Bell pepper endornavirus: molecular and biologicalproperties, and occurrence in the genus Capsicum

Ryo Okada,1 Eri Kiyota,1 Sead Sabanadzovic,2 Hiromitsu Moriyama,1

Toshiyuki Fukuhara,1 Prasenjit Saha,3 Marilyn J. Roossinck,3 Ake Severin4

and Rodrigo A. Valverde5

Correspondence

Rodrigo A. Valverde

ravalve@lsu.edu

Received 31 May 2011

Accepted 19 July 2011

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

2Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology,Mississippi State University, Mississippi State, MS 39762, USA

3Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA

4Laboratoire de Physiologie Vegetale, Universite de Cocody-Abidjan, UFR Biosciences, 22 BP,582 Abidjan 22, Cote d’Ivoire

5Department of Plant Pathology & Crop Physiology, Louisiana State University Agricultural Center,Baton Rouge, LA 70803, USA

Bell peppers (Capsicum annuum) harbour a large dsRNA virus. The linear genome (14.7 kbp) of

two isolates from Japanese and USA bell pepper cultivars were completely sequenced and

compared. They shared extensive sequence identity and contained a single, long ORF encoding a

4815 aa protein. This polyprotein contained conserved motifs of putative viral methyltransferase

(MTR), helicase 1 (Hel-1), UDP-glycosyltransferase and RNA-dependent RNA polymerase. This

unique arrangement of conserved domains has not been reported in any of the known

endornaviruses. Hence this virus, for which the name Bell pepper endornavirus (BPEV) is

proposed, is a distinct species in the genus Endornavirus (family Endornaviridae). The BPEV-

encoded polyprotein contains a cysteine-rich region between the MTR and Hel-1 domains, with

conserved CXCC motifs shared among several endornaviruses, suggesting an additional

functional domain. In agreement with general endornavirus features, BPEV contains a nick in the

positive-strand RNA molecule. The virus was detected in all bell pepper cultivars tested and

transmitted through seed but not by graft inoculations. Analysis of dsRNA patterns and RT-PCR

using degenerate primers revealed putative variants of BPEV, or closely related species, infecting

other C. annuum genotypes and three other Capsicum species (C. baccatum, C. chinense and

C. frutescens).

INTRODUCTION

Endornaviruses have a single linear dsRNA genome (9.8–17.6 kbp), infect plants, fungi and oomycetes and aretransmitted at a high rate through seeds and spores(Fukuhara et al., 2006; Gibbs et al., 2000). They infecteconomically important crops, such as rice (Moriyamaet al., 1995), French bean (Wakarchuk & Hamilton, 1990),broad bean (Pfeiffer, 1998), barley (Zabalgogeazcoa &Gildow, 1992), cucurbits (Coutts, 2005) and pepper

(Valverde & Gutierrez, 2007), as well as some plant-pathogenic fungi and the oomycete Phytophthora (Hackeret al., 2005). The presence of endornaviruses in phenotypi-cally normal plants is a unique feature of particulargenotypes of these crops. The full sequence of the genomeof approved or putative Endornavirus species from culti-vated rice [Oryza sativa endornavirus (OSV)] (Moriyamaet al., 1995), wild rice [Oryza rufipogon endornavirus (ORV)]

(Moriyama et al., 1999), broad bean [Vicia faba endornavirus(VFV)] (Pfeiffer, 1998), Phytophthora spp. [Phytophthoraendornavirus 1 (PEV-1)] (Hacker et al., 2005), Helicoba-sidium mompa [Helicobasidium mompa endornavirus 1(HmEV-1)] (Osaki et al., 2006), Gremmeniella abietina[Gremmeniella abietina type B RNA virus XL (GaBRV-XL)]

(Tuomivirta et al., 2009) and Tuber aestivum [Tuberaestivum endornavirus (TaEV) (Stielow et al., 2011) have

The GenBank/EMBL/DDBJ accession numbers for the bell pepperendornavirus sequences reported in this paper are: AB597230,JN019858, JN019859, JN019860, JN019861, JN019862 andJN019863.

Three supplementary figures are available with the online version of thispaper.

Journal of General Virology (2011), 92, 2664–2673 DOI 10.1099/vir.0.034686-0

2664 034686 G 2011 SGM Printed in Great Britain

been reported. Moreover, endornavirus-like dsRNAs havebeen isolated from bottle gourd (Lagenaria siceraria),Malabar spinach (Basella alba) and eel grass (Zosteramarina) (Fukuhara et al., 2006).

Endornaviruses encode a single polypeptide, which ispresumed to be processed by virus-encoded proteinases.Genomes of all completely sequenced endornavirusescontain conserved motifs of a viral RNA-dependent RNApolymerase (RdRp_2, pfam00978), similar to the alpha-likevirus superfamily of positive-stranded RNA viruses (Gibbset al., 2000).

