Virology 435 (2013) 201–209

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Virology

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Rapid Communication

Evidence for negative-strand RNA virus infection in fungi

Hideki Kondo a,n, Sotaro Chiba a, Kazuhiro Toyoda b, Nobuhiro Suzuki a,nn

a Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki 710-0046, Japanb Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan

a r t i c l e i n f o

Article history:

Received 11 August 2012

Returned to author for revisions

27 September 2012

Accepted 2 October 2012Available online 23 October 2012

Keywords:

Endogenous viral element

Transcriptome shotgun assembly

Negative-strand RNA virus

Mononegavirales

Fungi

Erysiphe pisi

Sclerotinia homoeocarpa

Evolution

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x.doi.org/10.1016/j.virol.2012.10.002

esponding author. Fax: þ82 86 434 1232.

responding author. Tel.: +81 86 434 1230; fa

ail addresses: hkondo@rib.okayama.u-ac.jp (H

@rib.okayama.u-ac.jp (N. Suzuki).

a b s t r a c t

Fungal viruses comprise two groups: a major group of five families with double-stranded RNA genomes

and a minor group with positive-sense single-stranded (ss)RNA genomes. Although many fungal

viruses have been identified, no negative-stranded (�)ssRNA mycoviruses have been reported. Here we

present two lines of evidence suggesting the presence of (�)ssRNA viruses in filamentous fungi based

on an exhaustive search using extant (�)ssRNA viruses as queries. This revealed (�)ssRNA virus

L protein-like sequences in the genome of a phytopathogenic obligate ascomycete, Erysiphe pisi. A

similar search for (�)ssRNA viruses in fungal transcriptome shotgun assembly libraries demonstrated

that two independent libraries from Sclerotinia homoeocarpa, another phytopathogenic ascomycete,

contained several sequences considered to correspond to the entire mononegavirus L gene and likely

originating from an infecting (�)ssRNA virus. These results provide strong evidence for both ancient

and extant (�)ssRNA virus infections in fungi.

& 2012 Elsevier Inc. All rights reserved.

Introduction

Fungal viruses (mycoviruses) are widespread in all majorgroups of fungi. Extensive searches for mycoviruses in endophytic,human-pathogenic, entomopathogenic and phytopathogenicfungi have revealed varying incidence rates. For example,approximately 21% of over 1000 field isolates of a soil-borneascomycetous plant pathogen, Rosellinia necatrix, were found tobe infected with viruses (Arakawa et al., 2002; Ikeda et al., 2004)while approximately 68% of isolates of another basidiomycetousphytopathogen, Helicobasidium mompa, carried viruses (Ikedaet al., 2004). As described by Ghabrial and Suzuki (2009), mostfungal viruses have double-stranded (ds)RNA genomes, althoughsome have single-stranded (ss)RNA genomes. This may beattributable to the method employed for dsRNA-based virusscreening. Yu et al. (2010) recently reported an ssDNA virus infect-ing a plant pathogenic fungus, Sclerotinia sclerotiorum. Accordingto the 9th report of the International Committee on Taxonomyof Viruses (ICTV), mycoviruses are now classified into fivedsRNA families, five ssRNA families, two reverse-transcribing(RT) families, unassigned ssDNA and dsDNA viruses (Kinget al., 2011). It has long been unclear whether mycoviruses with

ll rights reserved.

x: +81 86 434 1232.

. Kondo),

negative-stranded (�)ssRNA (i.e., complementary to the mRNA)genomes exist in fungi, as in other eukaryotic organisms (Doljaand Koonin, 2011).

Nucleotide sequence databases for viral and cellular genomesand transcriptomes have been growing rapidly. Database searcheshave led to the discovery of non-retrovirus RNA viral sequences(NRVSs) integrated into the genomes of diverse eukaryotic organ-isms (Belyi et al., 2010; Chiba et al., 2011; Fort et al., 2012; Horieet al., 2010; Katzourakis and Gifford, 2010; Liu et al., 2010;Taylor et al., 2010, 2011), a phenomenon that was thoughtpreviously not to occur. Viruses integrated into host chromo-somes include partitiviruses (members of the family Partitiviridae)with dsRNA genomes (plant), mononegaviruses (members of theorder Mononegavirales) with non-segmented (�)ssRNA genomes,such as rhabdoviruses (plant, invertebrate, and vertebrate),bornaviruses (vertebrate), or filoviruses (vertebrate). In compar-ison with dsRNA and (�)ssRNA viruses, there are fewer examplesof integration of positive-sense ssRNA viruses (plant and inverte-brate) (Chiba et al., 2011; Crochu et al., 2004; Cui and Holmes,2012). In fungi, Taylor and Bruenn (2009) and Liu et al. (2010)found totivirus (dsRNA virus)-like sequences in several fungalnuclear genomes, while Liu et al. (2011) reported the presenceof ssDNA virus sequences in fungal chromosomes. A series ofthese studies has provided interesting insights into molecularevolution, virus/host interactions, and ancient virus geneticstructures. It remains unknown how such sequences were endo-genized or what their biological significance may be.

