Molecular characterization of two evolutionarilydistinct endornaviruses co-infecting common bean(Phaseolus vulgaris)

Ryo Okada,13 Chee Keat Yong,13 Rodrigo A. Valverde,2

Sead Sabanadzovic,3 Nanako Aoki,1 Shunsuke Hotate,1 Eri Kiyota,1

Hiromitsu Moriyama1 and Toshiyuki Fukuhara1

Correspondence

Hiromitsu Moriyama

hmori714@cc.tuat.ac.jp

Received 21 May 2012

Accepted 24 September 2012

1Laboratory of Molecular and Cellular Biology, Faculty of Agriculture, Tokyo University of Agricultureand Technology, Tokyo 183-8509, Japan

2Department of Plant Pathology and Crop Physiology, Louisiana State University AgriculturalCenter, Baton Rouge, 70803, USA

3Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi StateUniversity, Mississippi State, MS 39762, USA

Two high-molecular-mass dsRNAs of approximately 14 and 15 kbp were isolated from the

common bean (Phaseolus vulgaris) cultivar Black Turtle Soup. These dsRNAs did not appear to

cause obvious disease symptoms, and were transmitted through seeds at nearly 100 % efficiency.

Sequence information indicates that they are the genomes of distinct endornavirus species, for

which the names Phaseolus vulgaris endornavirus 1 (PvEV-1) and Phaseolus vulgaris

endornavirus 2 (PvEV-2) are proposed. The PvEV-1 genome consists of 13 908 bp and

potentially encodes a single polyprotein of 4496 aa, while that of PvEV-2 consists of 14 820 bp

and potentially encodes a single ORF of 4851 aa. PvEV-1 is more similar to Oryza sativa

endornavirus, while PvEV-2 is more similar to bell pepper endornavirus. Both viruses have a site-

specific nick near the 59 region of the coding strand, which is a common property of the

endornaviruses. Their polyproteins contain domains of RNA helicase, UDP-glycosyltransferase

and RNA-dependent RNA polymerase, which are conserved in other endornaviruses. However, a

viral methyltransferase domain was found in the N-terminal region of PvEV-2, but was absent in

PvEV-1. Results of cell-fractionation studies suggested that their subcellular localizations were

different. Most endornavirus-infected bean cultivars tested were co-infected with both viruses.

INTRODUCTION

The family Endornaviridae contains dsRNA viruses thatinfect plants, fungi and oomycetes and are transmittedvertically via gametes, but not horizontally (Fukuhara &Moriyama, 2008; Okada et al., 2011; Valverde & Gutierrez,2007). Endornaviruses consist of a non-encapsidated,single, linear dsRNA that ranges in length from 9.8 to17.6 kbp. These viruses have been reported to infecteconomically important crops, such as rice (Moriyamaet al., 1995), common bean (Wakarchuk & Hamilton,1990), broad bean (Pfeiffer, 1998), barley (Zabalgogeazcoa& Gildow, 1992), cucurbits (Coutts, 2005), pepper (Okadaet al., 2011) and avocado (Villanueva et al., 2012), as well as

some plant-pathogenic fungi and the oomycete Phytophthoraspp. (Hacker et al., 2005). With the exception of Viciafava endornavirus (VfEV), which is associated with malesterility, most endornaviruses do not appear to affect thephenotype of the host and are generally found at constantconcentrations per cell in every tissue and at everydevelopmental stage (Moriyama et al., 1996).

The full genomic sequences of approved or putativeendornavirus species from cultivated rice [Oryza sativaendornavirus (OsEV)] (Moriyama et al., 1995), broad bean(VfEV) (Pfeiffer, 1998), wild rice [Oryza rufipogon endorna-virus (OrEV)] (Moriyama et al., 1999a), Phytophthora spp.[Phytophthora endornavirus 1 (PEV-1)] (Hacker et al., 2005),Helicobasidium mompa [Helicobasidium mompa endorna-virus 1 (HmEV-1)] (Osaki et al., 2006), Gremmeniellaabietina [Gremmeniella abietina type B RNA virus XL(GaBRV-XL)] (Tuomivirta et al., 2009), Tuber aestivum[Tuber aestivum endornavirus (TaEV)] (Stielow et al., 2011),bell pepper [bell pepper endornavirus (BPEV)] (Okada et al.,

3These authors contributed equally to this work.

The GenBank/EMBL/DDBJ accession numbers for the sequencesreported in this paper are AB719397 and AB719398.

Three supplementary tables, seven supplementary figures and a colourversion of Fig. 4 are available with the online version of this paper.

Journal of General Virology (2013), 94, 220–229 DOI 10.1099/vir.0.044487-0

220 044487 G 2013 SGM Printed in Great Britain

2011) and avocado [Persea americana endornavirus (PaEV)](Villanueva et al., 2012) have been reported.

Endornaviruses encode a single polypeptide that ispresumed to be processed into several proteins of differentfunctions by virus-encoded proteases. Based on conserveddomain database (CDD) comparison, the genomes of allcompletely sequenced endornaviruses contain conservedmotifs of an RNA-dependent RNA polymerase (RdRp,pfam00978) similar to the alpha-like virus superfamily ofpositive-stranded RNA viruses (Roossinck et al., 2011).

Two dsRNAs of approximately 13 and 15 kbp have beenreported in the common bean (Phaseolus vulgaris) cultivarBlack Turtle Soup (BTS) (Wakarchuk & Hamilton, 1985,1990). These dsRNAs were not correlated with cytoplasmicmale sterility in common bean and were associated withthe mitochondria (Mackenzie et al., 1988). Based on thenucleic acid type, size and partial sequence information forone of the two putative dsRNAs (GenBank accession no.AB185246), Phaseolus vulgaris endornavirus was accepted asa member of the family Endornaviridae (Fukuhara & Gibbs,2012). However, from which of the two dsRNA the partialgenomic sequence was obtained was not determined, andnothing is known about the other dsRNA molecule.

In this investigation, we detected two dsRNAs (14 and 15 kbp)in 11 common bean cultivars. The dsRNAs, from BTS, wereisolated, characterized, and determined that they consist of thegenome of two distinct endornaviruses. We propose thenames of Phaseolus vulgaris endornavirus 1 (PvEV-1) andPhaseolus vulgaris endornavirus 2 (PvEV-2) for these viruseswith dsRNA genomes of 14 and 15 kbp, respectively. Here, wereport their complete nucleotide sequences, genome organ-ization, detection, subcellular localizations, and phylogeneticrelationships with other endornaviruses.

