Discovery and Genomic Function of a Novel Rice Dwarf-Associated Bunya-like Virus

Bunyaviruses cause diseases in vertebrates, arthropods, and plants. Here, we used high-throughput RNA-seq to identify a bunya-like virus in rice plants showing the dwarfing symptom, which was tentatively named rice dwarf-associated bunya-like virus (RDaBV). The RDaBV genome consists of L, M, and S segments. The L segment has 6562 nt, and encodes an RdRp with a conserved Bunya_RdRp super family domain. The M segment has 1667 nt and encodes a nonstructural protein (NS). The complementary strand of the 1120 nt S segment encodes a nucleocapsid protein (N), while its viral strand encodes a small nonstructural protein (NSs). The amino acid (aa) sequence identities of RdRp, NS, and N between RDaBV and viruses from the family Discoviridae were the highest. Surprisingly, the RDaBV NSs protein did not match any viral proteins. Phylogenetic analysis based on RdRp indicated that RDaBV is evolutionarily close to viruses in the family Discoviridae. The PVX-expressed system indicated that RDaBV N and NS may be symptom determinants of RDaBV. Our movement complementation and callose staining experiment results confirmed that RDaBV NSs is a viral movement protein in plants, while an agro-infiltration experiment found that RDaBV NS is an RNA silencing suppressor. Thus, we determined that RDaBV is a novel rice-infecting bunya-like virus.


Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and 3 and 5 Rapid
Amplification of the cDNA Ends (3 /5 RACE) For viral genome amplification, total RNA was extracted from the collected rice samples and reverse-transcribed into cDNAs using the reverse transcriptase M-MLV (TaKaRa, Kusatsu, Japan) according to the manufacturer's instructions. Super-fidelity PCR was performed using the KOD One PCR Master Mix (TOYOBO, Osaka, Japan) and specific primers (Table S1). The rapid amplification of cDNA ends (RACE) was performed using the HiScript-TS 5/3 RACE Kit (Vazyme, Nanjing, China), according to the manufacturer's instructions, to amplify the 3 and 5 end sequences of the newly identified virus. The PCR products were individually sequenced and used to assemble the complete viral genome.
To survey viral infections in the paddy fields, total RNA from field rice plants was reverse-transcribed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. PCR reactions were performed to detect RDaBV using the Green Taq Mix (Vazyme, Nanjing, China) and the specific forward and reverse primer pair, RDaBV-Detection-F/R (Table S1). Each PCR reaction system consisted of 10 µL of 2 × Green Taq Mix, 1 µL of each primer (10 µmol/L), 7 µL of sterile deionized water, and 1 µL of cDNA. PCR reaction conditions were 2 min at 94 • C; 30 cycles of amplification at 94 • C for 30 s, 54 • C for 30 s, and 72 • C for 30 s; the final extension step was performed at 72 • C for 5 min.

Analyses of Viral Genome and Proteins
Open reading frames (ORFs) in the viral genomic RNA of the new virus were predicted using the NCBI ORFfinder software (http://www.ncbi.nlm.nih.gov/orffinder, accessed on 18 December 2019) and using the sequence of the Plasmopara viticola lesion-associated mycobunyavirales-like virus 8 as a model [25]. Three viral genome segments of the new rice virus obtained in this study were used to perform BLASTx and BLASTp searches in the NCBI databases. In addition, the conserved domains in the viral proteins were identified using the Conserved Domain Search Service (CD-Search) software in the NCBI (https: //www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 10 January 2020) [26].
For homology analyses of genomic RNAs or predicted proteins of the new virus, we downloaded the nucleotide and amino acid sequences of representative species from all fourteen families of the order Bunyavirales. Detailed information on these sequences is provided in Table S2. The sequence identities were aligned using the DNAMAN7.0 software (https://www.lynnon.com/dnaman.html, accessed on 10 January 2021).

Phylogenetic Analysis
Genome sequences of representative viruses in different families of the order Bunyavirales were downloaded from the GenBank database. Detailed information on these viruses is provided in Table S2. Amino acid (aa) sequences of RdRps of these viruses, including RDaBV, were aligned using the ClustalW method in the MEGA X software. The phylogenetic analysis was performed using the maximum-likelihood method in the MEGA X software with 1000 bootstrap replicates [27]. The resulting phylogenetic tree was edited using the iTOL online tool (https://itol.embl.de/, accessed on 10 January 2021).