Peppers (Capsicum spp.) are perennial plants native toSouth America and today they are cultivated throughoutthe world for their nutritional and medicinal value and foruse as a cooking condiment (DeWitt & Bosland, 1996). AdsRNA was reported from tissue extracts of apparentlyhealthy bell pepper (Capsicum annuum) cultivars (Valverdeet al., 1990b). Partial sequence information suggested thatthe dsRNA constitutes the genome of an endornavirus(Valverde & Gutierrez, 2007).

In this investigation, we report the full nucleotidesequence, genome organization and some biologicalproperties of the putative endornavirus, tentatively desig-nated Bell pepper endornavirus (BPEV), isolated from YoloWonder (YW) bell pepper reported by Valverde &Gutierrez (2007) and an isolate from Kyousuzu (KS) bellpepper from Japan. Furthermore, we analysed a number ofadditional pepper cultivars for endornavirus-sized dsRNAsand we report its occurrence in other C. annuum genotypesand three other Capsicum species. Partial sequence analysisof the RdRp(s) from additional pepper isolates suggeststhat its evolution is congruent with that of its host.

RESULTS AND DISCUSSION

Screening Capsicum spp. and cultivars for BPEV

BPEV dsRNA was detected in all seven bell pepper cultivarsfrom Japan. Testing for BPEV dsRNA in Capsicumgenotypes from the USA, including bell peppers and otherhorticultural types, resulted in 40 additional positivegenotypes, shown in Table 1. BPEV was detected in alltested bell peppers and Capsicum baccatum lines. SomeC. annuum, Capsicum chinense and Capsicum frutescensgenotypes were BPEV-positive while others were negative(Tables 1 and 2). A sample of PAGE results is shown inFig. 1. Several dsRNAs with a lower molecular mass thanBPEV were associated with some Capsicum genotypes. Themajority of these dsRNAs were determined to constitutethe genome of plant partitiviruses (Sabanadzovic &Valverde, 2011). BPEV-KS dsRNA was detected in pollen,callus and cultured cells and the relative concentration wassimilar to that of the dsRNA in leaf tissue (data notshown), unlike OSV and ORV that were reported to betenfold more concentrated in pollen than in the leaf tissueof rice (Moriyama et al., 1999). BPEV appears to be

common among domesticated Capsicum species, particu-larly in cultivated peppers (C. annuum, C. frutescens and C.chinense) (Valverde & Fontenot, 1991) and in C. baccatum.These closely related species share a mutual ancestral genepool (Moscone et al., 2007). One can speculate that thegene pool from which the domesticated peppers originatedincluded pepper genotypes that were infected with BPEV.Nevertheless the origin of BPEV in pepper remains unclear.Two lines of the most primitive Capsicum species tested inthis study (Capsicum chacoense) were free of BPEV. Inaddition, five accessions of C. annuum var. glabriusculum,also known as chiltepepin, were negative. This pepper iseaten by indigenous peoples in Mexico, and is thought bysome to be an ancestor of many cultivated peppers.

As in previous investigations, all tested bell peppercontained BPEV (Valverde & Fontenot, 1991; Valverdeet al., 1990b). Bell pepper cultivars have a narrow genepool, and, while making crosses to develop new cultivars,breeders may have inadvertently introduced and spreadBPEV among bell peppers. Since Japanese bell pepperbreeders have used Capsicum germ plasm from theAmericas to develop Japanese cultivars, it was notsurprising to find that bell pepper cultivars from Japanwere also infected with BPEV.

Occurrence of BPEV in bell pepper seedlings

The level of vertical transmission of BPEV is higherthrough the ovule than through pollen, thus it is very highwhen both parents are infected (Valverde & Gutierrez,2007). Screening of seedlings from two bell peppercultivars, Yolo Wonder (YW) and Marengo (MR), withboth parents being BPEV-infected, resulted in 50 of 50 and136 of 137 positive plants, respectively. These resultsconfirm that high rates of vertical transmission areobtained when both parents are virus carriers. Screeningof seedlings of three other bell pepper cultivars, originatingfrom seeds purchased at a local store, resulted in differentpercentages of BPEV infections. A high proportion ofseedling of KS (47 of 49) and California Wonder (CW, 36of 36) were infected by BPEV, indicating that both parentscontained the virus. In contrast, only 8 of 28 seedlingsof Capsicum annuum L. ‘Kyounami’ (KY) tested BPEVpositive, suggesting that the virus was not present in one ofthe parents.

In agreement with the general properties of endorna-viruses, the BPEV-free and BPEV-infected lines of MRpepper were phenotypically undistinguishable, revealinga lack of visible effect on the host. Furthermore, inpreliminary experiments, co-infections of BPEV with fouracute ssRNA viruses [pepper mild mottle virus, potatovirus Y (PVY), cucumber mosaic virus and tomato spottedwilt virus] in MR pepper did not have a visible effect on thesymptoms caused by these viruses when compared withsingle infections of the BPEV-free MR line by any of theseviruses (not shown).