RESULTS

BTS contains two distinct dsRNA species

Two dsRNAs of approximately 14 and 15 kbp, which havebeen reported previously in common bean, were originally

detected in BTS and, later in the work, in several other

common bean cultivars (Fig. 1, Table 1). These dsRNAs

were clearly resolved after agarose gel electrophoresis and

ethidium bromide staining (Fig. 1). Experiments of vertical

transmission of these dsRNAs using 161 individual BTS

plants from accession no. 48724 (National Institute of

Agrobiological Resources, Tsukuba, Japan) indicated that

they all contained the two dsRNAs (Table S1, available in

JGV Online). However, screening 50 BTS plants from a

1984 seed lot provided by R. Provvidenti (Cornell

University, Ithaca, NY, USA) resulted in three dsRNA-free

plants. Self-pollination of a selected dsRNA-free BTS plant

resulted in a dsRNA-free BTS line. Observations of the

phenotype of the dsRNA-free and dsRNA-infected BTS

lines did not reveal detectable differences. These results

indicate that the dsRNAs were transmissible via seed at

nearly 100 % efficiency.

As shown in Fig. 1, the two dsRNAs were detected in both

developmental stages of BTS. In 10-day-old seedlings, the

relative copy number of the 15 kbp dsRNA was lower than

that of the 14 kbp dsRNA. However, the concentrations of

both dsRNAs were similar when 60-day-old plants were

tested. These results were obtained after .30 independent

dsRNA extractions.

Nucleotide sequences of PvEV-1 and PvEV-2

The full sequences of the two dsRNAs designated PvEV-1

and PvEV-2 were 13 908 and 14 820 bp respectively (Fig.

2). Both sequences are available from GenBank/DDBJ

under accession numbers AB719397 and AB719398,

respectively. As the 59 region of both dsRNAs contained

multiple candidate AUG initiation codons, the most

favourable initiation codon was determined according to

the consensus sequence AA(G)CAAUGGGC (Lutcke et al.,

1987). In silico analysis showed that a favourable context

for translation initiation was found at nt 375 and 231 of

PvEV-1 and PvEV-2, respectively; therefore, the estimated

59 UTRs of PvEV-1 and PvEV-2 were 374 and 230 nt long,

respectively. The 39 UTRs of PvEV-1 and PvEV-2 consisted

23.6 kbp

7.7 kbp

15 kbp14 kbp

M 1 2 3 4 5 6 7 8 9 10 11 12

6.2 kbp

Fig. 1. Detection of dsRNAs in seeds and attwo developmental stages of the commonbean cultivar BTS. dsRNAs were isolated fromdry seeds (lanes 1–4), 10-day-old seedlings(lanes 5–8) and mature leaves of 60-day-oldplants (lanes 9–12) by the SDS-phenolmethod followed by DNase I treatment. ThedsRNAs derived from 20 mg of each tissuewere electrophoresed on 0.5 % agarose gelfor 40 h at 30 V, and stained with ethidiumbromide. Arrows indicate the positions of the14 and 15 kbp dsRNAs. Lane M, DNA sizemarkers.

Two endornaviruses co-infecting common bean

http://vir.sgmjournals.org 221

of 46 and 37 nt, respectively. In both cases the 39 terminus

ended in poly(C) sequences of 12 and 11 nt, respectively.

No other significant ORFs were found in either the coding

or non-coding strand.

PvEV-1 and PvEV-2 contain a single large ORFwith several enzyme motifs

Both PvEV-1 and PvEV-2 contain a single ORF in thecoding strand of 4496 and 4851 codons, respectively.

Table 1. Detection of dsRNA in various cultivars of common bean

+, Single infection; ++, double infection; 2, negative; NT, not tested; CDBN, National Cooperative Bean

Nursery, USA; GE, agarose and/or PAGE.

Cultivar Market class Origin/source GE RT-PCR

Black Turtle Soup Black Heirloom Seed ++ ++

Black Turtle Soup Black Cornell University ++ ++

Black Turtle Soup Black Brazil ++ ++

Black Turtle Soup (locus I) Black Cornell University ++ ++

Black Turtle Soup (locus i) Black Cornell University ++ ++

Frijol Negro Black Mexico ++ ++

Condor Black CDBN ++ ++

Bandit Black CDBN ++ ++

T-39 Black CDBN ++ ++

Matterhorn Great Northern CDBN ++ NT

Sedona Pink CDBN ++ ++

Croissant Pinto CDBN ++ ++

Lariat Pinto CDBN ++ ++

Max Pinto CDBN 2 NT

Avalanche Navy CDBN ++ NT

Blush Light Red Kidney CDBN 2 NT

Majesty Dark Red Kidney CDBN + +

Bellagio Cranberry CDBN + +

USRM 20 Small red CDBN ++ NT

Black Valentine Green bean Heirloom Seeds 2 2

Contender Green bean Heirloom Seeds 2 2

Kuro Kinugasa Japan 2 2

Fagiolo Nano Brittle Wax Green bean Italy 2 2

Fagiolo Rampicante Trionfo Violetto Italy 2 2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 kbp

Phaseolus vulgaris endornavirus 2

Phaseolus vulgaris endornavirus 1

1

Oryza sativa endornavirus

Bell pepper endornavirus

56831573

Hel-1 UGT RdRp

(nt 1112) ORF (4496 aa)

CPS 13908

58831761

Hel-1 UGT RdRp

(nt 1211) ORF (4572 aa)

CPS1 13952

231 ORF (4851 aa) 14786

Hel-1MTR RdRp

(nt 881)

UGT1 14820

225 ORF (4815 aa) 14673

Hel-1 UGTMTR RdRp

(nt 880)

1 14727

Fig. 2. Schematic representation of the genomeorganizations of PvEV-1 and PvEV-2, OsEV andBPEV with key nucleotide and amino acidreferences, including the position of the nick inthe coding strand (.). The box represents thelarge ORF, whereas lines depict UTRs. MTR,Viral methyltransferase; Hel-1, viral helicase 1;CPS, capsular polysaccharide synthase; UGT,UDP-glycosyltransferase; RdRp, viral RNA-dependent RNA polymerase.

R. Okada and others

222 Journal of General Virology 94

Comparison of amino acid sequences of the polyproteinsencoded by these ORFs revealed low levels of mutualidentity and similarity (5 and 17 %, respectively).

A BLAST search (NCBI database) using deduced amino acidsequences from both ORFs encoded by PvEV-1 and PvEV-2 detected conserved domains of a putative RNA helicase-1(Hel-1), UDP-glucose glycosyltransferase (UGT) and anRdRp. A putative viral methyltransferase (MTR) domainwas only found in PvEV-2, whereas a conserved putativecapsular polysaccharide synthase (CPS)-like domain wasonly present in PvEV-1 (Fig. 2).

A putative Hel-1, a conserved enzyme essential for virusreplication, was found in PvEV-1 and PvEV-2 (aa 1368–1618 and 1338–1585, respectively). Important motifs I–VI(Koonin et al., 1993) in the Hel-1-like region wereconserved (Fig. S1). Phylogenetic analysis of the Hel-1

domain indicated that PvEV-1 and PvEV-2 were distantlyrelated (Fig. 3a).

