Archives of Virology https://doi.org/10.1007/s00705-018-3831-9 ANNOTATED SEQUENCE RECORD A novel endornavirus isolated from cluster bean (Cyamopsis tetragonoloba) Ricardo Iván Alcalá‑Briseño1 · Ryo Okada2 · Favio Herrera3 · Rodrigo A. Valverde3 Received: 29 January 2018 / Accepted: 22 February 2018 © Springer-Verlag GmbH Austria, part of Springer Nature 2018 Abstract Cluster bean (Cyamopsis tetragonoloba), also called guar, is a drought-tolerant annual legume. We conducted investigations to characterize a large dsRNA (~13-14 kbp) detected in a symptomless cluster bean genotype. The dsRNA was gel-purified and used for Illumina MiSeq sequencing. Reads were assembled, and BLASTx search results showed sequence similarity with viruses classified within the family Endornaviridae. The complete sequence of the putative endornavirus consisted of 12,895 nt and contained an open reading frame which coded for a polyprotein of 4,207 aa with conserved domains for methyltransferase, helicase, and RNA-dependent RNA polymerase. The virus was named cluster bean endornavirus 1 (CBEV-1). A BLASTx search using the polyprotein sequence showed that the closest endornavirus to CBEV-1 was Hordeum vulgare endornavirus. Cluster bean (Cyamopsis tetragonoloba), also called guar, is a drought-tolerant annual legume in the Fabaceae family grown mainly in India, Pakistan, and the United States [12]. Unlike the seeds of other legumes, the cluster bean seed has a large endosperm which contains significant amounts of galactomannan gum which is the primary marketable product of the plant [8]. Viruses classified within the family Endornaviridae have linear RNA genomes that range from 9.8 to 17.6 kb and have been reported as infecting plants, fungi, and oomycetes [4, 9]. The family Endornaviridae contains two genera, Alphaendornavirus, which classifies viruses that infect Handling Editor: Robert H. A. Coutts. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00705-018-3831-9) contains supplementary material, which is available to authorized users. * Rodrigo A. Valverde ravalve@lsu.edu 1 Department of Plant Pathology, University of Florida, Gainesville, FL, USA 2 Laboratory of Molecular and Cellular Biology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan 3 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA, USA plants, fungi, and oomycetes, and Betaendornavirus, which classifies viruses of ascomycete fungi [1]. Endornaviruses have been reported in several economically important crops [4]. We detected, by gel electrophoresis, a large dsRNA (~1315 kbp) from symptomless cluster bean plant introduction (PI) line 593049 (National Plant Germplasm System Unit, Griffin, GA, USA). This dsRNA was similar in size to dsRNAs of plant endornaviruses. RT-PCR testing with endornavirus-degenerate primers [10], followed by sequence analysis, suggested that the dsRNA was the replicative form of a putative endornavirus; therefore, we conducted investigations to characterize it. In this paper, we report the genomic properties of a putative endornavirus from cluster bean. The large dsRNA was extracted from foliar tissue of PI line 593049 as described by Khankhum et al [6]. The dsRNA was gel-purified and sequenced by Illumina MiSeq (pairend 2 x 250). A total of 168,338 reads were obtained and assembled with SPAdes 3.8.0 [2] yielding 355 contigs which were subjected to BLASTx searches using a local viral database retrieved from NCBI. Most contigs had homology to sequences of endornaviruses. The de novo assembled putative endornavirus was named cluster bean endornavirus 1 (CBEV-1). The virus terminal sequences were obtained using the SMARTer® RACE 5′/3′ kit (Takara, Japan). The complete genome was 12,895nt in length containing an open reading frame which coded for a polyprotein of 4,207 aa (12,621 nt) (GenBank Accession No. MG764084). The 5′ 13 Vol.:(0123456789) R. I. Alcalá-Briseño et al. a 1 1000 2000 3000 4000 5000 6000 7000 8000 9000 Hel MTR 10000 11000 12000 12895 RdRp MTR 253 nt 12,876 nt 4,207 aa b LsEV 92 CmEV 61 PvEV-1 64 PaEV-1 YmEV-1 100 OrEV BPEV 99 88 HPEV PvEV-2 100 WBEV-1 94 CBEV-1 69 Alphaendornavirus OsEV 100 100 82 HvEV PEV-1 VfEV 100 GEEV RcEV-1 HmEV-1 GaEV-1 100 SsEV-1 99 TaEV 100 AbEV 0.3 Fig. 1 a. Genome organization of cluster bean endornavirus 1 (CBEV-1). The box represents the large ORF, whereas dark boxes depict the methyl transferase (MTR), helicase 1(Hel), and RNAdependent RNA polymerase (RdRp). b. Phylogenetic tree of the viral RdRp of CBEV-1 and selected endornaviruses from plants, fungi, and oomycetes. The alignment was generated using MAFFT [5], and substitution model selection and a maximum likelihood phylogenetic tree was calculated using IQ-TREE [7]. The best