Arch Virol DOI 10.1007/s00705-013-1893-2
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a novel endornavirus in the wheat sharp 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 Ó Springer-Verlag Wien 2013
Abstract We report here the presence of a novel doublestranded 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 contains 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 filamentous fungi, with a large number of its members being
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: [email protected] 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
soil-borne plant pathogens. Based on the number of nuclei in the young cell, these fungi can be divided into multinucleate, binucleate and uninucleate Rhizoctonia. The binucleate species Rhizoctonia cerealis Van der Hoeven (=Ceratobasidium cereale Murray & Burpee, Basidiomycota) 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 discernible phenotypic changes [11]. Double-stranded RNA (dsRNA) viruses are commonly detected in natural populations 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 endornaviruses 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 polymerase (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 endornaviruses, such as Phaseolus vulgaris endornavirus 2 [13], containing the characteristic motifs for the MTR, Hel1, and RdRp.
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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
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,
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
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
BPEV
HmEV1
Isolate
Host
Sequence identity*
GenBank**
Vmet %
Hel1%
RdRp %
Overall (aa) %
Nucleotide
Length (bp)
Protein
Length (aa)
BPEVYW
Capsicum annuum
33.04
25.54
48.28
17.21
JN019858
14728
AEK22062
4815
BPEV-2 HmEV1670
C. annuum Helicobasidium mompa
33.93 *
25.54 *
47.78 58.46
17.13 14.06
AB597230 NC_013447
14727 16614
BAK52155 YP_003280846
4815 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 VfEV-2
Vicia faba V. faba
* *
31.8 31.8
53.85 53.85
14.82 14.8
NC_007648 AJ000929
17635 17635
YP_438201 CAA04392
5825 5825
CeEV1
CeEV1
Thielaviopsis basicola
*
27.66
41.22
14.3
GQ494150
11602
ADN43901
3858
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
GaBRVXL1
GaBRVXL1
Gremmeniella abietina
22.32
14.78
28.43
12.93
DQ399289
10375
ABD73305
3429
GaBRVXL2
GaBRVXL2
G. abietina
22.32
14.78
33.58
12.96
DQ399290
10374
ABD73306
3429
GEEV
PaEV TaEV
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-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
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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 oligonucleotide-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 corresponding 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 sequencing at least three independent overlapping clones. The sequence of the virus genome was assembled and analysed using the DNASTAR software package (Madison, Wisconsin, 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 (residues 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 residues 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
Fig. 2 Phylogenetic tree based on the amino acid sequences of putative RdRp regions of the endornaviruses using the neighborjoining method with 1,000 bootstrap replicates. The sequences of two chrysoviruses, PcV (Penicillium chrysogenum virus, GenBank accession 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
grapevine endophyte endornavirus (GEEV) [4]. A comparison between the genomes of all available endornaviruses 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 classified 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 relationships of the host [16], while in this study, which was performed with representatives of a larger number of species, 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 endornavirus in R. cerealis and the first known full-length
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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 Agriculture 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 analysis 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 approaches 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. Gonza´lez Garcı´a 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 endornaviruses and contains a putative UDP glycosyltransferase gene. J Gen Virol 86:1561–1570 7. Jian J, Lakshman DK, Tavantzis SM (1997) Association of distinct double-stranded RNAs with enhanced or diminished virulence 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
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9. Morris TJ, Dodds JA (1979) Isolation and analysis of double stranded RNA from virus-infected plant and fungal tissue. Phytopathology 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 coinfecting 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 parsimony 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
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a novel endornavirus in the wheat sharp 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 Ó Springer-Verlag Wien 2013
Abstract We report here the presence of a novel doublestranded 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 contains 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 filamentous fungi, with a large number of its members being
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: [email protected] 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
soil-borne plant pathogens. Based on the number of nuclei in the young cell, these fungi can be divided into multinucleate, binucleate and uninucleate Rhizoctonia. The binucleate species Rhizoctonia cerealis Van der Hoeven (=Ceratobasidium cereale Murray & Burpee, Basidiomycota) 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 discernible phenotypic changes [11]. Double-stranded RNA (dsRNA) viruses are commonly detected in natural populations 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 endornaviruses 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 polymerase (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 endornaviruses, such as Phaseolus vulgaris endornavirus 2 [13], containing the characteristic motifs for the MTR, Hel1, and RdRp.
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W. Li et al.
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
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,
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
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
BPEV
HmEV1
Isolate
Host
Sequence identity*
GenBank**
Vmet %
Hel1%
RdRp %
Overall (aa) %
Nucleotide
Length (bp)
Protein
Length (aa)
BPEVYW
Capsicum annuum
33.04
25.54
48.28
17.21
JN019858
14728
AEK22062
4815
BPEV-2 HmEV1670
C. annuum Helicobasidium mompa
33.93 *
25.54 *
47.78 58.46
17.13 14.06
AB597230 NC_013447
14727 16614
BAK52155 YP_003280846
4815 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 VfEV-2
Vicia faba V. faba
* *
31.8 31.8
53.85 53.85
14.82 14.8
NC_007648 AJ000929
17635 17635
YP_438201 CAA04392
5825 5825
CeEV1
CeEV1
Thielaviopsis basicola
*
27.66
41.22
14.3
GQ494150
11602
ADN43901
3858
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
GaBRVXL1
GaBRVXL1
Gremmeniella abietina
22.32
14.78
28.43
12.93
DQ399289
10375
ABD73305
3429
GaBRVXL2
GaBRVXL2
G. abietina
22.32
14.78
33.58
12.96
DQ399290
10374
ABD73306
3429
GEEV
PaEV TaEV
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-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
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Novel endornavirus from Rhizoctonia cerealis
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 oligonucleotide-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 corresponding 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 sequencing at least three independent overlapping clones. The sequence of the virus genome was assembled and analysed using the DNASTAR software package (Madison, Wisconsin, 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 (residues 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 residues 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
Fig. 2 Phylogenetic tree based on the amino acid sequences of putative RdRp regions of the endornaviruses using the neighborjoining method with 1,000 bootstrap replicates. The sequences of two chrysoviruses, PcV (Penicillium chrysogenum virus, GenBank accession 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
grapevine endophyte endornavirus (GEEV) [4]. A comparison between the genomes of all available endornaviruses 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 classified 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 relationships of the host [16], while in this study, which was performed with representatives of a larger number of species, 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 endornavirus in R. cerealis and the first known full-length
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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 Agriculture 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.
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