Plasmid Construction
NS, N, and NSs protein genes of the newly identified virus were individually amplified from the diseased rice plants with RT-PCR using the specific primers listed in Table S1. The resulting genes were subcloned into the expression vector pCambia 1300-Flag, pCambia 1300-GFP, or pGR106 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) according to the manufacturer's instructions.

Agroinfiltration Assay
To identify the RNA silencing suppressor of RDaBV, the constructed vectors were confirmed by nucleotide sequencing and then transformed into the Agrobacterium tumefaciens strain EHA105. Leaves of line 16c transgenic Nicotiana benthamiana were co-infiltrated with A. tumefaciens harboring the GFP gene and A. tumefaciens harboring the N, NSs, NS, tomato bushy stunt virus (TBSV) P19 or GUS gene, respectively. GFP fluorescence in co-infiltrated leaves was observed and photographed under a UV lamp (UV Products, Upland, CA, USA).
To identify the symptom determinants, RDaBV N, NSs, and NS were separately subcloned into the PVX heterologous expression vector pGR106. The resulting recombinant expression vectors were transformed into the A. tumefaciens strain GV3101 using the freezethaw method as previously described [28]. N. benthamiana leaves were infiltrated with A. tumefaciens harboring the PVX-base expression vector. At 7 days post-agro-infiltration (dpi), the agro-infiltrated N. benthamiana and its leaves were observed and photographed.

Agrobacterium-Mediated Movement Complementation Experiment
An expression vector expressing a movement-defective potato virus X (i.e., PVX-GFP∆p25) was obtained from a previously published source [29]. The A. tumefaciens strain GV3101 harboring the vector PVX-GFP∆p25 was prepared and diluted to OD 600 = 0.001 in an infiltration buffer (10 mM MgCl 2 , 10 mM MES (pH 5.8) and 100 µM acetosyringone), as previously described [30], and then mixed 1:1:1 (v/v/v) with A. tumefaciens cells harboring a vector expressing the TBSV P19 protein (OD 600 = 0.6) and A. tumefaciens cells harboring a vector expressing the rice stripe virus (RSV)-encoded NSvc4 protein (OD 600 = 0.6). For other treatments, A. tumefaciens harboring the vector expressing RSV NSvc4 was replaced by A. tumefaciens harboring the vector expressing the new virus NS, N, or NSs protein, or A. tumefaciens harboring the vector expressing β-glucuronidase (GUS). The mixed A. tumefaciens cells were infiltrated into N. benthamiana leaves. At 6 dpi, the infiltrated leaves were examined under a UV light or a confocal microscope.

Western Blot Analysis
The total protein of N. benthamiana leaves was extracted in a protein extraction buffer [30], followed by Western blot analysis as previously described [31]. In brief, the resulting protein samples were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After protein bands were transferred to nitrocellulose membranes, the membranes were blocked for 1 h in a 0.01 M phosphate-buffered saline (PBS) containing 5% skimmed milk at room temperature, followed by a 60 min incubation in a primary antibody solution at 37 • C. After three rinses in PBS containing 0.05% Tween-20, the membranes were incubated for 60 min in the horseradish peroxidase (HRP)-labeled secondary antibody solution at 37 • C. The detection signal was visualized by adding the Immobilon Western Chemiluminescent HRP Substrate, according to the manufacturer's instructions (Millipore, WBKLS0100).

Transmission Electron Microscopy
Field-collected rice plants were analyzed for viral infections using RT-PCR, after which leaf tissues were sampled from the diseased rice plants and fixed for 4 h in a 2.5% glutaraldehyde solution (pH 7.0) at room temperature. The fixed leaf samples were dehydrated in serially diluted ethanol solutions. After 20 min of incubation in acetone, the tissues were individually embedded in the Spurr resin (SPI Supplies, Westchester, PA, USA). Ultra-thin sections were cut from the embedded samples and examined under a Hitachi H-7650 transmission electron microscope.

