Complete Nucleotide Sequence of a Partitivirus from Rhizoctonia solani AG-1 IA Strain C24

The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 (1787 nucleotides). DsRNA-1 has an open reading frame (ORF) 1 that potentially codes for a protein of 584 amino acid (aa) containing the conserved motifs of a RNA-dependent RNA polymerase (RdRp), and dsRNA-2 also contains a ORF 2, encoding a putative capsid protein (CP) of 513 aa. Phylogenetic analysis revealed that RsPV5 clustered together with six other viruses in an independent clade of the genus Alphapartitivirus, indicating that RsPV5 was a new member of the genus Alphapartitivirus, within the family Partitiviridae.

Rhizoctonia solani Kühn [teleomorph: Thanatephorus cucumeris (Frank) Donk] is an economically important soil-borne fungal pathogen that causes severe plant diseases and disastrous economic losses in a wide variety of commercial crops including rice, maize and wheat [11,12]. R. solani is a common mycovirus host [7,13]. Investigations concerning the association of dsRNA with Rhizoctonia decline revitalised research on R. solani mycoviruses [14] which in turn revealed that mycoviruses are ubiquitous in natural R. solani isolates [7,13,[15][16][17][18][19]. Subsequently complete genome sequences of several R. solani mycoviruses have been documented and their sequence properties and phylogene have been analysed. Thus far the R.
Investigations on the AG-1 IA isolate of R. solani, the causal agent of rice sheath blight revealed the presence of three novel mycoviruses in the authors' laboratory [7,13,18]. In this study we describe the complete nucleotide sequence of another partitivirus nominated Rhizoctonia solani partitivirus 5 (RsPV5), isolated from R. solani AG-1 IA strain C24. The sequences of the two genomic components of RsPV5 were analysed and a phylogenetic tree was constructed based on the derived amino acid sequence of the putative RNA-dependent RNA polymerase (RdRp) to clarify the phylogenetic status of RsPV5. The phylogenetic analysis indicated that RsPV5 has the closest relationship with members of the genus Alphapartitivirus.

Fungal Strain
The C24 strain of R. solani AG-1 IA was used in this study, which was isolated from rice leaves with typical symptoms of rice sheath blight collected from Zhangzhou city, Fujian province, China, in 1999 and stored at −20 • C.

Isolation and Sequencing of Mycovirus dsRNA
Mycelia of the strain C24 were cultured on cellophane covered on potato dextrose agar (PDA) plates at 28 • C. After cultivation for 5 days, the mycelia were harvested and stored at −80 • C for further use. The lyophilized mycelia were ground into a fine powder with a mortar and pestle in the presence of liquid nitrogen. Viral dsRNAs were extracted using a slightly modified version of a CF-11 cellulose chromatography method as described by Morris and Dodds [26]. To remove contaminating DNA and single stranded RNA, the extracts were treated with DNase I and SI nuclease, and viral dsRNAs were separated and analysed by gel electrophoresis and visualization with ethidium bromide staining. The cDNA library was constructed using random primer (5 -CCTGAATTCGGATCCTCCNNNNNN-3 ) along with reverse transcriptase, and amplified with specific primer (5 -CCTGAATTCGGATCCTCC-3 ). To sequence the 5 and 3 -termini of the dsRNA, a RACE procedure modified from that described by Potgieter et al. [27] was used. All PCR amplicons were cloned into the pMD18-T vector and transformed into Escherichia coli strain JM109. Plasmid DNA from recombinant clones was isolated and at least three clones for each fragment of sequence were sequenced in both directions. The complete nucleotide sequences of the two genomic components of RsPV5 were assembled and deposited in GenBank database with the accession numbers of MH715946 and MH715947.

Data Analysis
Sequence analysis and multiple alignments were actualized by DNAMAN and ClustalX. A phylogenetic tree was constructed on the basis of neighbor-joining (NJ) method using MEGA 6 with 1000 replicates.