H. Kondo et al. / Virology 435 (2013) 201–209202

Negative-strand RNA viral sequences can be detected in all themajor eukaryotic kingdoms except for fungi (Belyi et al., 2010;Chiba et al., 2011; Fort et al., 2012; Horie et al., 2010; Katzourakisand Gifford, 2010; Taylor et al., 2010). This fact prompted usto search available sequence databases for (�)ssRNA virusesin fungi. In the present study, we performed an extensive searchof publicly available fungal genome and transcriptome shotgunassembly sequence databases, and found evidence stronglysuggesting the presence of both ancient (�)ssRNA virusesand probably extant (�)ssRNA viruses in phytopathogenicfilamentous fungi.

Results and discussion

Genome sequence database search

Because many mononegavirus-related sequences are present inplant and animal chromosomes, we searched the available NCBIdatabases by using L-polymerase (RNA-dependent RNA polymerase,RdRp) sequences encoded by mononegaviruses or related viruses ofdifferent genera as queries (Table S1). We identified nine significantmatches to (�)ssRNA viruses in the whole-genome shotgun (WGS)assemblies of the chromosomes of a plant pathogenic obligateascomycete, Erysiphe pisi (pea powdery mildew fungus) (Table S2).These WGS sequences assembled into four independent contigs.According to our system of nomenclature for integrated NRVSs(Chiba et al., 2011), these sequences were termed E. pisi mono-negavirus L protein-like sequences 1–4 (EpMLLS1-EpMLLS4) (Fig. 1).A further BLAST search against the EMBL (WGS) database (http://www.ebi.ac.uk/Tools/sss/fasta/wgs.html) using mononegaviralL-proteins or EpMLLSs as queries resulted in identification of eightother E. pisi WGS sequences of candidate EpMLLSs (Table S2). TheseWGS sequences assembled into another four contigs (EpMLLS5–EpMLLS8), as shown in Fig. 1. Reverse BLAST analysis revealedthat EpMLLSs were distantly related to members of a recentlyproposed novel genus Nyavirus (Midway and Nyamanini viruses)(Mihindukulasuriya et al., 2009), and several other mononegaviruses(Table S2 and data not shown).

EpMLLSs possess truncated L-like genes and some of themhave ORF disruptions (Fig. 1). Their potentially encoded proteinsdiffer from one another in the corresponding region of nyavirusL (Fig. 1) and show significant levels of amino acid sequenceidentity (20–80%) (Table S3). EpMLLS4 contains a possiblerearranged virus sequence, and EpMLLS5 has a large internaldeletion (Fig. 1). Some EpMLLSs have a domain found in RdRp(Pfam00946) of species belonging to the order Mononegavirales

(Poch et al., 1990) (Table S2). Fig. S1 shows the multiple align-ment of the hypothetical RdRp core module of EpMLLS2, EpMLLS3and other mononegavirus L protein-like sequences (see below).The EpMLLS2 and EpMLLS3 deduced amino acid sequencescontain the highly conserved motifs A to D and a typical catalyticmotif GDNQ within C regions that are found in almost allmononegavirus L proteins, with the exception that the firstresidue in EpMLLS2 is Ser (S) instead of Gly (G) (Fig. S1).

EpMLLSs have distinguishable flanking sequences and some ofthem contain trace putative fungal transposable elements (Fig. 1).Together with the fact that the E. pisi shotgun library is derivedfrom a single fungal strain, these results suggest that EpMLLS1–EpMLLS8 reside at different fungal genome positions. Notably, theflanking ORFs of EpMLLS6 show significant similarity to a TE1a(a class of LINE-1 retrotransposons, CgT1-like) retrotransposon inBlumeria graminis (a powdery mildew fungus in barley andwheat) and a powdery mildew-specific avirulence (AVR) effectorgene, AVRk1. TE1a and AVRk1 are believed to be expressed asa single transcript and to be retrotransposed as a single unit

(Sacristan et al., 2009). Therefore, we speculated that EpMLLS6might have been integrated after template switching from thisunique transcript to mRNA of (�)ssRNA virus (progenitor virus ofEpMLLS6) during reverse transcription of the retrotransposon(Ballinger et al., 2012; Geuking et al., 2009).

