Identification and characterization of water chestnut Soymovirus-1 (WCSV-1), a novel Soymovirus in water chestnuts (Eleocharis dulcis)

Abstract

Background: A disease of unknown etiology in water chestnut plants (Eleocharis dulcis) was reported in China between 2012 and 2014. High throughput sequencing of small RNA (sRNA) combined with bioinformatics, and molecular identification based on PCR detection with virus-specific primers and DNA sequencing is a desirable approach to identify an unknown infectious agent. In this study, we employed this approach to identify viral sequences in water chestnut plants and to explore the molecular interaction of the identified viral pathogen and its natural plant host.

Results: Based on high throughput sequencing of virus-derived small RNAs (vsRNA), we identified the sequence a new-to-science double-strand DNA virus isolated from water chestnut cv. 'Tuanfeng' samples, a widely grown cultivar in Hubei province, China, and analyzed its genomic organization. The complete genomic sequence is 7535 base-pairs in length, and shares 42-52% nucleotide sequence identity with viruses in the Caulimoviridae family. The virus contains nine predicated open reading frames (ORFs) encoding nine hypothetical proteins, with conserved domains characteristic of caulimoviruses. Phylogenetic analyses at the nucleotide and amino acid levels indicated that the virus belongs to the genus Soymovirus. The virus is tentatively named Water chestnut soymovirus-1 (WCSV-1). Phylogenetic analysis of the putative viral polymerase protein suggested that WCSV-1 is distinct to other well established species in the Soymovirus genus. This conclusion was supported by phylogenetic analyses of the amino acid sequences encoded by ORFs I, IV, VI, or VII. The sRNA bioinformatics showed that the majority of the vsRNAs are 22-nt in length with a preference for U at the 5'-terminal nucleotide. The vsRNAs are unevenly distributed over both strands of the entire WCSV-1 circular genome, and are clustered into small defined regions. In addition, we detected WCSV-1 in asymptomatic and symptomatic water chestnut samples collected from different regions of China by using PCR. RNA-seq assays further confirmed the presence of WCSV-1-derived viral RNA in infected plants.

Conclusions: This is the first discovery of a dsDNA virus in the genus Soymovirus infecting water chestnuts. Data presented also add new information towards a better understanding of the co-evolutionary mechanisms between the virus and its natural plant host.

Keywords: Caulimoviridae; Pararetrovirus; RNA sequencing; Soymovirus; Virus-derived small RNA (vsRNA); Water chestnut.

Fig. 1

Illumina sequencing results. A Graph showing the size distribution of the small RNAs sequence from water chestnut samples with WCSV-1. B The relative frequency of each nucleotide A (blue), C (red), G (green), and U (purple) in the 5′ terminal nucleotide position in sRNA molecules 18–26 nt in length-only high quality reads shown

Fig. 2

The cloning strategy for the WCSV-1 full-length genome. a A linear genomic map of WCSV-1 is shown, in which the putative ORFs are represented by rectangles; b the 12 contigs (H1-H12) obtained from small RNA sequencing are presented as gray lines (B1), and the four fragments (labeled A-D) amplified by the first PCR reaction using primers based on the contigs are indicated by black lines (B2); c three fragments e–g amplified by the second PCR using primers from the obtained clones to fill in the gaps are shown as double arrows. The relative positions of primers are also indicated

Fig. 3

Phylogenetic tree showing the relationships between WCSV-1 and selected numbers of the Caulimoviridae family. a A phylogenetic tree was constructed based on full-length genomic sequences for WCSV-1 and other Caulimoviridae specified as virus names (abbreviation) followed by sequence accession numbers. b Phylogenetic tree based on the amino acid sequences encoded by ORF V. The phylogenetic trees were estimated using the neighbor-joining method with 1000 bootstrap replicates. Bootstrap values > 50% are shown at branch numbers. The bar represents 0.1 substitutions per site. The following abbreviations are used to indicate virus names in the phylogenetic tree analysis. BRRV, Blueberry red ringspot virus; SbCMV, Soybean chlorotic mottle virus; PCSV, Peanut chlorotic streak virus; CmYLCV, Cestrum yellow leaf curling virus; DMV-Portland, Dahlia mosaic virus-Portland; FMV, Figwort mosaic virus; CaMV-XinJiang, cauliflower mosaic virus-XinJiang; CERV, Carnation etched ring virus; BSV-GF, Banana streak virus-GF; CSSV, Cacao swollen shoot virus; ComYMV, commelina yellow mottle virus; CsVMV, Cassava vein mosaic virus; SPVCV-Dom1, Sweet potato vein clearing virus-Dom1; PVCV, Petunia vein clearing virus; RTBV-PH, Rice tungro bacilliform virus-PH. The position of WCSV-1 is indicated by the triangle

Fig. 4

Phylogenetic tree analysis of WCSV-1 compared with selected numbers of Caulimoviridae family based on ORF I, IV, VI and VII. Phylogenetic trees were constructed based on the amino acid sequence encoded by each of the ORFs as described in Fig. 3 and using the same abbreviations. The position of WCSV-1 is indicated by the triangle

Fig. 5

WCSV-1 genomic organization based on the cloned viral DNA sequences. The circle represents the dsDNA genome. The outer arrows indicate the position of nine ORFs, designated as ORF I, IV, V, VI, VII and A, B, C, D, respectively. “▼” indicates the starting position of the 12 nt primer binding site

Fig. 6

Alignments of highly conserved motifs encoded by WCSV-1 ORF V (a), ORF I (b), and ORF VI (c) with the corresponding regions from other viruses in the Caulimoviridae family. The abbreviated name of the virus, ORF designation, and the amino acid position starting from the N terminal (numerical) are indicated for each sequence in the left hand columns. The spacing between amino acid sequences is shown in square bracket. The abbreviations are defined in Fig. 3

Fig. 7

Characteristics of the WCSV-1 vsRNA pool from water chestnut samples. a The length of the WCSV-1 vsRNA was determined by sequencing and the number of vsRNAs of each length is shown. The vsRNA pool is divided into the positive (blue) and negative (red) strands. b Genome-wide map of WCSV-1 vsRNA at the single-nucleotide resolution. A linear genomic map of WCSV-1 is shown. The positive strand is shown in blue and the negative strand is shown in red

Fig. 8

The relative frequency of 5′ terminal nucleotides in water chestnut vsRNA pool. The relative frequency of each nucleotide A (blue), C (red), G (green), and U (purple) in the 5′ position on the positive strand (a) and negative strand (b) of WCSV-1 vsRNAs is indicated and sorted by length of the vsRNA

Fig. 9

Overview of water chestnut transcriptome reads distribution along the WCSV-1 genomic sequence. The color is shown according to the overlapping of genomics coordinates between the predicted ORFs and read counts