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Attempts to transmit BPEV

None of the BPEV-free MR scions grafted onto BPEV-infected rootstocks tested positive for BPEV. ELISAdetection of PVY in the scions confirmed successful grafts.

The presence of PVY in the rootstocks and subsequentsystemic infection did not aid the movement of BPEV fromthe rootstocks into the scions. The failure to transmitBPEV by grafting supports the idea that endornaviruseslack systemic movement. Infection of the BPEV-infected

Table 1. Capsicum spp. and cultivars that tested positive for bell pepper endornavirus by RT-PCR and/or agarose gelelectrophoresis (GE) analyses

+, Positive; NT, not tested; LSU, Louisiana State University; AVRDC, Asian Vegetable Research and Development Center.

Capsicum species Type Cultivar or PI line Origin GE RT-PCR

C. annuum Bell California Wonder USA + +

C. annuum Bell Yolo Wonder USA + +

C. annuum Bell Kyousuzu Japan + +

C. annuum Bell Kyounami Japan + +

C. annuum Bell Kyoumidori Japan + +

C. annuum Bell Ace Japan + +

C. annuum Bell Suigyokunigou Japan + +

C. annuum Bell High Green Japan + +

C. annuum Bell Jumbo Colour Japan + +

C. annuum Bell Marengo USA + +

C. annuum Bell Avelar USA + NT

C. annuum Bell Casca Dura Brazil + NT

C. annuum Bell King Arthur USA + NT

C. annuum Bell VR-4 USA + NT

C. annuum Bell Magda USA + NT

C. annuum Bell Bonnie’s Green Bell, USA + +

C. annuum Bell Red Bell USA + +

C. annuum Bell Chocolate Beauty Sweet USA + +

C. annuum Pimento Pimento Sweet USA + +

C. annuum Cayenne Cayenne Long Red Thick USA + +

C. annuum Cayenne Super Cayenne USA + +

C. frutescens Tabasco Greenleaf USA + NT

C. frutescens Tabasco LSU USA + +

C. frutescens Tabasco PI 159239 USA + NT

C. frutescens Tabasco PI 193470 Ethiopia + NT

C. baccatum Aji Monk’s Hat USA + +

C. baccatum Aji PI 238061 Bolivia + NT

C. baccatum Aji PI 633752 Paraguay + NT

C. baccatum Aji PI 260549 Peru + NT

C. baccatum Aji PI 215699 Peru + NT

C. baccatum Aji PI 260590 Peru + NT

C. baccatum Aji PI 260543 Brazil + NT

C. baccatum Aji PI 441589 Brazil + NT

C. baccatum Aji PI 257135 Ecuador + NT

C. baccatum Aji PI 337524 USA + NT

C. baccatum Aji PI 337522 Argentina + NT

C. baccatum Aji C00754 (AVRDC) Argentina + NT

C. baccatum Aji C01218 (AVRDC) Egypt + NT

C. baccatum Aji C01527 (AVRDC) USA + NT

C. baccatum Aji C01300 (AVRDC) Germany + NT

C. chinense Habanero PI 159236 USA + +

C. chinense Habanero PI 315008 Peru + NT

C. chinense Habanero PI 315023 Peru + +

C. chinense Habanero PI 315024 Peru + NT

C. chinense Habanero PI 273426 USA + NT

C. chinense Habanero C00943 (AVRDC) Peru + +

C. chinense Habanero C00949 (AVRDC) England + NT

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rootstock by PVY did not aid transmission. Similar resultswere reported for a partitivirus infecting Jalapeno Mpepper (Valverde & Gutierrez, 2008).

Nucleotide sequence analysis of BPEV-YW andBPEV-KS

The complete sequences of BPEV-KS (14 727 nt) andBPEV-YW (14 728 nt) were determined. The two genomeswere very similar, except for the presence of a guanine atthe 59 end in the case of BPEV-YW. They shared 88.2 %identical nucleotide sequences in the whole genome, withhighly conserved 59 and 39 UTRs, and only singlenucleotide differences in each of the UTRs, which under-scores their functional importance in virus replication.

A single ORF was found on the plus strand of both BPEV-KS and BPEV-YW, starting at nucleotide 225 in the case ofBPEV-KS (nt 226 in the case of BPEV-YW) and ending atnucleotide 14 670, which could encode a polyprotein of4815 aa of with an estimated molecular mass of 545 kDa(Fig. 2a). The genome size and protein-encoding strategyare typical of endornaviruses. The putative proteinproducts of BPEV-KS and BPEV-YW shared 92 % iden-tical amino acid residues (96 % similarity). Nevertheless,

Table 2. Capsicum spp. and cultivars that tested negative for bell pepper endornavirus by PAGE analyses