Conversely, although the most numerous mononegavirus-likesequences in plant and animal genomes were related to thenucleoprotein (or nucleocapsid protein) N (Belyi et al., 2010;Chiba et al., 2011; Fort et al., 2012; Horie et al., 2010; Katzourakisand Gifford, 2010; Taylor et al., 2010, 2011), we found no hit forN proteins and other viral proteins such as glycoproteins (G) inthe fungal genome (data not shown), perhaps as a result of theirlow or moderate levels of conservation among mononegaviruses.

During the course of these analyses we noticed that WGSassembly sequences of another powdery mildew fungus,Golovinomyces orontii (formerly Erysiphe orontii, order Erysi-phales), are available from the Max Planck Institute for PlantBreeding Research Powdery Mildew Genome Project site(http://www.mpiz-koeln.mpg.de/24322/Project_Description) (Spanuet al., 2010), but not from the NCBI website. Further BLASTanalysis was therefore conducted against the G. orontii WGSlibrary using EpMLLSs as queries. Consequently, in our preli-minary database search, we found several significant matches,although most were short (data not shown). This finding stronglysuggests that endogenization of mononegaviruses into the fungalgenome might have occurred not only in the pea powdery mildewfungus (E. pisi), but also in another powdery mildew fungus(G. orontii), which has a broad host range including Arabidopsis

thaliana (Spanu, 2012).

Detection of EpMLLSs by genomic PCR

In order to confirm the presence of EpMLLSs in fungalchromosomes, we harvested two samples of powdery mildewfrom pea (Pisum sativum L.) and red clover (Triticum aestivum L.)plants in the experimental field at the Institute of Plant Scienceand Resources (IPSR). The fungal genomic DNA was extracted andsubjected to genomic PCR. Based on internal transcribed spacer(ITS) sequencing, the pea plants appeared to harbor a doubleinfection with E. pisi and E. trifolii, whereas the red clover plantswere infected only with E. trifolii. A total of 24 clones from peasamples were sequenced. While three clones showed over 99%sequence identity to the ITS sequences reported for E. pisi, theremaining clones were identical to those reported for E. trifolii

(Fig. S2). The ITS sequence obtained by direct sequencing from redclover samples was identical to the reported ITS sequence forE. trifolii isolates (Fig. S2). Co-infection with two powdery mildewspecies on the same host plant had been reported previously(Glawe, 2008).

Genomic PCR using a different set of primers corresponding toeach of the EpMLLS sequences (see Table S4 for the primersequences) revealed that DNA fragments of the expected sizeswere amplified on fungal genomic DNA from the pea sample(E. pisi and E. trifolii), but not on those from the red clover sample(E. trifolii) (Fig. 2). No DNA fragment was amplified by PCR onpea or red clover genomic DNA (Fig. 2 and data not shown).The sequences of the resulting DNA fragments from the peasample were identical to the relevant regions of the E. pisi

genome, with the exception of a single-nucleotide deletion anda substitution found in sequenced EpMLLS4 (data not shown).These results demonstrated that the EpMLLSs were not contami-nants during the WGS sequencing, and are probably widelypresent in field isolates of E. pisi.

Therefore, the EpMLLSs might have been inserted into chromo-somes after divergence of the two Erysiphe species. The geneticdistance between E. pisi (GenBank accession numbers of the

Fig. 1. Schematic representation of Erysiphe pisi mononegavirus L protein-like sequences (EpMLLSs) and their flanking regions. EpMLLSs found in the whole-genome

shotgun (WGS) database of E. pisi are shown to match L-polymerase (RNA-dependent RNA polymerase, RdRp; pfam00946) from an insect mononegavirus, Nyamanini virus

(a prototypic member of Nyavirus). Top row shows a diagrammatic representation of the 30-to-50 arrangement of the ORFs (I, nucleoprotein N; II, putative phosphoprotein;

III, unknown; IV, putative matrix protein; V, glycoprotein G; VI, large protein L or RNA-dependent RNA polymerase) of Nyamanini virus (virus complementary strand)