Confocal Microscope
The agro-infiltrated leaves were cut from the N. benthamiana plants and observed under a confocal microscope (model FLUOVIEW FV3000; Olympus, Tokyo, Japan). The excitation wavelengths of GFP, RFP, and aniline blue were 488, 555, and 405 nm, respectively, and the emission wavelength was set at 510 nm for GFP, 583 nm for RFP, and 461 nm for aniline blue.

Identification of a Novel Rice Dwarf-Associated Bunya-like Virus
During the surveys of rice viral diseases in 2019, rice plants showing dwarfing, reduced tillering, and blighted grain symptoms were found in paddy fields in the Zhejiang province of China, and samples were obtained ( Figure 1A). Using the total RNA from these fieldcollected rice plants as the template and primers designed according to the known riceinfecting viruses in China, common rice viruses, including rice dwarf virus (RDV), rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf virus (SRBSDV), rice gall dwarf virus (RGDV), rice stripe mosaic virus (RSMV), rice ragged stunt virus (RRSV), and rice stripe virus (RSV) were separately monitored by RT-PCR. RT-PCR results found no known rice virus in the sampled rice plants ( Figure S1). Therefore, we conducted further analyses using RNA-seq and RT-PCR technologies. Analyses of the resulting sequencing reads with the TopHat software v2.1.1 produced three long contigs with 1093, 1681, and 6483 nucleotides (nt), respectively. BLASTx searching the GenBank database with these three long contigs identified a new virus belonging to the order Bunyavirales. These three long contigs corresponded to the large (L), medium (M), and small (S) segments of bunyaviruses, respectively. To confirm this RNA-seq result, we amplified partial segments of the above three long contigs using RT-PCR, and all gene segments could be amplified ( Figure 1B-D). We then performed 5 and 3 RACEs to obtain the full-length viral genome. The 5 and 3 RACEs and sequencing results showed that these three full-length viral genomic RNAs contained 6562 (L), 1667 (M), and 1120 (S) nt, respectively. Complete genome sequences of RDaBV have been deposited in the GenBank under the accession numbers ON366507 (L), ON366508 (M), and ON366509 (S). Based on the typical viral symptoms and its genome, we tentatively named this new virus rice dwarf-associated bunya-like virus (RDaBV). In the 2021 rice-growing season, we also detected RDaBV and known rice viruses (RBSDV, SRBSDV, RDV, RRSV, RGDV, RSMV, and RSV) in rice plants in paddy fields in the Zhejiang province of China. We found that 4 of 30 field-collected rice plants showing dwarfing symptoms were only infected with RDaBV, with an infection rate of RDaBV reaching 13.3% ( Figure S2). This indicates that RDaBV is already one of the prevalent rice viruses in Zhejiang province, China.
An analysis of leaf tissues from the RDaBV-infected rice plants through transmission electron microscopy (TEM) showed that these leaf samples contained enveloped spherical virus-like particles approximately 125 nm in diameter ( Figure 1E), similar to the morphology of some known bunyaviruses. The diameters of most bunyavirus virions range from 80 to 120 nm, but some bunyaviruses have larger virions. For instance, the High Plains wheat mosaic virus in the family Fimoviridae has virions up to 200 nm in diameter [32] and the Hantaan virus in the family Hantaviridae has virions 80-200 nm in diameter [33].

Viral Genome and Protein Prediction
Similar to other plant-infecting bunyaviruses, the RDaBV genome was found to have three RNA segments ( Figure 2A). The RDaBV L segment shares 21.96-51.24% nucleotide homology with viruses in the family Discoviridae (Table 1). The L segment contains a large ORF (nucleotide position 85 to 6483) that is predicted to encode a protein with 2132 amino acids (aa). Analysis of this protein using the Conserved Domain Database (CDD) tool in the NCBI showed that this protein contains a conserved Bunya_RdRp super family domain (cl20265, aa residue 684 to 1238). Therefore, we considered that the RDaBV L segment also encodes an RNA-dependent RNA polymerase (RdRp). The RdRp protein encoded by RDaBV shares 27.69-57.39% aa identity with that encoded by the viruses in the family Discoviridae (Table 1). The BLASTx search using the RDaBV L segment query

