ANNOTATED SEQUENCE RECORD

Complete genome sequence of a novel endornavirus in the wheatsharp eyespot pathogen Rhizoctonia cerealis

Wei Li • Tao Zhang • Haiyan Sun • Yuanyu Deng •

Aixiang Zhang • Huaigu Chen • Kerong Wang

Received: 6 August 2013 / Accepted: 9 October 2013

� Springer-Verlag Wien 2013

Abstract We report here the presence of a novel double-

stranded RNA (dsRNA) virus in an isolate (R0959) of the

fungus Rhizoctonia cerealis, the causal agent of sharp

eyespot of wheat in China. Sequence analysis showed that

the dsRNA segment is 17,486 bp long and contains a single

open reading frame (ORF) with the potential to encode a

protein of 5,747 amino acids. The predicted protein con-

tains conserved motifs of putative viral methyltransferase,

helicase 1, and RNA-dependent RNA polymerase.

Sequence similarity and phylogenetic analysis clearly place

it in a distinct species within the genus Endornavirus,

family Endornaviridae, and therefore we propose its name

to Rhizoctonia cerealis endornavirus 1 (RcEV1). This is

the first report of the full-length genomic sequence of a

dsRNA mycovirus in R. cerealis.

Introduction

The genus Rhizoctonia includes a complex group of fila-

mentous fungi, with a large number of its members being

soil-borne plant pathogens. Based on the number of nuclei

in the young cell, these fungi can be divided into multi-

nucleate, binucleate and uninucleate Rhizoctonia. The

binucleate species Rhizoctonia cerealis Van der Hoeven

(=Ceratobasidium cereale Murray & Burpee, Basidiomy-

cota) belongs to the Rhizoctonia AG-D anastomosis group

and is the causal pathogen of sharp eyespot in wheat in

China [2, 5]. Mycoviruses are widespread throughout the

major fungal groups, usually without causing any dis-

cernible phenotypic changes [11]. Double-stranded RNA

(dsRNA) viruses are commonly detected in natural popu-

lations of multinucleate R. solani isolates AG-1 to -13 [1,

21], and some of these dsRNA suppress the virulence of

their host [7]. However, it is still not clear whether dsRNA

mycoviruses are also present in binucleate R. cerealis.

In this study, we report the complete genomic sequence

of a virus from R. cerealis belonging to a putatively novel

species in the genus Endornavirus from. Accordingly, we

propose the name R. cerealis endornavirus 1 (RcEV1). The

endornaviruses are large dsRNA viruses that are found in

plants, fungi and oomycetes [16]. The genomes of endor-

naviruses consist of linear dsRNA with a characteristic

single ORF of up to 18 kbp in length, often with a nick in

the plus strand at the 5’ end [4, 6, 10, 12–15, 17, 19, 20].

The viral ORF often contains four similar domains, the

viral methyltransferase (MTR), viral RNA helicase (Hel),

glycosyltransferase (GT) and RNA-dependent RNA poly-

merase (RdRp) domains. However, only the RdRp is

clearly homologous among all species, and the other

domains are found in some, but not all members of this

family [16]. The RcEV1 genome structure is highly similar

to that of other fungus-infecting and plant-infecting en-

dornaviruses, such as Phaseolus vulgaris endornavirus 2

[13], containing the characteristic motifs for the MTR, Hel-

1, and RdRp.