(Mihindukulasuriya et al., 2009). Other conserved domains in the L-protein (mRNA-capping enzyme motif: pfam14318 and TIGR04198) found on the NCBI website

(Marchler-Bauer et al., 2011) are also shown. The potential coding regions of EpMLLSs and flanking small ORFs are shown as boxes, and EpMMLSs are placed in the

corresponding regions of the Nyamanini virus-L ORF. Retrotransposon-like sequences are shown by thick black lines. The positions of the primers used for genomic PCR

(Table S4 and Fig. 2) are shown by arrows. The WGS assembled sequences and undetermined sequences are shown by solid and dashed lines, respectively. Symbols

referring to mutations are also shown; kinked line, major deletion; asterisk, internal stop codon; F, frame-shift.

H. Kondo et al. / Virology 435 (2013) 201–209 203

Fig. 2. Genomic PCR analysis of EpMLLSs. EpMLLSs of the Erysiphe spp. field

samples isolated from pea (upper panel) and red clover (middle panel) were

amplified using a primer set specific for each EpMLLS. Leaf DNA samples from pea

(bottom panel) and red clover (data not shown) were subjected to PCR. Two

primer sets, EryF and EryR and At-IRS-FW and At-IRS-RV, were used for

amplification of the Erysiphe spp. and plant ITS regions, respectively. For primer

sequences and positions, please refer to Table S4 and Fig. 1 (named arrows, except

for ITS primers).

H. Kondo et al. / Virology 435 (2013) 201–209204

representative isolates: FJ378869, FJ378870 and FJ378872) andE. trifolii (representative isolates: AB015913, FJ378884 andFJ378878), calculated on the basis of the Kimura 2-parametercriterion using MEGA (Kimura, 1980), was 0.026 for the ITSregion. Based on the molecular clock of the ITS (2.52�10�9

substitutions per site per year) (Takamatsu and Matsuda, 2004),the split between E. pisi and E. trifolii was roughly estimated tohave occurred 5.2 million years ago (Fig. S2). Therefore, EpMLLSsintegration may have occurred more recently than this. Furtherstudies will be needed to examine this possibility. It was alsosuggested that NRVSs in fungal genomes can serve as a geneticmarker for clarifying phylogenetic relationships that are other-wise unclear.

The discovery of these EpMLLSs provides the first example of(�)ssRNA virus sequences detected in the genomes of fungi. Thesesequences might have integrated into the genome of E. pisi followinginfection by ancestral fungal (�)ssRNA viruses related to possibleextant fungal (�)ssRNA viruses (see below). Nevertheless, wecannot rule out the possibility that intimate interactions betweenobligate pathogens and plant hosts may have allowed integration ofancestral plant (�)ssRNA virus sequences into the fungal genome.Partitiviruses (dsRNA viruses) are assumed to be transferredlaterally between fungi and plants, and these virus sequences mightbe integrated relatively frequently into plant host genomes asdescribed above (Chiba et al., 2011; Liu et al., 2010).

Transcriptome shotgun library search

Several studies have demonstrated that pyrosequencing-basedtranscriptome analysis is a useful new research tool for discoveryof (�)ssRNA viruses (Bekal et al., 2011; Honkavuori et al., 2008;Valles et al., 2012). Therefore, we further expanded our searchto fungal transcriptome shotgun assembly (TSA) libraries in asearch for unknown (�)ssRNA viruses. Interestingly, four TSAsequences showing moderate similarity to (�)ssRNA virus

L protein sequences were found in the two independent librariesfrom Sclerotinia homoeocarpa, an ascomycete fungus causingdollar spot in grass plants (Fig. 3a and Table S5). Two TSAs,JU091017 and JU091016, originated from the same library(Orshinsky et al., 2012) and were almost identical to each other,apart from a short terminal heterologous sequence. Therefore, wetermed their contig ShTSA1 (S. homoeocarpa TSA contig 1). Thesecond library of TSAs (Hulvey et al., 2012), JW826636 (ShTSA2)and JW828891 (ShTSA3), showed moderate or low identity (73%and 53% nucleotide identity, respectively) over the whole of theShTSA1 sequences. Each ShTSA has a long ORF (L-like ORF)considered to correspond to the entire region of the nyavirusL protein coding region (Table S5). The ShTSA L-like ORF proteins(ShTSA1-L–ShTSA3-L) show significant levels of amino acidsequence identity among these sequences (56–86% amino acididentity) (Table S6). These potentially encoded proteins have twotypical domains, RdRp (Pfam00946) and virus-capping methyl-transferase (pfam14318), found in most mononegaviruses (Fig. 3aand Table S5). ShTSAs have another two or three short ORFs(ORF1-3) (Fig. 3a). While the products of these small ORFs did nothave any detectable similarity to the NCBI protein database, therewas remarkable conservation among ShTSAs in each of thesesmall ORF proteins (ORF1 70%, ORF2 42–87 % and ORF3 30%amino acid identity) (Table S6).