As with most endornaviruses, a UGT domain was locatedbetween Hel-1 and RdRp regions in both viruses. TheUGT-like region consists of four important motifs and aputative steroid-binding domain (Warnecke et al., 1999).The putative UDP-sugar-binding domain (motif IV) washighly conserved in both PvEV-1 and PvEV-2 (Fig. S2).However, the putative steroid-binding domain and motif IIexhibited low similarity to the typical UGTs of mostendornaviruses. The putative UGT of PvEV-1 was relatedto the UGT of OsEV, OrEV and PaEV, while that of PvEV-2 was related to that of BPEV (Fig. 3b).

RdRps encoded by PvEV-1 and PvEV-2, which are located inthe C-terminal regions of their polyproteins (aa 4013–4476and 4373–4828, respectively), exhibited high similarity to

Medicago truncatulaArabidopsis thaliana Glomerella cingulata

Pichia angusta

Saccharomyces cerevisiae

Ashbya gossypii

BPEV

OsEV

OrEV

PaEV

PEV-1

PvEV-1

PvEV-2

Plant

Yeast

Endornavirus

Fungus

BPEV

OsEV

OrEV

PaEV

PEV-1

PvEV-1

GLRaV

GaBRV-XL

HmEV-1

PvEV-2

CeEV

VfEV

(c)

BPEV

OsEV

OrEV

PaEV

PEV-1

PvEV-1

GLRaV

GaBRV-XL

HmEV-1

PvEV-2

CeEV

VfEV

TaEV

BPEV

PepRSV

VTRV

ObPV

BSBV

GaBRV-XL

AMV

PvEV-2

PMMoV

TMV

CMV

BMV

(a)

(b) (d)Oryza sativa japonica

Fig. 3. Maximum likelihood-based phylogenetic trees of viral Hel-1 (a), UGT (b), RdRp (c) and MTR (d) of endornaviruses,related taxa and corresponding regions encoded by some representative viruses, fungi and plants inferred by using the methodbased on the WAG substitution model (Whelan & Goldman, 2001). A discrete gamma distribution was used to modelevolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable (G+I) in(a) and (c). Consensus trees obtained after 1000 replicates are presented. The percentage of trees in which taxa clusteredtogether is shown. Branches with 50 % bootstrap values are collapsed, as they are considered untrustworthy. GenBankaccession numbers of genes are provided in Table S2. Grapevine leafroll-associated virus (GLRaV) is a closterovirus and wasused as an outgroup in (a) and (c). AMV, Alfalfa mosaic virus; BSBV, beet soil-borne virus; BMV, brome mosaic virus; CeEV,Chalara elegans endornavirus; CMV, cucumber mosaic virus; ObPV, Obuda pepper virus; PepRSV, pepper ringspot virus;PMMoV, pepper mild mottle virus; TMV, tobacco mosaic virus; TRV, tobacco rattle virus.

Two endornaviruses co-infecting common bean

http://vir.sgmjournals.org 223

the RdRps encoded by known endornaviruses. Multiplesequence alignment of these sequences and RdRp derivedfrom ssRNA viruses showed that motifs A–E (Poch et al.,1989) were conserved in PvEV-1 and PvEV-2 (Fig. S3). Aneighbour-joining phylogenetic tree showed that they wererelated closely to endornaviruses (Fig. 3c). PvEV-1 wasrelated closely to OsEV and OrEV in addition to PaEV, whilePvEV-2 was related closely to BPEV. PvEV-1 and PvEV-2were only distantly related to each other. This phylogeny wassimilar to that derived from the Hel-1 domain.

A viral MTR domain was found only in PvEV-2, but not inPvEV-1. The MTR domain is also found in the N terminusof two recently reported endornaviruses, GaBRV-XL andBPEV (Tuomivirta et al., 2009; Okada et al., 2011). ThePvEV-2 viral MTR domain was found in the N-terminalregion (aa 250–608). The MTR is involved in 59-capping tomRNAs to enhance their stability, and this process occursin the nucleus. Therefore, many viruses that replicate in thecytoplasm encode their own MTR enzymes. Because theMTR is commonly found in ssRNA viruses of the alpha-like virus superfamily, the MTR-like region of PvEV-2 wasaligned with the corresponding region of selected ssRNAviruses of the families Virgaviridae and Bromoviridae. Amultiple sequence alignment revealed conserved motifs I–IV of the ‘Sindbis-like’ supergroup (Rozanov et al., 1992)in PvEV-2. The invariant amino acid residues for MTRactivity, a histidine in motif I, the DXXR signature in motifII and a tyrosine in motif IV, were conserved (Fig. S4). Theclosest relative of this domain was that from the plantendornavirus, BPEV (65 % identity, 79 % similarity).Phylogenetic analysis showed that the putative MTR inendornaviruses formed an independent clade in the tree(Fig. 3d).

A CPS domain was found in PvEV-1, but not in PvEV-2.It was located upstream of the UGT domain (aa 2623–2904). According to a BLASTP search, the CPS domain ofPaEV had the highest similarity to that of PvEV-1 (35 %identity and 55 % similarity), whilst the mannosyltrans-ferase of Shigella boydii, which is a Gram-negative bacteriathat causes dysentery in humans, also has the secondhighest similarity (31 % identity and 48 % similarity),followed by those of OsEV (28 % identity and 43 %similarity) and OrEV (27 % identity and 48 % similarity).All of the rest were bacterial mannosyltransferase andCPS. This CPS domain was previously identified in some,but not all, endornaviruses.

A cysteine-rich domain (CRR), identified in otherendornaviruses and suggested as a candidate for a protease(Hacker et al. 2005; Tuomivirta et al., 2009), was found inboth PvEV-1 and PvEV-2. Multiple sequence alignmentshowed that most endornaviruses, including PvEV-2, hadfour CXCC signatures, although cysteine at the thirdcharacter in signatures 2 and 4 was often substituted (Fig.S5). In contrast, rice endornaviruses (OsEV and OrEV),PaEV and PvEV-1 lacked signatures 1 and 2, and containedtwo conserved CXCC signatures (Fig. S5).