W. Li, T. Zhang contributed equally to this work.

W. Li � K. Wang (&)

Department of Plant Pathology, College of Plant Protection,

Nanjing Agricultural University, Nanjing, Jiangsu 210095,

People’s Republic of China

e-mail: wkr01@njau.edu.cn

W. Li � T. Zhang � H. Sun � Y. Deng � A. Zhang � H. Chen

Institute of Plant Protection, Jiangsu Academy of Agricultural

Sciences, Nanjing, Jiangsu 210014, People’s Republic of China

T. Zhang

College of Life Sciences, Nanjing Agricultural University,

Nanjing, Jiangsu 210095, People’s Republic of China

123

Arch Virol

DOI 10.1007/s00705-013-1893-2

Provenance of the virus material

The virus from R. cerealis strain R0959 was isolated from

wheat sheath with sharp eyespot symptom in Anhui

Province, China, in 2009. The viral dsRNAs of the fungus

were extracted by the cellulose chromatography method

[9]. After purification, the dsRNAs were treated with

DNase I and S1 nuclease to eliminate contaminating DNA

and single-stranded RNA. Complementary DNAs were

synthesized using purified dsRNA as a template and

amplified using the tagged random primer dN6 (5’-CCT

GAA TTC GGA TCC TCC NNN NNN-3’) [3]. The

amplified cDNA products were ligated with pMD18-T

vector (Takara) and introduced by transformation into

Fig. 1 The genome organization of RcEV1. The long rectangular box

represents the open reading frame (ORF). The smaller boxes indicate

the positions of the viral methyltransferase (MTR), helicase 1 (Hel-1),

and RNA-dependent RNA polymerase (RdRp) conserved domains,

respectively. Amino acid numbers in the protein are given above the

box, and the corresponding nucleotides in the genome are below the

box

Table 1 Percent amino acid sequence identity of RcEV1 domains to those of other endornaviruses

Virus Isolate Host Sequence identity* GenBank**

Vmet % Hel-

1 %

RdRp % Overall

(aa) %

Nucleotide Length

(bp)

Protein Length

(aa)

BPEV BPEV-

YW

Capsicum

annuum

33.04 25.54 48.28 17.21 JN019858 14728 AEK22062 4815

BPEV-2 C. annuum 33.93 25.54 47.78 17.13 AB597230 14727 BAK52155 4815

HmEV1 HmEV1-

670

Helicobasidium

mompa

* * 58.46 14.06 NC_013447 16614 YP_003280846 5373

OrEV OrEV Oryza rufipogon * * 50.38 15.51 NC_007649 13936 YP_438202 4627

OsEV OsEV O. sativa * * 51.54 15.91 D32136 13952 BAA06862 4572

PvEV1 PvEV1 Phaseolus

vulgaris

* 24.03 51.91 14.22 AB719397 13908 BAM68539 4496

PvEV2 PvEV2 P. vulgaris 35.64 26.62 49.75 16.64 AB719398 14820 BAM68540 4851

PEV1 PEV1 Phytophthora sp. * 28.94 48.09 15.55 NC_007069 13883 YP_241110 4612

VfEV VfEV-1 Vicia faba * 31.8 53.85 14.82 NC_007648 17635 YP_438201 5825

VfEV-2 V. faba * 31.8 53.85 14.8 AJ000929 17635 CAA04392 5825

CeEV1 CeEV1 Thielaviopsis

basicola

* 27.66 41.22 14.3 GQ494150 11602 ADN43901 3858

GEEV GEEV-1 Chalara elegans * 32.34 41.22 15.63 NC_019493 12154 YP_007003829 4027

GEEV-2 C. elegans * 32.34 41.22 15.63 JX678977 12154 AFV91541 4027

GaBRV-

XL1

GaBRV-

XL1

Gremmeniella

abietina

22.32 14.78 28.43 12.93 DQ399289 10375 ABD73305 3429

GaBRV-

XL2

GaBRV-

XL2

G. abietina 22.32 14.78 33.58 12.96 DQ399290 10374 ABD73306 3429

PaEV PaEV-1 Persea americana * * 47.73 15.38 NC_016648 13459 YP_005086952 4393

PaEV-2 P. americana * * 47.73 15.38 JN880414 13459 AEX28369 4393

TaEV TaEV-1 Tuber aestivum 28.41 * 35.61 13.64 NC_014904 9760 YP_004123950 3217

TaEV-2 T. aestivum 28.41 * 35.61 13.64 HQ380014 9760 ADU64759 3217

*,* indicates that this domain was not present in the virus isolate

** The accession no. of the nucleotide or protein sequence in GenBank and the length of the viral genomic RNA (bp) and polyprtein (aa).

Available genomic sequence and polyproteins are limited for CeEV1, as no complete CeEV1 genome sequence is available yet

W. Li et al.

123

Escherichia coli DH5a for sequencing. Based on the

sequences obtained, dsRNA-specific primers were

designed and used for RT-PCR.