In addition, a semi-consensus sequence (30-AAUAAAUAUU-UUUGAAUCCUC-50) was found in the putative untranslatedsequences between the ORFs of each ShTSA sequence (Figs. 3and S3). These are probably ‘gene-junction’ sequences, which arebroadly conserved among mononegaviruses (Conzelmann, 1998).Mononegaviral gene-junction sequences contain a gene-endsequence, which is required for transcription termination andpolyadenylation of upstream genes, and a gene-start sequence,which plays an important role in initiating the transcriptionof downstream genes (Conzelmann, 1998). The AU-rich region(30-AAUAAAUAUUUUU-50) is reminiscent of the gene-endsequences found in plant rhabdoviruses and some other mono-negaviruses, comprising an AU-rich region and a poly(U) tract(Jackson et al., 2008) (Fig. 3b). This sequence was followed by amoderate-consensus sequence GAAUCCUC, which may beincluded in the untranscribed intergenic spacer sequences (thefirst nucleotide sequence is G in many rhabdoviruses) and theinitiation of transcription of downstream genes (the first threenucleotide sequences are CUN or CUA in plant cytorhabdovirusesand varicosaviruses) (Heim et al., 2008; Sasaya et al., 2004)(Fig. 3b).

Each of these ShTSAs most likely originates from multipletranscripts corresponding to genes of an infecting virus that is notof host chromosomal origin, for the reasons stated below. Theassembled sequences from two independent TSA libraries resemblethe 50-terminal half of a (�)ssRNA virus genome encoding the entireL protein, and each ShTSA ORF possesses putative gene-junctionsequences as described above. These characteristics are typical of amononega-like virus genome structure. Furthermore, we found nohit for ShTSA-Ls in a BLAST search against the S. homoeocarpa WGSdatabase (8-fold redundancy) at the EMBL site (Hulvey et al., 2012).If ShTAS1 to ShTAS3 were transcripts from the same locus inundetermined regions of the S. homoeocarpa genome, they wouldbe expected to show closer similarity to each other. In addition,NRVSs in host genomes frequently harbor ORFs that have beeninterrupted due to spontaneous mutations during the courseof evolution, as is the case for EpMLLSs (Fig. 1). However, allL-like ORFs in the ShTSAs appear to have remained intact. Takentogether, our findings provide strong evidence for extant (�)ssRNAvirus infection in fungi. Interestingly, reverse BLAST analysisusing ShTSA3-L yielded a significant match (GeneBank accessionGR225954; e-value¼3e�12, identities¼83/342¼24%) in the EST

Fig. 3. Schematic representation of mononegavirus-related L protein-like sequences and their flanking regions present in Sclerotinia homoeocarpa transcriptome shotgun

assembly (ShTSA) libraries. (a) ShTSA L protein-like sequences (ShTSA-Ls) are shown to be similar to the L-polymerase (containing RdRp motif: pfam00946 and mRNA-

capping enzyme motifs: pfam14318 and TIGR04198) from Nyamanini virus (Mihindukulasuriya et al., 2009). Small boxes indicate potential ORFs adjacent to the ShTSA-Ls.

ShTSA1 consists of two independent TSA sequences (JUO91017 and JU091016), as shown below the map, while the other two ShTSAs are composed of single TSA

sequences. The solid line is the TSA sequence, and the dashed line is the undetermined sequence. A putative gene junction sequence (Flag) is also indicated. (b) Comparison

of putative gene-junction regions between the predicted ORFs in ShTSAs. Alignment of the putative gene-junction sequences (denoted by flags in A) is shown in the 30-to-50

orientation (potential viral genomic strand, see Fig. S3). Conserved sequences are highlighted.

H. Kondo et al. / Virology 435 (2013) 201–209 205

library of a fungal pathogen of caterpillars (medicinal fungus),Cordyceps militaris (Xiong et al., 2010), which belongs to an orderdifferent from that of S. homoeocarpa. This may be another similarexample of a filamentous fungus infected with a (�)ssRNA virus.

