Identification and Characterization of Two Novel Noda-like Viruses from Rice Plants Showing the Dwarfing Symptom

Abstract

Nodaviruses are small bipartite RNA viruses and are considered animal viruses. Here, we identified two novel noda-like viruses (referred to as rice-associated noda-like virus 1 (RNLV1) and rice-associated noda-like virus 2 (RNLV2)) in field-collected rice plants showing a dwarfing phenotype through RNA-seq. RNLV1 genome consists of 3335 nt RNA1 and 1769 nt RNA2, and RNLV2 genome consists of 3279 nt RNA1 and 1525 nt RNA2. Three conserved ORFs were identified in each genome of the two novel viruses, encoding an RNA-dependent RNA polymerase, an RNA silencing suppressor, and a capsid protein, respectively. The results of sequence alignment, protein domain prediction, and evolutionary analysis indicate that these two novel viruses are clearly different from the known nodaviruses, especially the CPs. We have also determined that the B2 protein encoded by the two new noda-like viruses can suppress RNA silencing in plants. Two reverse genetic systems were constructed and used to show that RNLV1 RNA1 can replicate in plant cells and RNLV1 can replicate in insect Sf9 cells. We have also found two unusual peptidase family A21 domains in the RNLV1 CP, and RNLV1 CP can self-cleave in acidic environments. These findings provide new knowledge of novel nodaviruses.

Keywords: RNA silencing suppressor; noda-like virus; nodavirus; reverse genetic system; rice.

Figure 1

Images of dwarfed rice plants in fields and RT-PCR detection of virus infections. (a) Images of rice plants showing dwarf symptoms in a rice field in Shanghai City (left image) and in the Zhejiang Province (right image). White arrows indicate the plants showing dwarf symptoms. (b) RT-PCR detection of virus infection in the rice plants shown in (a) using primers designed according to the contig3 sequence (384 bp), the contig15 sequence (303 bp), the contig3206 sequence (366 bp), and the contig3545 sequence (279 bp), respectively.

Figure 2

Genome organizations and protein domains of RNLV1 and RNLV2. (a) Genome organization of RNLV1. The predicted ORFs are shown in rectangles with different colors. (b) Genome organization of RNLV2. The predicted ORFs are shown in rectangles with different colors. (c) Two predicted domains in the RdRps of RNLV1 and RNLV2. The predicted domains are presented in rectangles with different colors. The domain accession numbers in CD database of the predicted domains are shown below the corresponding rectangles. SP, Signal peptide; MT, nodavirus vmethyltransferase domain; RdRp, RNA-dependent RNA polymerase domain; GDD, the conserved GDD box in the RdRp domain. (d) Predicted domains in the RNLV1 and RNLV2 CPs. The predicted CPs and domains are shown in rectangles with different colors. The domain accession numbers in CD and InterPro database are indicated above or below the corresponding rectangles. Peptidase A21, Peptidase family A21 domain; CP, coat protein domain; and CPS, coat protein s domain.

Figure 3

Phylogenetic relationships between RNLV1, RNLV2, and other related viruses. The phylogenetic trees were constructed using the RdRp sequences (a) or the CP sequences (b) and the maximum likelihood method with 1000 bootstraps. Accession numbers of these sequences are listed in Table S3. The bootstrap values are indicated adjacent to the nodes. Taxonomy and hosts of these analyzed viruses are indicated.

Figure 4

Both RNLV1 and RNLV2 B2 proteins can suppress RNA silencing in plants. (a) Images of transgenic 16c N. benthamiana leaves co-expressing mGFP5 and RNLV1 B2, mGFP5, and RNLV2 B2, mGFP5 and TBSV P19 (the positive control), or mGFP5 and the empty vector (EV, the negative control). These leaves were examined and photographed under a UV light at 5 dpi. Weak green fluorescence in the leaf tissues co-expressing mGFP5 and EV indicated the silencing of mGFP5 expression. (b) Western blot analysis of mGFP5 accumulation in the infiltrated leaf tissues at 5 dpi. The Ponceau S-stained large RuBisCO subunit gel is used to show sample loadings. (c) Images of PVX-RNLV1 B2-, PVX-, and mock-inoculated N. benthamiana plants at 6 and 9 dpi. White arrows indicate the leaves showing disease symptoms. (d) By 11 dpi, the PVX-RNLV2 B2-inoculated plants also developed necrosis in their systemic leaves, but not the PVX-inoculated or the mock-inoculated N. benthamiana plants. White arrows indicate the leaves showing disease symptoms.

Figure 5

RNLV1 RNA1 can replicate in N. benthamiana leaf cells. (a) Insertion site of an eGFP gene in RNLV1 RNA1 in the pCB301-RNLV1 RNA1(wt/opt)-B2:eGFP vector. 2 × 35S, doubled CaMV 35S promoter; RZ, HDV ribozyme; NOS, NOS terminator. (b) The infiltrated N. benthamiana leaves were photographed at 3 dpi under a fluorescence microscope. (c) RT-PCR analysis of RNLV1 RNA1 replication in the infiltrated leaf tissues shown in (b) using primers specific for RNLV1 RNA1wt or RNA1opt (560 bp). (d) Analysis of fusion protein accumulation in the infiltrated leaf tissues shown in (b) through Western blot assay using a GFP specific antibody at 3 dpi.

Figure 6

RNLV1 can replicate in insect Sf9 cells. (a) An electron micrograph showing negatively stained RNLV1 virions from the transfected Sf9 cells. Bars = 200 nm. (b) RT-PCR analysis of RNLV1 replication in Sf9 cells. Sf9 cells were transfected with in vitro transcribed RNLV1 RNA transcripts, double distilled water (ddH₂O, the negative control), or inoculated with the P1-supernatant from the transfected Sf9 cells. After three days, these cells were analyzed for RNLV1 infection through RT-PCR using primers specific for the negative strand RNA1 (422 bp, left panel) or RNA2 (509 bp, right panel).

Figure 7

Analysis of RNLV1 CP self-cleavage. (a) Sequence alignments of RNLV1 CP and NωV CP using Phyre2 service. (b) Western blot assay analyzing the self-cleavage of RNLV1 CP from the transfected Sf9 cells. Extracts were incubated in a weakly alkaline (pH 7.4) or acidic environment (pH 5.0) at room temperature for 1 d and then analyzed through Western blot assay using a polyclonal antibody against RNLV1 CP. (c) Western blot assay analyzing the self-cleavage of RNLV1 CP^WT and the mutant RNLV1 CP^N480A from N. benthamiana leaves agro-infiltrated with pGD-RNLV1 CP^WT and pGD-RNLV1 CP^N480A. At 3 dpi, extracts were incubated in a weakly alkaline (pH 7.4) or acidic environment (pH 5.0) at room temperature for 1 d and then analyzed through Western blot assay using a polyclonal antibody against RNLV1 CP.