdownstreamstopcodon.SincetheRNAsequencewas
almostidenticaltothecorrespondingregionofFbLFV1andthejunctionoftheupstream/downstream
partswasuniform, thisD-RNAofFbLFV1mighthavearisenveryrecently, butatleastbeforethe
isolationofEp-BL13. Thesegmenthasthepotentialtofacilitateviralreplicationbecausethecoded
proteinretainedpartialRdRpmotifs(122aaresiduesofC-terminalend; Figure2A);however,itis
alsopossiblethatitmayinterferewithreplication. AsBVFisreportedtocarrysimilarD-RNAspecies
withunknownfunction[39],itisinterestingtoinvestigatethesignificanceofD-RNAsinlifecyclesof
mycovirusesintheorderTymovirales.
3.5. PredictedRNAStructureoftheTerminalRegionsofFbLFV1Genome
TheUTRsofRNAvirusesgenerallycarryimportantcis-elementsthatformsecondary/tertiary
structures and/or distant base-pairing. In many cases, these are essential for encapsidation,
transcription,translationandreplication. ToinvestigatewhetherFbLFV1containssuchstructures,
UTRsequencesweresubjectedtosecondarystructurepredictionusingmfoldsoftware. The5(cid:48) UTR
of FbLFV1 did not consistently exhibit stem loops in several outputs, except for the one shown
in Figure 2C. Similarly, those of the 3(cid:48) UTR were mostly inconsistent but a stem-loop predicted
nearthe3(cid:48)-endwasthemostprobable(Figure2C).ItisknownthatsomemembersofTymoviridae,
such as turnip yellow mosaic virus (TYMV), have tRNA-like structures (TLS) in their 3(cid:48)-UTR
that play an important role in viral replication [63,64]. Given that TLSs are difficult to predict
(e.g.,theTYMV-TRL(6234–6318nt, NC_004063)wasnot detected bytRNAscan-SEsoftware [65]),
moreextensivebase-pairinganalysisoftheFbLFV13(cid:48)-endshouldbeconductedbeforedrawingany
definitiveconclusions.
3.6. MolecularPhylogeneticAnalysisofFbLFV1
AminoacidsequencesofwholeFbLFV1replicaseandrepresentativemycovirusesintheorder
Tymovirales,includingtaxonomicallyunassignedmembers,werealignedandtheMet,HelandRdRp
domainswereextracted(Table1andFigure3). Thesethreedomainswerefoundtobewellconserved
inthededucedproteinsequenceofFbLFV1replicase. Theevolutionarilyconservedrelationshipof
FbLFV1totheTymoviralesmemberswascharacterisedbymaximumlikelihood(ML)phylogenetic
analysesbasedonthealignmentsofeachofthethreedomains(Figure4). AsexpectedfromBLAST
analyses, theFbLFV1MetHelandRdRpdomainsweredistantlyassociatedwiththoseofviruses
in the order Tymovirales, with relatively close relationship to a gammaflexivirus (Figure 4A–C).
TheconcatenatesequenceofthesedomainsfinallyrevealedthatFbLFV1wasplacedindependently
fromthecladesofestablishedfamiliesintheorderTymovirales,althoughthebranchingsupportvalue
wasnotsufficientlyhighintheMLtree(Figure4D).Basedonthesephylogeneticanalyses,FbLFV1is
expectedtobelongtoanovelvirusspecies“Fusariumboothiilargeflexivirus1”thatmayforman
independentgenus,orevenanewvirusfamily,intheorderTymovirales.
Viruses2018,10,584 10of20

Figure3.AminoacidalignmentofMet,HetandRdRpdomainsofFbLFV1.Thepositionsofthemotifs

areindicatedbylinesabovethesequencealignmentswiththemotifnumbersfromItoVI.Identical

residuesarecolour-highlightedwithblueandindicatedbyasterisks;conservedandsemi-conserved

aminoacidresiduesarecolour-highlightedwithyellowandgray,andbycolonsanddots,respectively.

(A–C)DeducedaasequencescorrespondingtoMet(A),Hel(B)andRdRp(C)domainswereextracted
fromtheFbLFV1largepolypeptideandalignedwiththosefrommycovirusesoftheorderTymovirales.
AbbreviationsofviralnamesarethesameasinTable1.
1