It is noteworthy that searches for other mononegaviral genessuch as N, which presumably would be most abundant amongall the gene products, yielded no hits in the S. homoeocarpa

TSA libraries (data not shown). Although the complete genomestructure of S. homoeocarpa (�)ssRNA viruses is still unknown, thisresult suggests an unusual particle structure that would make anymorphology-based identification of a subset of fungal (�)ssRNAviruses more difficult. In this connection, it should be noted thatmany fungal positive-sense ssRNA and dsRNA viruses form noparticles. The candidate ORF2 gene of ShTSAs (Fig. 3a), whoseposition corresponds to that of a glycoprotein G for mononegavirus,has no putative signal peptide or C-terminal hydrophobic trans-membrane anchor domain, which are common structural features of

mononegavirus G protein (data not shown). Similarly, in lettucebig-vein associated virus (LBVaV, floating genus Varicosavirus),which has a rod-shaped virion (not enveloped) containing a bipartite(�)ssRNA with a plant rhabdovirus-like genome structure and istransmitted by a soil-inhabiting fungus (Olpidium virulentus), theLBVaV protein 5, whose position also corresponds to G protein,appeared to lack any direct (sequence) relatedness to the rhabdo-viral G protein (Kormelink et al., 2011; Sasaya et al., 2002, 2004).In general, the G protein forms the membrane spikes of mono-negaviral virions and is thought to play a critical role in virusmembrane fusion during cell entry (Albertini et al., 2012). However,as mycoviruses have no known extracellular mode of transmissionin nature (Ghabrial and Suzuki, 2009), (�)ssRNA viruses adapted tofungal hosts may lack the G protein gene. It is also speculated thatdifferences among other virally encoded proteins and/or genomicstructure may represent another unique history of co-evolutionbetween (�)ssRNA virus and the fungus (and/or plant).

Fig. 4. Maximum likelihood phylogeny of the L protein sequences of mononegaviruses, EpMLLSs and ShTSA-Ls. A phylogenetic tree was constructed using PhyML 3.0 based

on the multiple amino acid sequence alignment of the RdRp polymerase core module shown in Fig. S1. Mononegavirus L protein-like sequences from fungi, plants, insects,

mammals and fishs (Tables S2, S7 and S8), and fungal TSA-derived sequences (Table S5) are shown in red, green, wine red, ochre, gray and blue boxes, respectively.

L proteins from mononegaviruses (filoviruses, paramyxoviruses, rhabdoviruses and bornaviruses, order Mononegavirales) and other related viruses (nyaviruses,

varicosavirus, dichorhabdovirus and others) (Table S1) are shown in black and gray letters, respectively. Asterisks show the viruses with the segmented (�)ssRNA

genome. Shaded cartoons on the gray dashed circle indicate outlines of host species that encode mononegavirus L proteins-like sequences in their genomes. Numbers at

the nodes represent aLRT values derived using an SH-like calculation (only values greater than 0.7 are shown).

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H. Kondo et al. / Virology 435 (2013) 201–209 207

Phylogenetic analysis of EpMLLSs and ShTSA-Ls

In order to assess the relationships among mononegavirusesand mononegavirus-like sequences (Figs. 1 to 3), we conductedphylogenetic analysis based on amino acid alignment of theirL and L protein-like sequences. In this analysis, we included thetype species for each genus in the Mononegavirales (four familiesBornaviridae, Filoviridae, Paramyxoviridae and Rhabdoviridae andthe proposed genus Nyavirus) and other related viruses withsegmented (�)ssRNA genomes infecting plants (i.e., prototypicmembers of the genus Varicosavirus and the proposed genusDicorhabdovirus) (King et al., 2011; Kondo et al., 2006;Mihindukulasuriya et al., 2009; Sasaya et al., 2002), as listed inTable S1, and the previously identified MLLSs (Belyi et al., 2010;Fort et al., 2012; Katzourakis and Gifford, 2010; Taylor et al.,2010), as listed in Table S7. We also included several novel MLLSsfrom plant and insect genomes identified in this study (Table S8and Fig. S4). The multiple alignment of these sequences isshown in Fig. S1. Phylogenetic analysis of the most known MLLSs(rhabdovirus L protein-like sequences [RLLSs], filovirus L protein-like sequence [FLLS] and bornavirus L protein-like sequences[BLLSs]) in vertebrate and invertebrate genomes are placed intothe modern mononegavirus clades (e.g., rhabdoviruses, filovirusesand bornaviruses) (Belyi et al., 2010; Fort et al., 2012; Katzourakisand Gifford, 2010; Taylor et al., 2010) (Fig. 4). Similarly, the novelMLLSs (nyavirus L protein-like sequences: NLLSs) in lettuce plant(Lactuca sativa) (LsNLLS), silkworm (Bombyx mori) (BmNLLS1),leafcutter bee (Megachile rotundata) (MrNLLS), and carpenter andfire ants (Camponotus pennsylvanicus and Solenopsis invicta)(CfNLLSs and SiNLLS) form a cluster with the modern insectviruses (nyaviruses) (sister clade of the BLLSs and bornaviruses).