Capsicum species Type Cultivar or PI line Country of origin

C. annuum Cayenne Thai Cluster Thailand

C. annuum Unknown Pico de Pajaro Honduras

C. annuum Unknown MI-2 Sri Lanka

C. annuum Serrano Criollo de Morelos Mexico

C. annuum Jalapeno Jaloro USA

C. annuum Unknown PBC-535 Taiwan

C. annuum Yellow wax Sweet Banana USA

C. annuum Yellow wax Rio Grande Gold USA

C. annuum Serrano Hidalgo USA

C. annuum Cayenne Thai Cluster Thailand

C. annuum var. glabriusculum Chiltepin PI 224405 Mexico

C. annuum var. glabriusculum Chiltepin PI 310488 Mexico

C. annuum var. glabriusculum Chiltepin PI 511887 Mexico

C. annuum var. glabriusculum Chiltepin PI 631135 Guatemala

C. annuum var. glabriusculum Chiltepin PI 632932 Guatemala

C. chacoense Unknown PI 260429 Argentina

C. chacoense Unknown PI 260435 Bolivia

C. chinense Habanero PI 152225 USA

C. frutescens Tabasco PI 368084 Malaysia

C. frutescens Tabasco PI 193470 Ethiopia

C. frutescens Tabasco PI 439502 Costa Rica

C. frutescens Tabasco PI 441650 Brazil

C. frutescens Tabasco PI 593924 Ecuador

C. pubescens Rocoto C00640 Guatemala

C. pubescens Rocoto C00778 Guatemala

C. pubescens Rocoto PI 593639 Guatemala

C. pubescens Rocoto PI 593630 Ecuador

C. pubescens Rocoto Grif 1614 Mexico

Fig. 1. Polyacrylamide gel electrophoresis (5 % acrylamide) of bellpepper endornavirus dsRNA from C. annuum YW (lane 1) andrelated endornavirus dsRNAs infecting various Capsicum species;lane 2, C. frutescens LSU Tabasco; lane 3, C. chinense PI315023; lane, 4, C. chinense PI 159236; lane 5, C. chinense PI273426; lane 6, C. chinense PI315008; lane 7, C. chinense PIC00943; lane 8; C. baccatum PI 260590; lane 9, dsRNAsextracted from Nicotiana benthamiana infected with grapevineleafroll-associated virus 2 (GLRaV-2); lane 10, dsRNA-free C.

annuum. Arrow indicates endorna-like dsRNAs. Sizes of visibleGLRaV-2 dsRNAs are indicated.

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detailed analyses revealed three variable regions in the twopolyproteins (Fig. 2b). Interestingly, this pattern roughlycorresponds to regions of low similarity between ORVand OSV (Moriyama et al., 1999). Considering that thegenomes of these two viral isolates shared all their majorcharacteristics, further discussion is based on the BPEV-KSgenome data (except when otherwise stated).

The estimated 59 UTR of BPEV-KS was 225 nt long (226 ntin the case of BPEV-YW). Although two AUG codons werefound at nucleotides 18–21 and 121–123, in silico analysesshowed that these two codons were probably not utilizedowing to their unfavourable initiation context. Comparedwith the consensus sequence for plants AA(G)CAAUG-GC (Lutcke et al., 1987), the third AUG codon(AGUGAUGGC) of BPEV is in the most favourablecontext for translation initiation and is assumed to bethe starting codon for the translation of a long ORF. The 39

end of BPEV consists of a 57 nt-long UTR, ending in the

unique sequence GGGGAGGGAGGGCCCCCCCCCCCCand lacking a poly(A) tail.

A BLAST search using the BPEV sequence detectedconserved domains of a putative viral methyltransferase(MTR), RNA helicase 1 (Hel-1), UDP-glucose–glycosyl-transferase (UGT) and an RdRp (Fig. 2a). The BPEVgenome organization is unique since it is the onlyendornavirus to contain these four domains (Roossinck,et al., 2011).

The search in the protein family (Pfam) database (Finnet al., 2010) revealed the presence of a highly conservedMTR (pfam01660) domain between amino acids 288 and482 with an expect (E) value of 1.1610210. Similar resultswere obtained in analyses performed with the conserveddomain database (CDD) database (Marchler-Bauer et al.,2007). Multiple-sequence alignment of the putative MTRregion of BPEV and several ssRNA viruses belonging to the

1 880

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Fig. 2. (a) Schematic representation of the genome organization of BPEV with key nucleotide and amino acid references,including the position of the nick in the coding plus strand (+). The box represents the large ORF, whereas lines depict UTRs.MTR, Viral methyltransferase; Hel-1, viral helicase 1; UGT, UDP-glycosyltransferase; RdRp, viral RNA-dependent RNApolymerase. (b) Graphic representation of pairwise comparison of amino acid sequences (every 40 aa) encoded by the largeORFs of BPEV isolates KS and YW. Note the presence of three variable regions between the two polyproteins (denoted as I, IIand III). The pairwise comparison was performed with a FASTA homology and similarity search of protein versus protein withGENETYX. The graph was generated with EXCEL by manually counting the similarity and identity of each 40 amino acid region.