fitted model according to BIC was LG+I+G. Values at the nodes indicated bootstrap values obtained for 10,000 replicates, branches with bootstrap values lower than 60 were collapsed. Lagenaria siceraria endornavirus (LsEV, YP_009010973), Cucumis melo endornavirus, (CmEV, YP_009222598), Phytophthora endornavirus 1 (PEV-1, YP_241110), Persea americana endornavirus 1 (PaEV-1, YP_005086952), yerba mate endornavirus (YmEV, YP_009046830), Oryza sativa endorna- 13 Betaendornavirus 61 virus (OsEV, YP_438200), Oryza rufipogon endornavirus (OrEV, YP_438202), bell pepper endornavirus (BPEV, YP_004765011), hot pepper endornavirus (HPEV, YP_009165596), Phaseolus vulgaris endornavirus 2 (PvEV-2, YP_241110), winged bean endornavirus 1 (WBEV-1, YP_009305414), Hordeum vulgare endornavirus (HvEV, YP_009212849), Phaseolus vulgaris endornavirus 1 (PvEV1, YP_009011062), grapevine endophyte endornavirus (GEEV, YP_007003829), Vicia faba endornavirus (VfEV, YP_438201), Rhizoctonia cerealis endornavirus 1 (RcEV-1, YP_008719905), Helicobasidium mompa endornavirus 1 (HmEV-1, YP_003280846), Gremmeniella abietina endornavirus 1 (GaEV-1, YP_529670), Sclerotinia sclerotiorum endornavirus 1 (SsEV-1, YP_008169851), Tuber aestivum endornavirus (TaEV, YP_004123950), Alternaria brassicicola endornavirus (AbEV, YP_009115493) A novel endornavirus in cluster bean Table 1 Comparison of the amino acid identity (%) of the polyprotein and conserved domains of cluster bean endornavirus 1 with selected endornaviruses Virus name Accession No. PP MTR (aa 272-498) Hel-1 (aa 1277-1527) RdRp (aa 3809-4133) Hordeum vulgare endornavirus Winged bean endornavirus 1 Bell pepper endornavirus Hot pepper endornavirus Gremmeniella abietina endornavirus 1 Sclerotinia sclerotiorum endornavirus 1 Tuber aestivum endornavirus YP_009212849 YP_009305414 YP_004765011 YP_009165596 YP_529670 YP_008169851 YP_004123950 23.5 19.5 19.1 19.3 10.7 11.2 9.0 32.5 31.0 37.0 37.0 15.6 15.7 15.5 36.5 36.1 32.2 32.6 16.7 17.1 ND 58.8 48.4 47.2 49.1 23.2 24.1 23.6 PP= Polyprotein; MTR= viral methyltransferase; Hel-1= viral helicase-1, RdRp = viral RNA-dependent RNA polymerase; ND= not detected and 3′ UTR, consisted of 252 and 19 nt respectively. The 3′UTR ended with eight cytosines which is a typical feature of many endornaviruses. Using the SMARTer® RACE 5′/3′ kit, a sequence discontinuity (nick) was detected near the 5’end of the positive strand of the dsRNA of CBEV-1 at nt 1278-1279. The protein sequence of CBEV-1 was analyzed with InterPro [3] resulting in three conserved domains, a methyltransferase (MTR) (272-498 aa), helicase (Hel-1) (1,275-1,527 aa) and RNA dependent RNA polymerase (RdRp) (3,809-4,133 aa) which are commonly found in endornaviruses (Fig 1a). Pairwise comparison analysis was conducted using the CBEV-1 polyprotein and conserved domains with corresponding sequences of selected endornaviruses (Table 1). Results showed that the closest endornavirus to CBEV-1 was Hordeum vulgare endornavirus. A phylogenetic tree constructed using the RdRp domain placed it in a clade with alphaendornaviruses (Fig 1b). The size of CBEV-1 is about 500nt smaller than Persea Americana endornavirus, [11]. This makes CBEV-1 the smallest plant endornavirus reported to date. Seeds of 23 C. tetragonoloba PI lines, and two other Cyamopsis species (C. senegalensis and C. serrata) (National Plant Germplasm System Unit) were planted and kept in the greenhouse with day/night temperatures averaging 25/18 °C respectively. Foliar tissues from at least two plants of each line were desiccated and large dsRNAs extracted, analyzed by agarose gel electrophoresis, and used in RT-PCR reactions [6, 10]. Results of both gel electrophoresis and RTPCR suggested that all 23 PI lines of C. tetragonoloba were endornavirus-infected; however the two other Cyamposis species were endornavirus-negative (Suppl. Table 1). Data presented in this investigation support the endornavirus nature of the large dsRNA isolated from the cluster bean PI line 593049. The genome organization and phylogenetic relationships with other endornaviruses supports this isolate, CBEV-1, as being classifiable within the genus Alphaendornavirus of the family Endornaviridae. Acknowledgements We wish to thank the National Plant Germplasm System, Plant Genetic Resources Conservation Unit, Griffin, GA, USA for providing cluster bean germplasm and the USDA National Institute of Food and Agriculture for partial support to RAV. Funding This study was partially funded by the National Institute of Food and Agriculture, USA. Compliance with ethical standards Conflict of interest Authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References 1. 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