Viral Genome and Protein Prediction
Similar to other plant-infecting bunyaviruses, the RDaBV genome was found to have three RNA segments ( Figure 2A). The RDaBV L segment shares 21.96-51.24% nucleotide homology with viruses in the family Discoviridae (Table 1). The L segment contains a large ORF (nucleotide position 85 to 6483) that is predicted to encode a protein with 2132 amino acids (aa). Analysis of this protein using the Conserved Domain Database (CDD) tool in the NCBI showed that this protein contains a conserved Bunya_RdRp super family domain (cl20265, aa residue 684 to 1238). Therefore, we considered that the RDaBV L segment also encodes an RNA-dependent RNA polymerase (RdRp). The RdRp protein encoded by RDaBV shares 27.69-57.39% aa identity with that encoded by the viruses in the family Discoviridae (Table 1). The BLASTx search using the RDaBV L segment query indicated that this RdRp shares the highest aa sequence similarity (57.86%) with that of the Botrytis cinerea negative-stranded RNA virus 2 (GenBank accession QJT73695.1) (Table S4).     The RDaBV M segment shares 33.23-47.83% nucleotide homology with viruses in the family Discoviridae (Table 1). The M segment of RDaBV contains 1667 nt with a single ORF that is predicted to encode an NS protein with 506 aa residues. The NS protein shares the aa sequence identity ranging from 10.67% to 35.48% with that of viruses in the family Discoviridae. The BLASTx search using this M segment query showed that all matched sequences are from viruses in the family Discoviridae (Table S5). The CDD search result showed that the RDaBV NS protein had no conserved protein domain.
The RDaBV S segment shares 37.7-40.76% nucleotide homology with viruses in the family Discoviridae (Table 1). As shown in Figure 2, the RDaBV S segment is ambisense and contains two ORFs, one on the viral RNA strand and another on the viral RNA complementary strand. This phenomenon also occurs in some other bunyaviruses. For example, S segments of viruses in both Phenuiviridae and Tospoviridae families are ambisense, encoding for a small nonstructural protein (NSs) and a nucleocapsid (N) protein [34]. The ORF1 on the viral RNA strand of the RDaBV S segment (nt position 95-442) was predicted to encode the NSs protein with 115 aa residues, and the ORF2 on the viral RNA complementary strand (nt position 174-1031) was predicted to encode the N protein with 285 aa residues (Figure 2A). The aa sequence identity of the N protein between RDaBV and the known viruses in Discoviridae ranged from 30.53 to 50.17% and was much higher than that between RDaBV and viruses of other families in the order Bunyavirales (Table 1). At the same time, the matched sequences by the BLASTp search using the RDaBV N protein query were all from the family Discoviridae (Table S6). The RDaBV NSs protein did not match any viral proteins in the NCBI protein database by the BLASTp search.
Moreover, the sequence alignment result of the RDaBV three genomic segments found that both the 3 and 5 ends of all three RDaBV genomic segments have a complementary conserved sequence CACACAAAG, and the same 14 and 16 nucleotides at their 3 and 5 ends, respectively ( Figure 2B). This characteristic is shared by some plant-infectious bunyaviruses, such as melon chlorotic spot virus (MeCSV) and rice grassy stunt virus (RGSV) [35,36].

Phylogenetic Analysis of RDaBV
To determine the evolutionary relationship of RDaBV, we retrieved 52 RdRp aa sequences of viruses from 14 families in the order Bunyavirales and one family (Rhabdoviridae) in the order Mononegavirales in the NCBI database (Table S3). We then constructed a phylogenetic tree using aa sequences of the downloaded 52 RdRps and RDaBV RdRp (Figure 3).
The phylogenetic tree indicated that the RDaBV is in the same branch as viruses from the family Discoviridae (Figure 3). RDaBV was evolutionarily closer to the Plasmopara viticola lesion-associated mycobunyavirales-like virus 8. This result is consistent with the viral protein identity analyses ( Table 1). The family Discoviridae was created by the ICTV in July 2021. The hosts of all six reported viruses in the new family Discoviridae are from the phylums Oomycota and Ascomycota. However, the host of RDaBV is rice.  Table S3.