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alpha-like superfamily showed the conserved motifs I–IVof the ‘Sindbis-like’ supergroup (Rozanov et al., 1992). Thenearest relative of this motif is the MTR domain from atobamovirus, Sunn hemp mosaic virus; other relatives arerelated to tymoviruses. The invariant amino acid residuesfor MTR activity, a histidine in motif I, the DXXRArgsignature in motif II and a tyrosine in motif IV, are foundin BPEV.

A putative Hel-1 domain was found between amino acids1342 and 1584 and contained conserved motifs from I toVI (Koonin et al., 1993). A BLAST search of this regionagainst the Pfam database produced significant matcheswith the consensus sequences of the viral superfamily 1helicase (pfam01443; E51.7610210). This type of helicaseis found in many ssRNA viruses (Roossinck, et al., 2011).

The presence of a region with significant similarity (Evalues from 961024 to 5610216) to cellular UDP-glycosyltranferases is located between amino acids 3049and 3362, and is in a similar location to glycosyltranserasedomains found in other endornaviruses (Fig. 3a). TheUGT-like region in the BPEV genome showed greatestsimilarity to motifs A and B, which were reported byHacker et al. (2005) for PEV-1 and are present in someother endornaviruses, hypoviruses and cellular UGTs.While no direct effect of endornaviruses on plants hasbeen reported (except for VFV being associated with thecytoplasmic male sterility; Pfeiffer, 1998), the fungalendornavirus HmEV-1 reduces the virulence of the hostfungus, H. mompa (Ikeda et al., 2003). Osaki et al. (2006)

suggested that the hypovirulence caused by HmEV-1 mightbe due to the UGT, as the cellular sterol UGT gene of thehost fungus, which is essential for pathogenesis in plant-pathogenic fungi, might be repressed by gene silencing.

The RdRp_2 domain encoded by BPEV is located in thecarboxy-terminal region of the ORF and containedconserved motifs I–IV (Poch et al., 1989). It sharessimilarity with the RdRp domains encoded by otherendornaviruses (E values ranging from 8610221 to5610271) and was slightly more related to PEV-1 (43 %identity, 63 % similarity) than to endornaviruses reportedfrom plants (40–42 % identity, 55–57 % similarity)(Supplementary Fig. S1, available in JGV Online). Thenearest non-endornavirus relatives are members of generaAmpelovirus and Closterovirus in the family Closteroviridae.

An additional putative domain of approximately 300 aawas identified by in silico analyses of BPEV-encodedpolyproteins. This domain, located downstream of theMTR, had significant matches with corresponding regionsin other endornaviruses (E values ranged between 561024

in the case of OSV to 1610215 in the case of HmEV-1).The carboxy-terminal part of this putative product wasidentified as a ‘cysteine-rich region’ (CRR), and is acandidate for a protease domain in PEV-1 (Hacker et al.,2005) and in GaBRV-XL (Tuomivirta et al., 2009). Inparticular, the region between amino acids 700 and 777 wasrich in cysteine residues (22 %) and characterized by thepresence of four CXCC motifs. Two of these CXCC motifs(motifs 1 and 3), along with two histidine residues, were

Fig. 3. (a) Multiple alignments of conserved motifs A and B of putative UDP glycotransferases encoded by endornaviruses.Positions of these motifs in each polyprotein are indicated along with numbers of amino acids between motifs A and B. (b)Cysteine-rich region (CRR) present in BPEV and in several approved or putative species of the genus Endornavirus. Note thepresence of several CXCC motifs (white lettering on a black background). Motifs conserved in all examined sequences areindicated by a solid line while those present only in selected viruses are indicated by a dotted line (motifs 2 and 4). Twoconserved histidine residues are also shown (grey background). Asterisks indicate conserved residues in all proteins used forcomparison. Colons indicate conserved substitutions.

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highly conserved in BPEV and several other recognizedand putative species of the genus Endornavirus, while ORVand OSV contained only conserved motifs 1 and 2 (Fig. 3band not shown). Although the importance of this region,shared among endornaviruses, is not yet clear, its conser-vation suggests an important role in the life cycle ofendornaviruses. The large endornavirus-encoded polypro-tein has been assumed to be processed by viral proteinase(s).This region could be a candidate region for a proteasefunction. However, the importance and function of the CRRregion is yet to be experimentally demonstrated.

RT-PCR

RT-PCR products (381 bp) were generated from thefollowing Capsicum species: C. annuum YW, KS, CayenneLong Red Thick and Super Cayenne; C. chinense linesC00943, PI 159236 and PI 315023; and C. frutescensTabasco-LSU and Greenleaf Tabasco. Sequence analysisshowed variation among them. Sequences of multipleclones generated from the same specimen were uniform(99–100 % amino acid identity), whereas amino acidvariation of up to 5 % was found among genotypes ofthe same Capsicum species (e.g. maximum level ofdifferences between clones generated from YW and fromSuper Cayenne pepper). The same level of amino aciddiversity was observed among clones generated from C.chinense and C. frutescens. However, the most significantdifferences in amino acid variation (up to 16 %) wereobserved between YW and C. chinense. The level of identitybetween BPEV and orthologous sequences of the endorna-virus amplified from C. chinense and C. frutescens (84–85 %) was lower than identities between ORV and OSV(94 %) in the same region. This suggests that theendornaviruses infecting these two species could bedistinct, BPEV-related species.