In contrast, the EpMLLSs and ShTSA-L sequences demonstratedthat they are the relatives closest to each other and distantlyrelated to the modern mononegaviruses and related viruses(Fig. 4). Notably, two MLLSs in the genomes of the yellow fevermosquito, Aedes aegypti (AaMLLS), and the zebrafish, Danio rerio

(DaMLLS), form a cluster distinct from the EpMLLSs and ShTSA-Ls(Belyi et al., 2010; Fort et al., 2012) (Fig. 4). It has beenhypothesized by Hillman et al. (2004) that fungus-infectingmycoreoviruses (the genus Mycoreovirus, family Reoviridae) andvertebrate-infecting coltiviruses (the genus Coltivirus, family Reo-

viridae) might have evolved from a common ancestor native inthe Acari. This hypothesis is based on the fact that some Acarimembers (mites) share their habitat with host fungi and others(ticks) transmit coltiviruses. Therefore, it is assumed that areovirus progenitor may have been transferred laterally fromAcari members to evolve into mycoreoviruses and coltiviruses.In this connection, a recent finding that horizontal transmissionof mycoviruses by vector organisms may occur in nature isnoteworthy (Yaegashi et al., 2012). Likewise, it may be relevantto speculate that a common progenitor of (�)ssRNA virusesmight have once infected fungi, mosquitoes and zebrafish. Theseorganisms share an interesting connection. Some mosquitoessuch as the anopheline species, Anopheles gambiae, feed on plantsthat could be infected by phytopathogenic fungi (Gouagna et al.,2010), while the diet of zebrafish can include mosquitoes (Spenceet al., 2008).

Conclusion

Viral genomes are classified into seven groups based onnucleic acid composition and genome expression strategy (Kinget al., 2011). However, the genome type shows a trend that isdependent on the virus host kingdom. For example, many morepositive-sense ssRNA viruses than dsDNA viruses are found in

plants, whereas an inverse trend is observed in bacteria.In contrast to the large number of reports on fungal dsRNAviruses, no previous report has documented fungal (�)ssRNAviruses. Our findings strongly suggest that mononegavirus infec-tion might have occurred in fungi, as recorded in the form ofendogenous virus-like sequences in their genomes, and also thatnovel mononegaviruses related to these virus-like elements aremost likely present in fungi. Our discovery may provide novelinsights into the origin and evolution of (�)ssRNA viruses.

Materials and methods

Database searches

To screen for (�)ssRNA virus-related DNA sequences, BLAST(tBLASTn) searches (Altschul et al., 1997) were conducted againstgenome sequence databases available from the NCBI (nucleotidecollection, nr/nt; genome survey sequences, GSS; high-throughputgenomic sequences, HTGS; whole-genome shotgun contigs, WGS;non-human, non-mouse expressed sequence tags, EST; transcrip-tome shotgun assembly, TSA, and others) (http://www.ncbi.nlm.nih.gov/). For this search, we used selected (�)ssRNA viruses fromdifferent families or genera as queries (as indicated in supplemen-tary Table S1). Fungal genome sequences that matched viral pep-tides with e-values of o0.01 were extracted (along with flankingsequences) and a putative ORF was restored by adding Ns asunknown sequences to obtain continuous amino acid sequences(edited residues are shown as Xs). These sequences were then usedto screen the non-redundant (nr) database in a reciprocal tBLASTnsearch. (�)ssRNA virus-related sequences were also selected fromS. homoeocarpa TSAs and the WGS sequences of other organisms(e.g., lettuce and several insect species). Transposable elementsequences were identified using the Censor (http://www.girinst.org/censor/index.php) (Kohany et al., 2006).