Detection and determination of the position of thenicks

The 59 region of the coding strand of most endornavirusescontains a site-specific nick, while the non-coding (minus)strand does not (Fukuhara & Moriyama, 2008). To identifythe presence of a nick in PvEV-1, denatured dsRNAblots were hybridized with PvEV-1-derived cDNA probeslocated between nt 34 and 633 (YpBT44) and nt 8390 and9374 (YpBT82) of the coding strand. A band ofapproximately 13 kb was detected by both probes, whilea fragment of approximately 1 kb was detected only withprobe YpBT44 (Fig. 4a). The presence of the 1 kb fragmentimplied that a discontinuity was located approximately1000 nt from the 59 end of the coding strand. These probescould detect both (coding and non-coding) strandsseparated by denaturing agarose gel electrophoresis, butthe 13 kb coding strand and intact non-coding strand(14 kb) were not separated by electrophoresis due to theirsimilar molecular mass. Therefore, signals of both mole-cules appeared as one band [dark grey and grey arrows inFig. 4(b)]. The same experiments were carried out todetermine the presence of a nick in PvEV-2 (Fig. 4). Afragment of approximately 1 kb was detected when probedwith YpBT104 (nt 258–692) for the 59 region, indicatingthat the nick was located at the 59 region of PvEV-2, as withPvEV-1, which was similar to other endornaviruses.

However, further 59 RACE experiments were carried out inorder to determine the exact sites of the nicks in bothPvEV-1 and PvEV-2. Sequence analyses of multiple RACE-generated clones indicated that the majority of cDNAsshowed a nick in the coding strand at nt 1111 for PvEV-1and nt 881 for PvEV-2 (Fig. 4a).

Subcellular localizations of PvEV-1 and PvEV-2

Subcellular fractionation was performed to determine thelocalization of PvEV-1 and PvEV-2 in host cells. PvEV-1was associated mainly with the microsomal fraction (Fig. 5,lanes 9–10), while the subcellular localization of PvEV-2was not clearly defined; PvEV-2 was associated mainly withthe crude chloroplast and mitochondrial fractions (Fig. 5,lanes 5–8). DNA was purified from the chloroplast fractionand digested with BamHI, EcoRI and HindIII. Electrophoresisof DNA digests with these enzymes confirmed that thisfraction was derived mainly from chloroplasts; neverthe-less, this fraction contained a small amount of the nuclearDNA (Fig. S6). Neither PvEV-1 nor PvEV-2 was found inthe cytosol fraction (Fig. 5, lanes 11–12). These resultssuggest different intracellular localizations for PvEV-1 andPvEV-2.

Presence of PvEV-1 and PvEV-2 in common beancultivars

Some common bean cultivars analysed were co-infectedwith both viruses, while others were virus-free (Table 1).Two cultivars, Bellagio and Majesty, were found to be

R. Okada and others

224 Journal of General Virology 94

infected with only a 14 kbp dsRNA. None of the cultivarstested was infected with the 15 kbp dsRNA alone.

A virus-specific duplex RT-PCR, designed and developedin the framework of this study, confirmed that the 14 kbpdsRNA present in cultivars Bellagio and Majesty representsthe genome of the same virus, PvEV-1, as was detected indouble infections in other cultivars (Fig. 6, lanes 2–3). In

the case of double infections, the test yielded two PCRproducts of distinct size, indicating the presence of twotarget viruses in reverse-transcribed RNA extracts (Fig. 6).

DISCUSSION

We have isolated and sequenced two dsRNAs of approxi-mately 14 and 15 kbp from the common bean cultivar BTSand determined that they represent the genomes of twodistinct species of endornavirus. Phylogenetic analyses of theputative Hel-1, UGT and RdRp showed that these twodsRNA viruses are members of the family Endornaviridae(Fig. 3). Therefore, we propose that both viruses be classifiedin the genus Endornavirus of the family Endornaviridaeand propose the names Phaseolus vulgaris endornavirus 1(PvEV-1) and Phaseolus vulgaris endornavirus 2 (PvEV-2)for the 14 and 15 kbp dsRNAs, respectively. The partialsequences (630 bp) of a dsRNA virus from the P. vulgariscultivar BTS have been published (Wakarchuk & Hamilton,1990) and, based on the sequences and other properties ofthe dsRNA, it was placed in the genus Endornavirus andnamed Phaseolus vulgaris endornavirus (PvEV) (Fukuhara etal., 2006). The genome sequences of PvEV-1 and PvEV-2revealed that the partial sequences reported by Wakarchuk &Hamilton (1990) were derived from PvEV-1. Therefore, wepropose that PvEV should be renamed PvEV-1 in order toclarify the nomenclature of these two endornaviruses co-infecting the same host.

With the exception of VfEV and TaEV, a UGT domain hasbeen found in all other sequenced endornaviruses. Hackeret al. (2005) reported that the putative UGT is probably aUDP-glucose : sterol glycosyltransferase, as it shows highsimilarity to those found in yeasts, fungi and plants. BothPvEV-1 and PvEV-2 have a conserved UGT domain, whichis highly unusual for RNA viruses. Although the exactfunction of the UGT in endornaviruses remains to beelucidated, it has been suggested that it could modify andenforce their vesicle membranes surrounding the nakeddsRNAs to protect them against cellular enzymes such asnucleases (Roossinck et al., 2011). The putative CRRdomain with multiple CXCC signatures, conserved among

PvEV-2 (15 kbp)PvEV-1 (14 kbp)

7.7 kbp

23.6 kbp

M 1 2 3 4 5 6 7 8 9 10 11 12 Fig. 5. Subcellular localizations of PvEV-1 andPvEV-2. Lanes 1 and 2, total nucleic acidsderived from the remains of the filtrate beforeand after DNase I treatment, respectively;lanes 3 and 4, nuclear fraction; lanes 5 and6, chloroplast fraction; lanes 7 and 8, mito-chondrial fraction; lanes 9 and 10, microsomalfraction; lanes 11 and 12, cytosol fraction.Samples were electrophoresed in 0.5 % agar-ose gel for 40 h at 30 V and stained withethidium bromide. Arrows represent the posi-tions of PvEV-1 and PvEV-2. Lane M, DNAsize markers.

PvEV-1

YpBT44 YpBT82

Nick (nt 1111)

(a)

YpBT13YpBT104

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 kbp

Nick (nt 881)PvEV-2

YpBT44 YpBT82

M 1 2 M 1 2 M 1 2

19.3 -

1.4 -

M 1 2 kbp

M 1 2

YpBT13YpBT104(b)

Fig. 4. Detection of the site-specific nicks in PvEV-1 and PvEV-2by a series of Northern hybridizations. (a) Schematic diagrams ofthe positions of the nick and probes used in hybridization. (b) Theleft panel shows the electrophoresis of dsRNA on a native agarosegel. Lane M, DNA size markers; lanes 1 and 2, purified dsRNAsderived from two different common bean plants (BTS). Results ofhybridization of denatured dsRNAs with probes YpBT44, YpBT82,YpBT104 and YpBT13 are shown in each panel. A colour versionof this figure is available in JGV Online.