Sequence differences among RT-PCR products generatedfrom different Capsicum species mirrors the taxonomicrelationships of the hosts. Phylogenetic analysis using theRdRp region of the various endornavirus sequences (Fig. 4)shows a tree that is congruent with what is known aboutrelationships in the Capsicum group, based on isozymedata (McLeod et al., 1982). These closely related speciesshare a mutual ancestral gene pool (Moscone et al., 2007).In the case of C. frutescens and C. chinense, some haveargued that they should be combined into one speciesowing to their minimal differences and easy interbreeding(Pickersgill, 1966, 1971). This supports our suggestion ofhost–virus relationship and that BPEV was present in thecommon ancestor of these three Capsicum species and co-evolved with its hosts. Nevertheless, the origin of BPEV inpepper remains unknown and could be elucidated bytesting more domesticated genotypes and wild Capsicumspecies. Not all lines of C. frutescens and C. chinense showedevidence of an endornavirus, and many C. annuumcultivars also lack the virus (Table 2). Assuming that thecommon ancestor of these Capsicum species was infected

with BPEV, these results suggest a rather inefficient verticaltransmission of the virus in certain genotypes. Curiously,two lines of the most primitive Capsicum species (C.chacoense), five of the non-cultivated C. annuum var.glabriusculum (Chiltepin pepper) and five of C. pubescens(Rocoto) were free of BPEV. The nucleotide-sequencediversity found among various isolates of BPEV fromdifferent Capsicum species may reflect how long this virushas been associated with these hosts.

Detection and characterization of the nick in thesequence of BPEV-KS

Purified BPEV-KS was subjected to electrophoresis on a0.8 % denaturing agarose gel, blotted onto a membraneand hybridized using three cDNA probes located betweennucleotides 805 and 1135 (Probe 1, Cads577), nucleotides906 and 1528 (Probe 2, Cads532) and nucleotides 1251 and1704 (Probe 3, Cads759-59) from the 59-end of the plusstrand of the dsRNA. These probes detected both (plus andminus) strands, which were separated by denaturingagarose gel electrophoresis. Approximately 14 and 15 kbRNA fragments were detected with all probes (Fig. 5). Afragment of approximately 1 kb was detected with probe 1but not with probe 3. With probe 2, the 1 kb fragment wasfaintly detected. These results indicate that BPEV-KS has anick in the plus strand between nucleotides 800 and 1100.Eighteen clones were obtained (by using 59 RACE) todetermine the position of the nick on the plus strand(Supplementary Fig. S2, available in JGV Online). Threeindependently obtained clones indicated a nick in the plusstrand at nucleotide 880. Of the remaining clones, nickswere detected at nucleotides 931, 937, 940, 941, 946, 954 or962, 1183, 1185 and 1187 or 1199. These results suggestthat BPEV-KS has a nick in the plus strand at nucleotide880, but it is likely that this virus has several nicks in the

Fig. 4. Bayesian phylogenetic analysis of the RdRp regions ofputative endornaviruses isolated from several Capsicum spp. andcultivars. Analysis is as described in the text. Relative branchlengths are shown above the lines, support for branches is shownat the nodes.

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plus strand, as reported for PEV-1 and OSV (Hacker et al.,2005; Fukuhara et al., 1995).

The biological properties, unique genomic organizationand phylogeny indicate that the dsRNAs isolated from thebell pepper cultivars KS and YW represent two closelyrelated isolates of a distinct and novel species of the genusEndornavirus (family Endornaviridae), for which the nameBell pepper endornavirus (BPEV) is proposed.

METHODS

Plant materials. Bell pepper plants Kyousuzu (KS), Kyounami

(KN), Kyoumidori and Ace were from TAKII Seed Company;

Suigyokunigou from Sakata Seed; High Green from Tohoku Seed;

and Jumbo Colour Bell Pepper from Kaneko Seeds. Seed of KS, KN

and bell pepper California Wonder (CW) were purchased from

commercial stores in Japan. Bell peppers Red Bell, Bonnie’s Green

Bell, Pimiento Sweet and Chocolate Beauty Sweet were purchased as

young plants from commercial garden stores in Ardmore, Oklahoma,

USA, and Marengo (MR) seeds were from Asgrow Seed. The source of

Yolo Wonder (YW) and all other C. annuum genotypes and Capsicum

species used in this investigation were reported in previous

publications (Valverde et al., 1990b; Valverde & Fontenot, 1991) or

were obtained from L. L. Black (Louisiana State University, Louisiana,

USA). Plant introductions (PI) and accessions that included

selections from different geographical locations of C. annuum, C.

baccatum, C. chacoense, C. chinense, C. frutescens and C. pubescens

were provided by R. L. Jarret (USDA-ARS, Griffin, Georgia, USA) and

L. M. Engle (AVRDC, Taiwan). Seed used in some experiments was

increased by self-pollination. All plants and seedlings were grown in a

greenhouse with day/night temperatures of about 25/18 uC, respect-

ively, and 16 h days.