Fungal materials, PCR amplification and DNA sequencing

Two samples of powdery mildew were collected from pea andred clover plants in the experimental fields at IPSR. Total genomicDNA was extracted from harvested powdery mildew (conidia andmycelia), pea plants, or red clover plants using the DNeasys

Blood and Tissue Kit (Qiagen) following the manufacturer’sinstructions. The resulting DNAs were stored at 4 1C until use.To amplify the ITS region of the ribosomal DNA, PCR wasperformed with total genomic DNA using the Erysiphe-specificITS primer pair (EryF and EryR) (Attanayake et al., 2009) or theuniversal primer pair (At-IRS-FW and At-IRS-RV) for plant ITS(Chiba et al., 2011) (see Table S4). To amplify the candidate fungalDNA fragment by PCR, primer pairs were designed based on thevirus-related sequences and/or their flanking sequences (seeTable S4). All PCR reactions were carried out in a final volumeof 50 ml containing 25 ml of Quick Taq HS DyeMix (TOYOBO) usingthe following conditions: an initial 94 1C for 2 min, followed by35 cycles of 94 1C for 10 s, 58 1C for 30 s, and 68 1C for 1 min,then 68 1C for 10 min. The PCR products were fractionatedby gel electrophoresis on 1% agarose gels and stained withethidium bromide. Purified PCR fragments were sequenced usingan ABI3100 DNA sequencer (Applied Biosystems, Foster City,CA, USA). To identify Erysiphe spp. co-infecting pea, ITS fragmentswere cloned in pGEM T-easy (Promega) and sequenced. Thesequences were assembled and analyzed using the DNA proces-sing software packages, AutoAssembler (Applied Biosystems) andGENETYX (SDC, Tokyo, Japan).

H. Kondo et al. / Virology 435 (2013) 201–209208

Phylogenetic analysis

The deduced amino acid sequences of (�)ssRNA virus-relatedsequences in the genomes of fungus and other selected organismswere aligned with MAFFT version 6 under the default parameters(Katoh and Toh, 2008) (http://mafft.cbrc.jp/alignment/server).The alignments were manually refined to remove gaps usingMEGA version 4.02 software (Tamura et al., 2007) (Fig. S1,highlighted region). The best-fit model of protein evolution(LGþGþF) was determined for maximum likelihood (ML) ana-lyses using ProtTest (Abascal et al., 2005) (http://darwin.uvigo.es/software/prottest_server.html) based on the Akaike informationcriterion (AIC). Phylogenetic trees were created in PhyML 3.0 usingthe appropriate substitution mode (Guindon et al., 2010) (http://www.atgc-montpellier.fr/phyml/). The following four categoriesof rate variation were used. As starting trees, we selected theBIONJ tree, and the type of tree improvement was subtreepruning and regrafting (SPR) (Hordijk and Gascuel, 2005).The branch support values were estimated by the approximatelikelihood ratio test (aLRT) with a Shimodaira–Hasegawa -like(SH-like) algorithm (Anisimova and Gascuel, 2006). Trees werevisualized using FigTree (version 1.3.1) (http://tree.bio.ed.ac.uk/software/figtree/).

For calculation of pairwise genetic distances, we used Kimura’stwo-parameter model for nucleotide substitution (Kimura, 1980)with MEGA version 4.02. The molecular clock was based on asubstitution rate 2.52�10�9 per site per year in the rDNA ITSsequences of the order Erysiphales (Takamatsu and Matsuda,2004). Estimate of the divergence time (T) was derived from themolecular clock (r) and genetic distance (K) using the simpleequation T¼K/2r (Fu and Li, 1997).

Acknowledgments

We thank Kazuyuki Maruyama, Sakae Hisano, Eri Umebayashiand Hideki Nishimura for technical assistance, Drs. Ida BagusAndika, Tetsuo Tamada and Yuki Ichinose for helpful advice, andDr. Yuichiro Yamaai for providing a field fungal material. We are alsograteful to the Max Planck Institute for Plant Breeding Research foruse of the G. orontii BLAST Server. This work was supported in partby a Grant-in-Aid for Scientific Research from the Japanese Ministryof Education, Culture, Sports, Science and Technology (KAKENHI toH.K. and N.S.), the Program for Promotion of Basic and AppliedResearches for Innovations in Bio-Oriented Industries (PROBRAIN toH. K.), and Yomogi Inc. (to N. S.).

Appendix A. Supplementary information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.virol.2012.10.002.

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