Two endornaviruses co-infecting common bean

http://vir.sgmjournals.org 225

endornaviruses, was found in both viruses. This region hasbeen suggested to be a candidate for a viral protease thatcan process the polyprotein to functional units (Hackeret al., 2005; Tuomivirta et al., 2009; Okada et al., 2011).

While conserved domains of Hel-1, UGT and RdRp werefound in both viruses, a viral MTR domain was only foundin PvEV-2. Among the endornaviruses identified to date,the MTR domain is only found in BPEV and GaBRV-XL.The MTR domain is characteristic of alpha-like virusesand, along with Hel-1 and RdRp, is implicated to be part ofthe replication machinery (Koonin et al., 1993). The orderof these three domains in PvEV-2 supports the hypothesisthat endornaviruses share a common ancestor with alpha-like viruses (Gibbs et al., 2000). The MTR in some ssRNAviruses is shown to have methyltransferase and guanylyl-transferase activities and to be involved in formation of acap structure at the 59 end of viral mRNAs, cooperatingwith Hel-1 (Huang et al., 2004). Conservation of theseimportant domains in PvEV-2 may suggest that PvEV-2would have the cap structure at the 59 end(s) of its dsRNA.In contrast to MTR, a CPS domain was found in PvEV-1,but not in PvEV-2. CPS is commonly produced by bacteriato help them adhere to surfaces and to prevent fromdesiccation. It is also a major virulence factor in Streptococcuspneumoniae (Jiang et al., 2001).

The genome organizations and nucleotide sequences ofPvEV-1 and PvEV-2 strongly support the notion that theyare distinct endornavirus species. Whilst PvEV-1 is relatedclosely to OsEV, PvEV-2 appears to be a close relative ofBPEV. As the topology of the trees from endornaviruses doesnot seem to follow the taxonomic relationship of their hosts(Fig. 3), it is probable that endornaviruses have a commonorigin, most likely in fungi, and have been transmittedhorizontally at some time in the past (Roossinck et al., 2011).

A unique molecular feature of members of the familyEndornaviridae is the presence of a site-specific nick in the 59

region of the coding (plus) strand RNA molecule. The 59

region of the coding strands of OsEV, OrEV, VfEV, PEV-1,HmEV-1 and BPEV contains a site-specific nick at nt 1211,

1197, 2735, 1215, 2552 and 880 from the 59 end, respectively(Fukuhara et al., 1995; Moriyama et al., 1999a; Pfeiffer, 1998;Hacker et al., 2005; Osaki et al., 2006; Okada et al., 2011).Similarly to other endornaviruses, PvEV-1 and PvEV-2contain a nick in the 59 region of their genome at nt 1111and 881 from the 59 end, respectively.

In this investigation, both PvEV-1 and PvEV-2 weretransmitted to the progeny plants at rates close to 100 %.However, virus-free plants were detected in a BTSaccession. This is not surprising, as similar results wereobtained with BPEV, which infects bell pepper cv. Marengo(Okada et al., 2011). This supports previous data on thehighly efficient transmission of other endornavirusesthrough seed (Moriyama et al., 1996; Valverde & Gutierrez,2007). Like other endornaviruses (Valverde et al., 1990;Fukuhara et al., 1993), both viruses were detected in everytissue tested (Figs 1 and S7). This property of endornavirusesis not observed with conventional plant viruses, but it iscommon in fungal viruses (mycoviruses). Nevertheless,unlike many mycoviruses (Rogers et al., 1986;Anagnostakis, 1988; Coenen et al., 1997; Chun & Lee,1997), endornaviruses can be maintained efficiently duringmeiosis via pollen and egg cells. Research to elucidate themechanism of transmission of fungal and plant endorna-viruses during meiosis will help to explain the efficient seedtransmission of plant endornaviruses. This unique propertyof endornaviruses can lead to the establishment of asymbiotic relationship with host plants (Roossinck, 2010).

The results of our investigation show that these twoendornaviruses are capable of maintaining stable co-infectionin common bean. Although it is known that multiplepartitiviruses can infect the same plant (Sabanadzovic &Valverde, 2011), this is the first report of co-infection of ahost by two distinct endornaviruses. Testing commonbean cultivars with the virus-specific one-step RT-PCRmethod developed in this study confirmed that dsRNAspresent in various bean cultivars consist of the genome ofthe same viruses. Furthermore, the results confirmed thatthe 14 kbp detected in the cultivars Bellagio and Majestyis the genome of the same virus (PvEV-1) detected inseveral double-infected cultivars. Testing more bean culti-vars and other Phaseolus species, together with pedigreeanalyses of infected cultivars, could help to unravel theorigin of these viruses in common bean.

In the case of rice, two endornaviruses infecting cultivated riceand wild rice (OsEV and OrEV, respectively), which share85 % identical amino acid sequence, have been reported(Moriyama et al., 1995, 1999a). Attempts to co-infect the ricewith these two viruses by interspecific crosses failed becauseapparently the two viruses were incompatible with each otherin hybrid rice progenies (Moriyama et al., 1999b). In contrast,PvEV-1 and PvEV-2 seem compatible in common bean,although they are apparently localized in different areas of thecell. VfEV, OsEV and OrEV are mainly found in themicrosomes of host cells (Lefebvre et al., 1990; Moriyamaet al., 1996), while BPEV was reported to be detected mainly

519 bp (PvEV-2)

303 bp (PvEV-1)

M 1 2 3 4 5 6 7

Fig. 6. Results of one-step duplex RT-PCR test performed onRNA extracts of different bean samples, visualized by agaroseelectrophoresis. Lane 1, dsRNA-free BTS; lane 2, Bellagio; lane 3,Majesty; lane 4, Croissant; lane 5, Lariat; lane 6, N3467; lane 7,dsRNA-positive BTS. M, DNA ladder (1 kb Plus DNA ladder,Invitrogen).

R. Okada and others

226 Journal of General Virology 94

in the chloroplast fraction of bell pepper (Valverde et al.,1990). Cell-fractionation experiments showed that PvEV-1was associated mainly with the microsomes and PvEV-2 wasfound mainly in the chloroplast and mitochondrial fraction(Fig. 5). The subcellular localizations of PvEV-1 and PvEV-2appeared to be similar to those of their phylogeneticallyrelated species, OsEV and BPEV, respectively (Fig. 3).Endornaviruses seem to have functional proteins, such asHel-1, UGT and RdRp. In the case of PvEV-1 and PvEV-2,these proteins are distantly related to each other (Fig. 3). TheMTR domain was present only in PvEV-2, while the CPSdomain was present only in PvEV-1. The differences in thepresence of these proteins might reflect the differentsubcellular localization of PvEV-1 and PvEV-2. The UGT inendornaviruses may have roles in membrane modificationand membrane binding. Future studies related to theintracellular localization of common bean endornavirusesshould involve these proteins.