In order to clone the termini of the dsRNAs, cDNA

amplification of the 5’ and 3’ ends was performed using the

RNA-ligase-mediated rapid amplification of cDNA ends

(RLM-RACE) method [8]. The 3’ terminus of each strand

of dsRNA was ligated with the 5’-end phosphorylated

oligonucleotide PR1 (5’-GCA TTG CAT CAT GAT CGA

TCG AAT TCT TTA GTG AGG GTT AAT TGC

C-(NH2)-3’) using T4 RNA ligase (TaKaRa). The oligo-

nucleotide-ligated dsRNA was denatured and used for the

RT reaction with a primer PR2 (5’-GGC AAT TAA CCC

TCA CTA AAG-3’). The cDNA was amplified with primer

PR3 (5’-TCA CTA AAG AAT TCG ATC GAT C-3’), a

nested PCR primer PR4 (5’-CGA TCG ATC ATG ATG

CAA TGC-3’), and a sequence-specific primer corre-

sponding to the 5’- and 3’- terminal sequences of the

dsRNA, respectively. The expected PCR products were

sequenced according to the method described above. In

both orientations, every base was determined by sequenc-

ing at least three independent overlapping clones. The

sequence of the virus genome was assembled and analysed

using the DNASTAR software package (Madison, Wis-

consin, USA).

The complete RcEV1 genome sequence has been

deposited in the GenBank database under the accession no.

KF311065. The amino acid sequence of the putative RdRp

gene was aligned with other viral RdRp amino acid

sequences using ClustalX2 and the EMBL-EBI MUSCLE

server (http://www.ebi.ac.uk/Tools/msa/muscle/). The

phylogenetic tree was inferred using MEGA 5.1 [18] with

1000 replicates of the neighbor-joining (NJ) procedure with

Poisson correction as the model.

Sequence properties

The entire genome of RcEV1 was 17,486 bp in length, with

a G?C content of 43.2 %. A 16-nucleotide (nt) 5’

untranslated region (UTR) is followed by the putative single

large ORF (17,246 bp), ending at nt position 17,260 and

coding for a 649.1-kDa protein (5747 aa). The 3’ UTR was

found to be 226 bp in length (Fig. 1). RcEV1 has the second

longest genome, after Vicia faba endornavirus (VfEV)

(17,638 bp), in the family Endornavirus [15], and until now

we have not found any nick in this genome. A MTR region

(cl03298) of the protein was found to be 112 aa long (res-

idues 830–941), and this aa sequence region of the protein

shares the highest degree of identity (35.64 %) with

Phaseolus vulgaris endornavirus 2 (PvEV2) [13]. At resi-

dues 1962-2200, we identified a 239-aa-long Hel-1 region

(pfam01443), and this aa sequence region of the protein

shares the highest degree of identity (32.34 %) with

grapevine endophyte endornavirus (GEEV) [4]. A com-

parison between the genomes of all available endornavi-

ruses infecting fungi, oomycetes and plants revealed that

only the common RdRp (cl03049) motif is shared among

members of all known taxa. The RdRp portion of the

RcEV1 protein is located at aa sequence position

5423-5625, sharing 58.46 % sequence identity with the

RdRp of Helicobasidium mompa endornavirus 1 (HmEV1)

[14]. A comparison of all protein-coding sequence regions

of RcEV1 with those of members of other endornavirus taxa

is shown in Table 1. The phylogenetic tree based on the

RdRp aa sequences suggests that RcEV1 should be classi-

fied as a member of a new distinct species within the genus

Endornavirus (Fig. 2). Previous studies have shown that the

topology of the phylogenetic tree does not follow the rela-

tionships of the host [16], while in this study, which was

performed with representatives of a larger number of spe-

cies, the phylogenetic tree showed that the viruses from the

same host (fungi or plants) have a tendency to cluster

together (Fig. 2), with some occasional exceptions. Further

sequence analysis of more endornavirus genomes may

provide more evidence and elucidate the evolution of this

interesting virus family.

In conclusion, we report the presence of a novel end-

ornavirus in R. cerealis and the first known full-length

Fig. 2 Phylogenetic tree based on the amino acid sequences of

putative RdRp regions of the endornaviruses using the neighbor-

joining method with 1,000 bootstrap replicates. The sequences of two

chrysoviruses, PcV (Penicillium chrysogenum virus, GenBank acces-

sion no. AAM95601) and HvV145S (Helminthosporium victoriae

virus 145S, GenBank accession no. YP-052858) were used as the

outgroup. The scale bar corresponds to a genetic distance of 0.1

amino acid substitutions per site

Novel endornavirus from Rhizoctonia cerealis

123

genome sequence of this virus. This dsRNA virus is

common among R. cereals isolates; however, it does not

appear to cause obvious disease symptoms. Further studies

are needed to determine the function and evolutionary

origin of this new dsRNA virus.

Acknowledgments This work was supported by Jiangsu Agricul-

ture Science and Technology Innovation Fund, CX(11)4015, National

Science Foundation of China (30900928), and the fund earmarked for

the China Agricultural Research System (CARS-3-1-17).

