Complete Genome Sequence of a Novel Latent Tobamovirus Infecting Hevea Brasiliensis in China

In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in rubber tree (Hevea brasiliensis) by high-throughput RNA-seq. The infected plants show no identiable symptoms, therefore this virus is tentatively named rubber tree latent virus 1 (RTLV1). The full genome of RTLV1 is 9,422 nt in length and contains three open reading frames (replicase, movement, and coat proteins) with 157 nt 5' UTR and 316 nt 3' UTR. The replicase encoded by ORF1 shares only 33.29% amino acid sequence identity with that of the closest related Frangipani mosaic virus. Phylogenetic analysis using the ORF1 amino acid sequence clusters RTLV1 with members of genus Tobamovirus (family Virgaviridae) at relative high bootstrap, suggesting that RTLV1 is a novel member of Tobamovirus.


Abstract
In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in rubber tree (Hevea brasiliensis) by high-throughput RNA-seq. The infected plants show no identi able symptoms, therefore this virus is tentatively named rubber tree latent virus 1 (RTLV1). The full genome of RTLV1 is 9,422 nt in length and contains three open reading frames (replicase, movement, and coat proteins) with 157 nt 5' UTR and 316 nt 3' UTR. The replicase encoded by ORF1 shares only 33.29% amino acid sequence identity with that of the closest related Frangipani mosaic virus. Phylogenetic analysis using the ORF1 amino acid sequence clusters RTLV1 with members of genus Tobamovirus (family Virgaviridae) at relative high bootstrap, suggesting that RTLV1 is a novel member of Tobamovirus.

Background
Rubber tree (Hevea brasiliensis) is an important crop in tropical area and a major commercial source of natural rubber. Vegetative propagation in traditional rubber tree plantation is prone to accumulating viruses, but only a few viruses have been identi ed in rubber tree so far [4,8]. Through RNA-seq, de novo assembly and annotation as previous description [8], several assembled unigenes identi ed in latex samples of rubber tree were annotated as tobacco mosaic virus (TMV), one of which was 9,361nt long, suggesting that the rubber tree sample was infected by a TMV-like virus. TMV is a member of tobamoviruses genus of family Virgaviridae. TMV has had an illustrious history for more than 100 years and been known to infect members of nine plant families, and at least 125 individual species, including a number of nightshades, cucurbits, beans and ornamental owers. However, TMV has never been reported in rubber tree. The identi ed virus might be a novel member of tobamovirus.
Virgaviridae is a plant virus family that currently includes 59 types of viruses in 7 genera: Furovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamvirus and Tobravirus [1,2]. Virgaviridae was named because its members have rod-shaped virions (Latin virga means rod). The seven genera are classi ed according to their distinct genome organisations and other features such as transmission modes.
Tobamovirus has no natural vector and the transmission is usually mechanical, whereas other six genera have biotical vectors such as pollen (Goravirus and Hordeivirus), or seed (Hordeivirus), or nematode (Tobravirus), or plasmodiophorid (Furovirus, Pomovirus and Pecluvirus). The genome of Tobamovirus is monopartite, linear, single stranded and positive sense RNA, which is distinct from multi-partites genome of other 6 genera [1,2].
In order to clone the entire genome of the virus, eight fragments nearly covering the complete genome were ampli ed by RT-PCR for subsequent Sanger sequencing, and the terminal fragments were obtained by 5' and 3' rapid ampli cation of cDNA ends (RACE) using a RACE 5'/3' kit , 2nd Generation (Roche, Indianapolis, IN) according to the manufacturer's instructions (Fig. 1). The ORFs were predicted using the ORF Finder (www.ncbi.nlm.nih.gov/or nder) and the conserved domains were analyzed online with default settings (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The complete genome consists of 9,422 nucleotides (Nt) (submission number: 244064), and the G/C content is 39%. RTLV1 has a typical tobamovirus organization with a 5' untranslated region (UTR, 157 nt) and 316-nt 3' UTRs ( Figure 1). The suggesting that RTLV1 is novel member of Virgaviridae virus. To further examine the relationship between RTLV1 and other Virgaviridae viruses, the amino acid sequences of replicase proteins from 36 members of seven genera in the family Virgaviridae were selected for phylogenetic tree construction and analysis ( Figure 2). The phylogenetic tree was constructed with MEGA6. RTLV1 was clustered in Tobamovirus with relative high bootstrap, suggesting that RTLV1 is much distantly related to other member and is a novel member of Tobamovirus.
Agrobacterium tumefaciens-mediated transformation has been widely used in plant genetics research [10,11] and has been successfully applied in rubber tree [5], but Agrobacterium-mediated transformation has rarely been used in the research of rubber tree due to the low transformation e ciency and time cost.
Virus-induced gene silencing (VIGS) allows the analysis of gene functions in species that are not amenable to stable genetic transformation, such as many non-model organisms including rubber tree [3].
Very recently, tobacco rattle virus (TRV) was reported to induce VIGS in rubber tree [7], which provides a mean to analyze the functions of candidate genes identi ed in rubber tree. However, rubber tree is not the natural host of TRV, whether serious side effects could be caused by TRV infection still requires further investigation. RTLV1 was detected in many "healthy" rubber trees without any visible symptoms; therefore RTLV1 was regarded as a latent virus of rubber tree. Like Apple latent spherical virus (ALSV) [6,9], RTLV1 has the potentiality to be developed as a vector for expression of foreign proteins, or VIGS, or virus-based vaccines in rubber tree. Figure 1 Schematic representation of RT-PCR fragments and the genome organization of rubber tree latent virus 1 (RTLV1). MTR, RNA methyltransferase; HEL, RNA helicase; RdRp, RNA-dependent RNA polymerase; CP, coat protein; MP, movement protein. Phylogenetic tree based on the deduced amino acid sequence of the RTLV1 ORF1 and that of 36 members of the family Virgaviridae. Multiple alignment was carried out with CLUSTALX 1.8.1 (http://www.clust al.org), and a phylogenetic tree was constructed using MEGA7.0.20. Accession numbers and virus names are given directly in the phylogenetic tree.