Rice Stripe Mosaic Virus, a Novel Cytorhabdovirus Infecting Rice via Leafhopper Transmission

Abstract

A new rice viral disease exhibiting distinct symptoms-yellow stripes, mosaic and twisted tips on leaves-was found in China. Electron microscopy of infected leaf cells revealed the presence of bacilliform virions and electron-translucent granular-fibrillar viroplasm in the cytoplasm. The enveloped viral particles were 300 to 375 nm long and 45 to 55 nm wide. The leafhopper Recilia dorsalis was able to transmit the virus to rice seedlings, which subsequently exhibited symptoms similar to those observed in fields. The complete genome of the virus was obtained by small-RNA deep sequencing and reverse transcription-PCR product sequencing. The anti-genome contains seven open reading frames (ORFs). The deduced amino acids of ORF1, ORF5, and ORF7 are, respectively, homologous to the nucleocapsid protein (N), glycoprotein (G), and large polymerase protein (L) of known rhabdoviruses. The predicted product of ORF2 is identified as a phosphoprotein (P) based on its multiple potential phosphorylation sites and 12.6 to 21.0% amino acid (aa) identities with the P proteins of plant rhabdoviruses. The product of ORF4 is presumed to be the viral matrix (M) protein for it shares 10.3 to 14.3% aa identities with those of other rhabdoviruses. The above five products were confirmed as the viral structural proteins by SDS-PAGE and aa sequencing analyses of purified virus preparation. ORF3 and ORF6 are considered to encode two nonstructural proteins with unknown functions. Phylogenetic analysis based on protein N, G, and L amino acid sequences indicated that the isolated virus, which we have tentatively named Rice stripe mosaic virus (RSMV), is a new species in the genus Cytorhabdovirus. To our knowledge, RSMV is the only cytorhabdovirus naturally infecting rice and the first reported leafhopper-transmitted cytorhabdovirus. Our surveys of rice fields indicate that RSMV occurs frequently in Guangdong Province, China. Although the disease incidence is low at present, it might become serious with the vector insect population increasing.

Keywords: Recilia dorsalis; Rice stripe mosaic virus; cytorhabdovirus; leafhopper; rice viral disease.

FIGURE 1

Symptoms of Rice stripe mosaic virus (RSMV)-infected rice. Infected rice in the field (A–C). Infected rice leaves at 21 days after inoculation by the viruliferous vector Recilia dorsalis (D,E) and negative controls (F). The black arrow indicates RSMV-infected rice plant whose symptoms was shown in (B,C). The rice cultivar is Wuyou 736 in (A–C), and Taichung Native 1 in (D–F).

FIGURE 2

Electron micrograph of infected rice leaves. Negative-stained sap of infected rice leaves (A,B). Ultrathin sections of infected rice leaves showing RSMV virions and cytopathological structures (C–G), the enlarged CS of (E) is shown in (F). The uninfected rice leaves cells are shown in (H). Black arrows indicate the RSMV virions with enveloped, red arrows indicate the vesicle structure with gathered virions in it. CW = cell wall, CH = chloroplast, CS = crystalline structure, M = mitochondrion, N = nucleus, V = Virion, and Vp = viroplasm.

FIGURE 3

Comparative genome organization of RSMV and plant-infecting rhabdoviruses. (A) Putative gene of RSMV are shown as black arrows on the anti-genomic strand. Fragments a, b, and c are contigs from small RNA sequencing that are similar to rhabdovirus N, G, and L genes, respectively. Contig fragments d, e, and f showed no homology with any known viruses in NCBI and were mapped to the complete genome of RSMV. (B) Comparison of the genome structure of plant rhabdoviruses. Open reading frames (ORFs) for the main structural protein genes N, P, M, G, and L are indicated as open arrows. Ancillary genes are shown as gray arrows.

FIGURE 4

SDS-PAGE pattern of purified SMRV preparation. The sample was resolved in 12% SDS-PAGE gel followed by staining with Coomassie brilliant blue-R250. Five protein bands (band 1 to 5) were analysis by LC-MS.

FIGURE 5

Molecular phylogenetic tree of N, G, and L genes of RSMV. The phylogenetic tree was constructed from N, G, and L gene sequences by neighbor-joining method based on poisson model. Bootstrap support (%) based on 1,000 bootstrap replicates is shown above and below nodes. RSMV is labeled with a black triangle.