The virus concentration of PvEV-2 in seedlings was lowerthan that in mature leaves (Fig. 1). In the case of OsEV, ithas been demonstrated that the concentration of virusdecreases or is lost completely in some Dicer-like protein(OsDCL2) knock-down plants during their developmentand, therefore, the rate of transmission of it is lower inthese plants than in the wild-type rice plants. This suggeststhat OsDCL2 regulates host factor(s) for maintenance ofOsEV in somatic and meiotic division (Urayama et al.,2010). In contrast, the maintenance system for PvEV-2may depend on developmental gene expression of commonbean, which could be elucidated if the exact subcellularlocalization for this virus is demonstrated.

Biological properties, genome organization and phylogenyindicate that the dsRNAs co-infecting the common beancultivar BTS represent the genomes of two novel, distantlyrelated species of the genus Endornavirus. As reportedpreviously, none of the double-infected BTS plantsexhibited disease symptoms (Wakarchuk & Hamilton,1985; Mackenzie et al., 1988). Moreover, phenotypicobservations of virus-free and virus-infected BTS linesdid not reveal detectable differences, although comparativestudies between virus-infected and virus-free lines need tobe conducted. The information reported here will behelpful to understand the relationships among endorna-viruses and to gain a better insight into the origin andevolution of this group of viruses.

METHODS

Plant materials. Seeds of common bean from various genotypeswere obtained from various sources (seed sources and cultivar namesare listed in Table 1). Plants were grown in a greenhouse at an averagetemperature of 28 uC.

Extraction and electrophoresis of dsRNAs. Plant tissues werepulverized with a mortar and pestle after being frozen in liquidnitrogen, and total nucleic acids were extracted with 26 STE buffer(200 mM NaCl, 20 mM Tris/HCl and 2 mM EDTA, pH 8.0). Nucleicacids were further purified with phenol and treated with DNase I

(TaKaRa). Alternatively, dsRNAs were fractionated from purifiedtotal nucleic acids by column chromatography on CF-11 cellulose(Whatman) as described by Morris & Dodds (1979) or as modified byValverde et al. (1990). For preliminary screening of dsRNAs incommon bean cultivars, purified dsRNAs were resolved in 0.8 %agarose gels (TAE buffer) at 50 V for 6 h or by PAGE (5 %acrylamide) at 100 V for 3 h. However, to determine and discrim-inate the presence of the individual viruses, 0.5 % agarose gels wererun at 20 V for 40 h at room temperature. The dsRNAs from BTSaccession no. 48724 (National Institute of Agrobiological Sciences,Tsukuba, Japan) were extracted and analysed from seed and at twodifferent developmental growth stages (10 and 60 days afteremergence); 20 mg foliar tissue was firstly cut off from a 10-day-old plant, and then 20 mg foliar tissue was cut off again from thesame individual plant after 50 days.

Transmission experiments. Eight BTS plants (accession no. 48724,National Institute of Agrobiological Resources, Tsukuba, Japan),which were co-infected with PvEV-1 and PvEV-2, were self-pollinatedand seed pods were collected. Seeds from each parental plant weregerminated and grown. Leaves from 2-month-old progeny plantswere harvested to determine the presence or absence of the dsRNAs.Extraction of dsRNAs was performed as described above.

Cloning, sequencing and sequence analyses. After phenolextraction, the two dsRNAs from BTS (14 and 15 kbp) thatcorresponded to PvEV-1 and PvEV-2 were purified by two cycles ofcolumn chromatography, and cDNA fragments were generated withrandom hexadeoxyribonucleotide primers (TaKaRa). The terminalsequences of both viruses were obtained with 59/39 RACE. 59 RACEwas performed as described previously (Okada et al., 2011). For 39

RACE, dsRNAs were polyadenylated on the 39 ends of both strands byEscherichia coli poly(A) polymerase (TaKaRa), and first-strand cDNAswere synthesized as described previously (Isogai et al., 1998) andamplified by PCR. cDNA fragments and 59/39 RACE products werecloned and sequenced as described by Okada et al. (2011). Obtainednucleotide sequences were analysed for ORFs, and any ORFs foundwere translated into amino acid sequences with GENETYX version 9(GENETYX). Deduced amino acid sequences with similarity to PvEV-1 and PvEV-2 were searched in the NCBI database with BLAST

(Altschul et al., 1997). A similarity search of protein versus proteinwas performed with GENETYX. Deduced amino acid sequences werealigned with CLUSTAL_X (Thompson et al., 1997) and GeneDoc v. 2.6(Nicholas et al., 1997). Phylogenetic analyses were performed onamino acid sequences using MEGA 5.05 (Tamura et al., 2011). Aftertesting each dataset for the best-fit substitution model, phylogeneticrelationships among sequences used for analyses were inferred by themaximum-likelihood method.

Detection and determination of the position of the nicks in

PvEV-1 and PvEV-2. For Northern blot analysis, 100 ng of both viraldsRNAs was separated on a 1.2 % agarose MOPS gel with 6 %formaldehyde and transferred to a nylon Zeta-Probe membrane (Bio-Rad) by capillary blotting (Okada et al., 2011). After UV cross-linkingand prehybridization in hybrisolution (250 mM phosphate bufferpH 7.2, 1 mM EDTA, 7 % SDS, 1 % BSA), blots were hybridized for16 h at 65 uC in the same solution with 32P-labelled DNA probesspecific for PvEV-1 clones YpBT44 (located between nt 34 and 633)and YpBT82 (between nt 8390 and 9374), or for PvEV-2-clonesYpBT104 (between nt 258 and 692) and YpBT13 (between nt 12 417and 14 255). Probes were synthesized with a random prime-labellingsystem (BcaBEST labeling kit, TaKaRa). Washing and detectionprocedures were as described previously (Okada et al., 2011).

Subcellular fractionation of PvEV-1 and PvEV-2. BTS leaves(20 g) were homogenized with a mixer for 5–10 s in 80 ml STC buffer(0.3 M sucrose, 50 mM Tris, 10 mM CaCl2, 2 mM EDTA, 0.1 %

Two endornaviruses co-infecting common bean

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2-mercaptoethanol, pH 7.5). The homogenate was filtered through a

four-layerednylon screen (80 mm pores). The filtrate was centrifuged

for 10 min at 1220 g using a swinging bucket rotor (Tomy,

Suprema21). The resulting pellet, containing nuclei and chloroplasts,

was resuspended in STC buffer and applied to a discontinuous

sucrose gradient (40–55 %), and centrifuged for 30 min at 74 700 gusing a swinging bucket rotor (Hitachi, CP800WX). A green layer

separated in the gradient was collected and considered to be the intact

chloroplast fraction. Nuclei were not observed after light microscopy

examinations. The pellet (from the gradient), which contained intact

nuclei, was washed with 0.5 % Triton X-100 in STC buffer to remove

chloroplast contaminants. After centrifugation at 1220 g using a

swinging bucket rotor, the pellet was saved and referred to as the

crude nuclear fraction. The supernatant obtained after the centrifu-

gation of the filtrate contained mitochondria, microsomes and

cytosol. The supernatant was first centrifuged for 20 min at

11 500 g using a fixed-angle rotor to obtain the crude mitochondrial

fraction from the resultant pellet. Then, the supernatant was

centrifuged for 1 h at 100 000 g with the RP65-733 fixed-angle rotor

(Hitachi, CP800WX). The resulting pellet or the supernatant were

referred to as microsomal or cytosol fractions, respectively. Nucleic

acids were extracted from these subcellular fractions as described

above. For enzymic analysis, total nucleic acids from the chloroplast

fraction were digested with RNase A (2 mg ml21) for 16 h and with

BamHI, EcoRI and HindIII.