Conflict of interest The authors declare no conflict of interest.

References

1. Bharathan N, Saso H, Gudipati L, Bharathan S, Whited K,

Anthony K (2005) Double-stranded RNA distribution and ana-

lysis among isolates of Rhizoctonia solani AG-2 to -13. Plant

Path 54:196–203

2. Chen Y, Li W, Zhang XX, Zhang BQ, Yu HS, Chen HG (2009)

Composition and virulence of pathogen of wheat sharp eyespot in

north latitude 33 of China (in Chinese). J Triticeae Crops

29:1110–1114

3. Darissa O, Willingmann P, Adam G (2010) Optimized approa-

ches for the sequence determination of double-stranded RNA

templates. J Virol Methods 169:397–403

4. Espach Y, Maree HJ, Burger JT (2012) Complete genome of a

novel endornavirus assembled from next-generation sequence

data. J Virol 86:13142

5. Gonzalez Garcıa V, Portal Onco MA, Rubio Susan V (2006)

Review: Biology and systematics of the form genus Rhizoctonia.

Span J Agric Res 4:55–79

6. Hacker CV, Brasier CM, Buck KW (2005) A double-stranded

RNA from a Phytophthora species is related to the plant endor-

naviruses and contains a putative UDP glycosyltransferase gene.

J Gen Virol 86:1561–1570

7. Jian J, Lakshman DK, Tavantzis SM (1997) Association of dis-

tinct double-stranded RNAs with enhanced or diminished viru-

lence in Rhizoctonia solani infecting potato. Mol Plant-Microbe

Interact 10:1002–1009

8. Liu H, Fu Y, Jiang D, Li G, Xie J, Peng Y, Yi X, Ghabrial SA

(2009) A novel mycovirus that is related to the human pathogen

Hepatitis E virus and rubi-like viruses. J Virol 83:1981–1991

9. Morris TJ, Dodds JA (1979) Isolation and analysis of double

stranded RNA from virus-infected plant and fungal tissue. Phy-

topathology 69:854–858

10. Moriyama H, Horiuchi H, Ntta T, Fukuhara T (1999) Unusual

inheritance of evolutionarily-related double-stranded RNAs in

interspecific hybrid between rice plants Oryza sativa and Oryza

rufipogon. Plant Mol Biol 39:1127–1136

11. Nuss DL (2005) Hypovirulence: mycoviruses at the fungal–plant

interface. Nat Rev Microbiol 3:632–642

12. Okada R, Kiyota E, Sabanadzovic S, Moriyama H, Fukuhara T,

Saha P, Roossinck MJ, Severin A, Valverde RA (2011) Bell

pepper endornavirus: molecular and biological properties, and

occurrence in the genus Capsicum. J Gen Virol 92:2664–2673

13. Okada R, Yong CK, Valverde RA, Sabanadzovic S, Aoki N,

Hotate S, Kiyota E, Moriyama H, Fukuhara T (2013) Molecular

characterization of two evolutionarily distinct endornaviruses co-

infecting common bean (Phaseolus vulgaris). J Gen Virol

94:220–229

14. Osaki H, Nakamura H, Sasaki A, Matsumoto N, Yoshida K

(2006) An endornavirus from a hypovirulent strain of the violet

root rot fungus, Helicobasidium mompa. Virus Res 118:143–149

15. Pfeiffer P (1998) Nucleotide sequence, genetic organization and

expression strategy of the double-stranded RNA associated with

the ‘447’ cytoplasmic male sterility trait in Vicia faba. J Gen

Virol 79:2349–2358

16. Roossinck MJ, Sabanadzovic S, Okada R, Valverde RA (2011)

The remarkable evolutionary history of endornaviruses. J Gen

Virol 92:2674–2678

17. Stielow B, Klenk HP, 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

18. 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 method. Mol Biol Evol 28:2731–2739

19. Tuomivirta TT, 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

20. Villanueva F, Sabanadzovic S, Valverde RA, Navas-Castillo J

(2012) Complete genome sequence of a double-stranded RNA

virus from avocado. J Virol 86:1282–1283

21. Zheng L, Liu H, Zhang M, Cao X, Zhou E (2013) The complete

genomic sequence of a novel mycovirus from Rhizoctonia solani

AG-1 IA strain B275. Arch Virol 158:1609–1612

W. Li et al.

123