Extraction, purification and analysis of dsRNA from Capsicum

spp. Leaf tissues were pulverized in a mortar and pestle after being

frozen in liquid nitrogen, and total nucleic acids were extracted by

using 26 STE buffer (200 mM NaCl, 20 mM Tris/HCl and 2 mM

EDTA, pH 8.0). Total nucleic acids were further purified with phenol

and subjected to agarose gel electrophoresis. Alternatively, dsRNA was

fractionated from total nucleic acid extracts by column chromato-

graphy on CF-11 cellulose (Whatman), as described by Morris &

Dodds (1979) or as modified by Valverde et al. (1990a), Marquez et al.

(2007) and Roossinck et al. (2010). The dsRNAs from all other C.

annuum genotypes and Capsicum species were extracted from 3.5 g of

leaf tissue and analysed on 5 % polyacrylamide gels. For each

Capsicum genotype and Capsicum species, foliar samples from at least

three plants were tested. In the case of KS pepper, dsRNA was also

extracted from pollen, callus and cultured cells. The nature of the

purified nucleic acids was ascertained by digestion with RNase-free

DNase I (Promega), followed by selective treatments with DNase-free

RNase A in high (26 SSC) and low (0.26 SSC) salt conditions (206SSC is 3 M sodium chloride, 0.3 M sodium citrate). Preparations

were further treated with Proteinase K (Sigma) followed by phenol/

chloroform purification and ethanol precipitation.

Occurrence of BPEV in bell pepper seedlings. Seeds from KS,

KN, CW, YW and MR bell peppers were planted and 8-week-old

plants screened for BPEV by RT-PCR or by electrophoretic analyses

of dsRNA. A total of 49, 28, 36, 50 and 137 plants of KS, KN, CW, YW

and MR, respectively, were screened. A single MR plant was found

free of BPEV and was self-pollinated in order to generate a virus-free

line for further use in graft-transmission experiments. Seed of CW,

YW and MR originated from self-pollinated BPEV-infected plants

while seed of KS and KN were purchased from commercial stores, and

thus with no knowledge of the parental status in regard to BPEV

infections.

Attempts to transmit BPEV. BPEV-infected MR was used as

rootstock and the BPEV-free line as the scion in graft inoculation

experiments. A total of eight rootstocks were used. Four were infected

with PVY, which was mechanically inoculated to the plants when they

were 6 weeks old. One month after the PVY inoculation, the stems of

all plants were cut and scions from the BPEV-free line graft were

inoculated (Supplementary Fig. S3, available in JGV Online). Six

weeks after grafting, foliar samples were taken and tested for BPEV by

electrophoresis and RT-PCR. Scions grafted on PVY inoculated plants

were tested for PVY by ELISA.

Cloning, sequencing and sequence analyses. BPEV dsRNA from

KS (BPEV-KS) and YW (BPEV-YW) were gel purified, heat

denatured and reverse transcribed with random hexadeoxynucleotide

primers (TaKaRa). Generated cDNA fragments were cloned into

pUC109 (TaKaRa) or pGEM-T Easy plasmid (Promega) and the

resulting recombinant plasmids were transferred into Escherichia coli

DH5a competent cells. DNAs from selected colonies were sequenced

by automated sequence analysis using a capillary sequencer, 3130xl

Genetic Analyzer (Applied Biosystems). After computer-assisted

analysis of the initial sequence data, specific primers were designed

in order to fill the gaps between adjacent clones by RT-PCR. Each

nucleotide in both genomes (BPEV-KS and -YW) was sequenced

from multiple clones from independent experiments in order to

ensure at least fivefold coverage.

For determination of the sequence of the 39 end of BPEV-KS, first-

strand cDNAs were synthesized by using 59-end-phosphorylated RT

primer and single-stranded cDNAs were circularized or concatamers

were formed at 16 h and at 15 uC by using RNA Ligase (TaKaRa).

Fig. 5. Detection of the nick in the coding strand of BPEV-KS byNorthern-blot analysis. BPEV-KS dsRNA was resolved byelectrophoresis in a denaturing 0.8 % agarose gel, transferred toa membrane and probed with probes Cads577 (corresponding tonucleotides 805—1135), Cads532 (corresponding to nucleotides906–1528) and Cads759-59 (corresponding to nt 1251–1704).While two bands of 14 and 15 kb were detected with all threeprobes, a band of 1 kb was clearly detected only when probedwith probe Cads577.