Occurrence of the two dsRNAs in selected common bean

cultivars. Seeds from common bean cultivars (listed in Table 1) were

planted, and 2–4-week-old seedlings were tested for the presence of

the 14 and 15 kbp dsRNAs by electrophoretic analyses. Furthermore,

a duplex, single-tube RT-PCR protocol for the simultaneous and

discriminatory detection of the two endornaviruses from plant tissue

was developed by applying specific primers for PvEV-1 and PvEV-2

(Table S2) designed to amplify genomic fragments of 303 and 519 bp,

respectively. Total RNAs were extracted from 0.1 g leaf tissue of

selected common bean cultivars with a Plant RNeasy kit (Qiagen) and

eluted with 100 ml TE buffer. An aliquot of 2.5 ml of each sample was

submitted to RT-PCR tests using a SuperScript III One Step kit

(Invitrogen) and slightly modifying the conditions recommended by

the manufacturer: aliquots of total RNA were mixed with 12.5 ml 26reaction buffer, 0.5 ml 5 mM MgSO4, 1 ml of each of the four primers

(100 ng ml21), 0.75 ml reverse transcriptase/Taq mix, and 2 ml RNase-

free water for a total volume of 25 ml. This mix was then subjected to

the following conditions: reverse transcription for 20 min at 53 uC,

denaturation for 2 min 30 s at 94 uC, and 35 cycles of PCR (94 uC for

20 s, 56 uC for 35 s, and 68 uC for 45 s) followed by a final extension

step at 68 uC for 5 min. The presence of virus-specific PCR products

was ascertained by electrophoresis in 1.5 % agarose gel in TAE buffer

and staining with ethidium bromide.

ACKNOWLEDGEMENTS

We wish to thank Dr M. A. Pastor-Corrales, of USDA-ARS, Beltsville,

MD, USA, Drs M. M. Jahn and R. Provvidenti, Cornell University,

Ithaca, NY, USA, and the National Institute of Agrobiological

Resources, Tsukuba, Japan, for providing selected common bean

germplasm, and Professor T. Natsuaki, Utsunomiya University,

Japan, for helpful advice.

REFERENCES

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller,W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation

of protein database search programs. Nucleic Acids Res 25, 3389–3402.

Anagnostakis, S. L. (1988). Cryphonectria parasitica, cause ofchestnut blight. Adv Plant Pathol 6, 123–136.

Chun, S. J. & Lee, Y. H. (1997). Inheritance of dsRNAs in the rice blastfungus, Magnaporthe grisea. FEMS Microbiol Lett 148, 159–162.

Coenen, A., Kevei, F. & Hoekstra, R. F. (1997). Factors affecting thespread of double-stranded RNA viruses in Aspergillus nidulans. GenetRes 69, 1–10.

Coutts, R. H. A. (2005). First report of an endornavirus in theCucurbitaceae. Virus Genes 31, 361–362.

Fukuhara, T. & Gibbs, M. J. (2012). Family Endornaviridae. In VirusTaxonomy: Ninth Report of the International Committee on Taxonomyof Viruses, pp. 519–521. Edited by A. M. Q. King, M. J. Adams,E. B. Carstens & E. J. Lefkowitz. Tokyo: Elsevier Academic Press.

Fukuhara, T. & Moriyama, H. (2008). Endornaviruses. In Encyclopedia ofVirology, 3rd edn, pp. 109–116. Edited by B. W. J. Mahy & M. H. V. vanRegenmortel. Oxford: Elsevier.

Fukuhara, T., Moriyama, H., Pak, J. Y., Hyakutake, H. & Nitta, T.(1993). Enigmatic double-stranded RNA in Japonica rice. Plant MolBiol 21, 1121–1130.

Fukuhara, T., Moriyama, H. & Nitta, T. (1995). The unusual structureof a novel RNA replicon in rice. J Biol Chem 270, 18147–18149.

Fukuhara, T., Koga, R., Aoki, N., Yuki, C., Yamamoto, N., Oyama, N.,Udagawa, T., Horiuchi, H., Miyazaki, S. & other authors (2006). Thewide distribution of endornaviruses, large double-stranded RNAreplicons with plasmid-like properties. Arch Virol 151, 995–1002.

Gibbs, M. J., Koga, R., Moriyama, H., Pfeiffer, P. & Fukuhara, T.(2000). Phylogenetic analysis of some large double-stranded RNAreplicons from plants suggests they evolved from a defective single-stranded RNA virus. J Gen Virol 81, 227–233.

Hacker, C. V., Brasier, C. M. & Buck, K. W. (2005). A double-strandedRNA from a Phytophthora species is related to the plant endorna-viruses and contains a putative UDP glycosyltransferase gene. J GenVirol 86, 1561–1570.

Huang, Y. L., Han, Y. T., Chang, Y. T., Hsu, Y. H. & Meng, M. (2004).Critical residues for GTP methylation and formation of the covalentm7GMP-enzyme intermediate in the capping enzyme domain ofBamboo mosaic virus. J Virol 78, 1271–1280.

Isogai, M., Uyeda, I. & Hataya, T. (1998). An efficient cloning strategyfor viral double-stranded RNAs with unknown sequences. AnnPhytopathological Soc Jpn 64, 244–248.

Jiang, S. M., Wang, L. & Reeves, P. R. (2001). Molecularcharacterization of Streptococcus pneumoniae type 4, 6B, 8, and 18Ccapsular polysaccharide gene clusters. Infect Immun 69, 1244–1255.

Koonin, E. V., Dolja, V. V. & Morris, T. J. (1993). Evolution and taxonomyof positive-strand RNA viruses: implications of comparative analysis ofamino acid sequences. Crit Rev Biochem Mol Biol 28, 375–430.

Lefebvre, A., Scalla, R. & Pfeiffer, P. (1990). The double-strandedRNA associated with the ‘447’ cytoplasmic male sterility in Vicia fabais packaged together with its replicase in cytoplasmic membranousvesicles. Plant Mol Biol 14, 477–490.

Lutcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, H. F. &Scheele, G. A. (1987). Selection of AUG initiation codons differs inplants and animals. EMBO J 6, 43–48.

Mackenzie, S. A., Pring, D. R. & Bassett, M. J. (1988). Large double-stranded RNA molecules in Phaseolus vulgaris L. are not associatedwith cytoplasmic male sterility. Theor Appl Genet 76, 59–63.

Moriyama, H., Nitta, T. & Fukuhara, T. (1995). Double-stranded RNAin rice: a novel RNA replicon in plants. Mol Gen Genet 248, 364–369.

Moriyama, H., Kanaya, K., Wang, J. Z., Nitta, T. & Fukuhara, T. (1996).Stringently and developmentally regulated levels of a cytoplasmic

R. Okada and others

228 Journal of General Virology 94

double-stranded RNA and its high-efficiency transmission via egg andpollen in rice. Plant Mol Biol 31, 713–719.

Moriyama, H., Horiuchi, H., Koga, R. & Fukuhara, T. (1999a).Molecular characterization of two endogenous double-stranded RNAsin rice and their inheritance by interspecific hybrids. J Biol Chem 274,6882–6888.

Moriyama, H., Horiuchi, H., Nitta, T. & Fukuhara, T. (1999b). Unusualinheritance of evolutionarily-related double-stranded RNAs ininterspecific hybrid between rice plants Oryza sativa and Oryzarufipogon. Plant Mol Biol 39, 1127–1136.

Morris, T. J. & Dodds, J. A. (1979). Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue.Phytopathology 69, 854–858.

Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997).GeneDoc: analysis and visualization of genetic variation. EMBnetNews 4 (2, ), 1–4. http://www.nrbsc.org/gfx/genedoc/ebinet.htm

Okada, R., Kiyota, E., Sabanadzovic, S., Moriyama, H., Fukuhara, T.,Saha, P., Roossinck, M. J., Severin, A. & Valverde, R. A. (2011). Bellpepper endornavirus: molecular and biological properties, andoccurrence in the genus Capsicum. J Gen Virol 92, 2664–2673.

Osaki, H., Nakamura, H., Sasaki, A., Matsumoto, N. & Yoshida, K.(2006). An endornavirus from a hypovirulent strain of the violet rootrot fungus, Helicobasidium mompa. Virus Res 118, 143–149.

Pfeiffer, P. (1998). Nucleotide sequence, genetic organization andexpression strategy of the double-stranded RNA associated with the‘447’ cytoplasmic male sterility trait in Vicia faba. J Gen Virol 79,2349–2358.

Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identificationof four conserved motifs among the RNA-dependent polymeraseencoding elements. EMBO J 8, 3867–3874.

Rogers, H. J., Buck, K. W. & Brasier, C. M. (1986). Transmission ofdouble-stranded RNA and a disease factor in Ophiostoma ulmi. PlantPathol 35, 277–287.

Roossinck, M. J. (2010). Lifestyles of plant viruses. Philos Trans R SocLond B Biol Sci 365, 1899–1905.

Roossinck, M. J., Sabanadzovic, S., Okada, R. & Valverde, R. A.(2011). The remarkable evolutionary history of endornaviruses. J GenVirol 92, 2674–2678.

Rozanov, M. N., Koonin, E. V. & Gorbalenya, A. E. (1992).Conservation of the putative methyltransferase domain: a hallmarkof the ‘Sindbis-like’ supergroup of positive-strand RNA viruses. J GenVirol 73, 2129–2134.

Sabanadzovic, S. & Valverde, R. A. (2011). Properties and detectionof two cryptoviruses from pepper (Capsicum annuum). Virus Genes43, 307–312.

Stielow, B., Klenk, H.-P. & Menzel, W. (2011). Complete genome

sequence of the first endornavirus from the ascocarp of the

ectomycorrhizal fungus Tuber aestivum Vittad. Arch Virol 156, 343–345.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar,S. (2011). MEGA5: molecular evolutionary genetics analysis using

maximum likelihood, evolutionary distance, and maximum par-

simony methods. Mol Biol Evol 28, 2731–2739.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible

strategies for multiple sequence alignment aided by quality analysis

tools. Nucleic Acids Res 25, 4876–4882.

Tuomivirta, T. T., Kaitera, J. & Hantula, J. (2009). A novel putative

virus of Gremmeniella abietina type B (Ascomycota: Helotiaceae) has

a composite genome with endornavirus affinities. J Gen Virol 90,

2299–2305.

Urayama, S., Moriyama, H., Aoki, N., Nakazawa, Y., Okada, R.,Kiyota, E., Miki, D., Shimamoto, K. & Fukuhara, T. (2010). Knock-

down of OsDCL2 in rice negatively affects maintenance of the

endogenous dsRNA virus, Oryza sativa endornavirus. Plant Cell

Physiol 51, 58–67.

Valverde, R. A. & Gutierrez, D. L. (2007). Transmission of a dsRNA in

bell pepper and evidence that it consists of the genome of an

endornavirus. Virus Genes 35, 399–403.

Valverde, R. A., Nameth, S., Abdallha, O., Al-Musa, O., Desjardins, P.& Dodds, J. A. (1990). Indigenous double-stranded RNA from pepper

(Capsicum annuum). Plant Sci 67, 195–201.

Villanueva, F., Sabanadzovic, S., Valverde, R. A. & Navas-Castillo, J.(2012). Complete genome sequence of a double-stranded RNA virus

from avocado. J Virol 86, 1282–1283.

Wakarchuk, D. A. & Hamilton, R. I. (1985). Cellular double-stranded

RNA in Phaseolus vulgaris. Plant Mol Biol 5, 55–63.

Wakarchuk, D. A. & Hamilton, R. I. (1990). Partial nucleotide

sequence from enigmatic dsRNAs in Phaseolus vulgaris. Plant Mol Biol

14, 637–639.

Warnecke, D., Erdmann, R., Fahl, A., Hube, B., Muller, F., Zank, T.,Zahringer, U. & Heinz, E. (1999). Cloning and functional expression

of UGT genes encoding sterol glucosyltransferases from

Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and

Dictyostelium discoideum. J Biol Chem 274, 13048–13059.

Whelan, S. & Goldman, N. (2001). A general empirical model of

protein evolution derived from multiple protein families using a

maximum-likelihood approach. Mol Biol Evol 18, 691–699.

Zabalgogeazcoa, I. A. & Gildow, F. E. (1992). Double-stranded

ribonucleic acid in ‘Barsoy’ barley. Plant Sci 83, 187–194.

Two endornaviruses co-infecting common bean

http://vir.sgmjournals.org 229