Genomic Structure Analysis
Sequence analysis revealed that R. solani strain C24 was infected by a novel virus, RsPV5, belonging the family Partitiviridae. The complete genome of RsPV5 is composed of two segments, designated dsRNA-1 and dsRNA-2, respectively (Figure 1a,b). A comparison of both dsRNA segments demonstrated that both 5 -and 3 -termini are conserved (Figure 1c). Additionally the 3 -ends of both dsRNAs were interrupted by poly(A) tails, a feature similar to some other members in the family Partitiviridae [13,28]. Analysis of the full-length cDNA sequence of dsRNA-1 indicated that it comprises 1899 nucleotides (nt), with a GC content of 45.98%, and contains an open reading frame 1 (ORF1), starting at nt 76 and terminating at nt 1830. ORF1 potentially encodes a 68.7 kDa protein of 584 amino acids (aa) containing sequence-conserved motifs characteristic for RNA-dependent RNA polymerase (RdRp; Figure 1a). The 5 -untranslated region (UTR) and 3 -UTR of dsRNA-1 consists of 75 nt and 69 nt, respectively. Homology searches with BLASTp confirmed that the protein was closely related to the RdRps of partitiviruses including Rhizoctonia solani dsRNA virus 3 (RsRV3, GenBank accession number: YP_009329886.2) with an aa identity of 82%, Heterobasidion partitivirus 12 (HetPV12, GenBank accession number: YP_009508051.1) with an aa identity of 61%, Heterobasidion partitivirus 13 (HetPV13, GenBank accession number: AHL25155.1) with an aa identity of 59%.
Analysis of the full-length cDNA sequence of dsRNA-2 indicated that it is 1787 bp in length with a GC content of 52.20% containing a single open reading frame 2 (ORF2) starting at nt 81 and terminating at nt 1622. DsRNA-2 potentially encodes a putative capsid protein (CP) of 513 aa that has an estimated molecular mass of 55.5 kDa (Figure 1b). The 5 -UTR and 3 -UTR of dsRNA2 are respectively 80 nt and 165 nt in length. BLASTp search revealed that this protein has 65% identity to RsRV3 CP gene (GenBank accession number: YP_009329885.2) and 30% identity to the HetPV13 CP gene (GenBank accession number: AHL25156.1).

Phylogenetic Analysis
To confirm the taxonomic status of RsPV5, a phylogenetic tree was constructed based on the aa sequences of RdRp regions of RsPV5 and 24 other selected viruses in the families Partitiviridae and Totiviridae as well as the unclassified viruses ( Figure 2). The result of phylogenetic analysis showed that RsPV5, Rhizoctonia solani dsRNA virus 3, Rhizoctonia solani dsRNA virus 2, Rhizoctonia solani partitivirus 3, Rhizoctonia solani partitivirus 4, White clover cryptic virus 1 and Carrot cryptic virus were clustered together in a distinct group belonging to the genus Alphapartitivirus. The phylogenetic tree illustrated that RsPV5 is a new member of the genus Alphapartitivirus in the family Partitiviridae. In addition, RsPV5 was placed in the same clade with RsRV3, a mycovirus of R. solani AG-1 IA previously identified in our laboratory [18], indicating that these two viruses have a close relationship. Furthermore, RsRV1 [7] and RsRV-HN008 [24] belong to the subclade of unclassified family. R. solani AG-1 IA appears to have an extensive virome which might expand further in the future with the advent of next generation sequencing.

Discussion
It can be seen from the above study, RsPV5, which infects R. solani AG-1 IA, is a novel dsRNA mycovirus of the genus Alphapartitivirus in the family Partitiviridae. So far, seven mycoviruses four of them were found in the authors' laboratory [7,13,18] infecting Rhizoctonia solani AG1-IA have been reported and have proved to belong to different viral family [7,13,18,19,24], indicating a rich diversity of mycoviruses in R. solani AG-1 IA.
Author Contributions: C.L. performed the experiments, analysed the data, sequenced and analysed the virus, wrote the draft paper; M.Z. (Miaolin Zeng) and M.Z. (Meiling Zhang) provided advices for experimental operation, assisted in data analysis and paper revision; C.S. and E.Z. conceived, designed and supervised the experiments, reviewed and revised the paper.
Funding: This study is supported by the National Natural Science Foundation of China, "Genome structure and function analysis of dsRNA mycoviruses of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight" (No. 31470247).

Conflicts of Interest:
The authors declare no conflict of interest.