An endornavirus from bell pepper

http://vir.sgmjournals.org 2671

Circular cDNAs or concatamers were amplified by PCR. The 39 viral

end of BPEV-YW was determined by the method described by

Lambden et al. (1992), involving the ligation of a 59-phosphorylated/

39-amino-blocked oligonucleotide (primer 1) to the target dsRNAs,

followed by cDNA synthesis primed by a complementary oligonu-

cleotide (primer 2) and amplification of the 39 end with a virus-

specific primer and primer 2. For determination of the sequence of

the 59 end of BPEV-KS, cDNAs were amplified by PCR after adding a

homopolymeric tail at the 39-end of the single-stranded cDNAs by

terminal deoxynucleotidyltransferase (TaKaRa). Amplification pro-

ducts were cloned and sequenced by using M13 RV-N (59-

TGTGGAATTGTGAGCGG-39) and M13 reverse primers (TaKaRa).

For MR pepper samples, dsRNA was isolated and converted to cDNA,

followed by multiplexing and sequence analysis on a 454/GS FLX

system (Roche), as described previously (Roossinck et al., 2010).

Amino acid sequences were assembled and analysed using GENETYX

version 9 (GENETYX) and/or LASERGENE (DNASTAR). Multiple se-

quence alignments were conducted by using CLUSTAL W (Thompson

et al., 1994), and alignments were edited manually using MESQUITE

(version 2.74) (Maddison & Maddison, 2010). Phylogenetic analyses

were done using MRBAYES (Huelsenbeck & Ronquist, 2001),

implemented via a plug-in for GENEIOUS. VFV (GenBank accession# CAA04392) was used as an out-group. Rate matrix was set to a

Poisson distribution with a gamma rate variation. Burn-in was

100 000 and total chain length was 1 100 000. Branch lengths were

unconstrained. PhyML (Guindon & Gascuel, 2003) and PAUP* version

4.0 beta 4b10 (Swofford, 2002) were used to confirm the tree topology

(not shown).

Molecular hybridization. For Northern blot analysis, 100 ng of

BPEV-KS dsRNA was separated on a 0.8 % agarose MOPS gel with

6 % formaldehyde and transferred to a nylon Zeta-Probe membrane

(Bio-Rad) by capillary blotting (Moriyama et al., 1999). After UV

cross-linking and prehybridization in hybrisolution (250 mM phos-

phate buffer, pH 7.2, 1 mM EDTA, 7 % SDS, 1 % BSA), blots were

hybridized for 16 h at 65 uC in the same solution with 32P-labelled

DNA-probes specific for BPEV-clones Cads577, Cads532 and

Cads759-59. Probes were synthesized with a random prime labelling

system, BcaBEST Labeling kit (TaKaRa). Membranes were washed

twice at 65 uC with 40 mM phosphate buffer containing 5 % SDS for

1 h and twice at 65 uC with 40 mM phosphate buffer, pH 7.2,

containing 1 % SDS for 1 h. Hybridization signals were detected with

a BAS 1500 system (Fujifilm). Images taken by the BAS 1500 system

were prepared with IMAGE GAUGE (Fujifilm).

RT-PCR. To confirm the presence of BPEV in C. annuum genotypes

and to investigate its occurrence in three other Capsicum species,

selected genotypes and species that yielded BPEV-like dsRNAs by gel

electrophoresis were subjected to RT-PCR using a pair of degenerate

primers: Endor-F (59-AAGSGAGAATWATHGTRTGGCA-39) and

Endor-R (59-CTAGWGCKGTBGTAGCTTGWCC-39), which were

designed to amplify a 381 nt fragment of the RdRp of plant

endornaviruses.

Aliquots of dsRNAs were heat denatured at 95 uC for 5 min, cooled

rapidly on ice and used for RT-PCR detection. RT-PCR was

performed using an Access one-tube, two-enzyme system RT-PCR

System (Promega), following the manufacturer instructions. The

following PCR conditions were applied for BPEV detection: (i) initial

denaturation at 94 uC for 2 min, (ii) denaturation for 1 min at 94 uC,

annealing for 45 s at 50 uC, extension for 45 s at 70 uC (40 cycles) and

(iii) final extension for 5 min at 70 uC. PCR products were cloned by

using a pGEM-T Easy Vector System. For each dsRNA sample, three

recombinant plasmids were sequenced and the amino acid sequences

derived were compared with sequences in the GenBank database by

using BLAST.

ACKNOWLEDGEMENTS

We wish to thank R. Jarret (USDA/ARS, Griffin, Georgia, USA)for providing seeds of Capsicum species; Guoan Shen (NobleFoundation) and Dina Gutierrez (Louisiana State University, USA)for helping with some experiments; Dr Tomohide Natsuaki(Utsunomiya University) for helpful comments; Estacion Ex-perimental ‘La Mayora’, CSIC, Spain for providing facilities for someexperiments. Approved for publication as journal article no. 12047 ofthe Mississippi Agricultural and Forestry Experiment Station, MSU.

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