Pathogenicity and Subcellular Localization of Viral Proteins
To clarify the symptom determinant of RDaBV, we constructed three PVX-based recombinant vectors: PVX-N, PVX-NS, and PVX-NSs. These three constructed vectors were individually agro-infiltrated into N. benthamiana leaves; A. tumefaciens carrying PVX was used as the negative control. After 7 days post-agro-infiltration (dpi), we found that PVX-N and PVX-NS caused plant leaf necrosis symptoms, while PVX-NSs showed the same mosaic symptoms as PVX ( Figure 4A). We further detected the infection of PVX through Western blot using the PVX CP specific antibody, and our results demonstrated that the accumulation levels of PVX in N. benthamiana leaves infiltrated with PVX-NSs or PVX-N were lower than that in leaves infiltrated with PVX, and the accumulation levels of PVX in N. benthamiana leaves infiltrated with PVX-NS were higher than that infiltrated with  Table S3.

Pathogenicity and Subcellular Localization of Viral Proteins
To clarify the symptom determinant of RDaBV, we constructed three PVX-based recombinant vectors: PVX-N, PVX-NS, and PVX-NSs. These three constructed vectors were individually agro-infiltrated into N. benthamiana leaves; A. tumefaciens carrying PVX was used as the negative control. After 7 days post-agro-infiltration (dpi), we found that PVX-N and PVX-NS caused plant leaf necrosis symptoms, while PVX-NSs showed the same mosaic symptoms as PVX ( Figure 4A). We further detected the infection of PVX through Western blot using the PVX CP specific antibody, and our results demonstrated that the accumulation levels of PVX in N. benthamiana leaves infiltrated with PVX-NSs or PVX-N were lower than that in leaves infiltrated with PVX, and the accumulation levels of PVX in N. benthamiana leaves infiltrated with PVX-NS were higher than that infiltrated with PVX ( Figure 4B). Furthermore, the accumulation level of PVX in the leaves infiltrated with PVX-N was significantly reduced ( Figure 4B). This suggests that proteins N and NS may be symptom determinants of RDaBV. To determine subcellular localization patterns in plant cells of viral proteins, RDaBV NS, N, and NSs genes were separately subcloned into the transient expression vector pCambia 1300-GFP to obtain pCambia 1300-NS-GFP (NS:GFP), pCambia 1300-N-GFP (N:GFP), and pCambia 1300-NSs-GFP (NSs:GFP). N. benthamiana leaves were individually agro-infiltrated with these expression vectors, and the green fluorescence was observed at 48 h post-agro-infiltration (hpi). The green fluorescence (from the NSs:GFP, NS:GFP, and N:GFP fusion proteins) was located at the cytomembranes, cytoplasm, and nuclei of N. benthamiana leaf cells, which is similar to that of GFP on its own ( Figure 5). Furthermore, NS:GFP and N:GFP fusion proteins formed puncta on the cytomembrane and in the cytoplasm ( Figure 5). To determine subcellular localization patterns in plant cells of viral proteins, RDaBV NS, N, and NSs genes were separately subcloned into the transient expression vector pCambia 1300-GFP to obtain pCambia 1300-NS-GFP (NS:GFP), pCambia 1300-N-GFP (N:GFP), and pCambia 1300-NSs-GFP (NSs:GFP). N. benthamiana leaves were individually agro-infiltrated with these expression vectors, and the green fluorescence was observed at 48 h post-agro-infiltration (hpi). The green fluorescence (from the NSs:GFP, NS:GFP, and N:GFP fusion proteins) was located at the cytomembranes, cytoplasm, and nuclei of N. benthamiana leaf cells, which is similar to that of GFP on its own ( Figure 5). Furthermore, NS:GFP and N:GFP fusion proteins formed puncta on the cytomembrane and in the cytoplasm ( Figure 5).

RDaBV NSs Protein Functions as the Viral Movement Protein
To determine which viral protein encoded by RDaBV can function as a viral mo ment protein in the plant, we produced three expression vectors that can express FLA tagged NSs (NSs-flag), FLAG-tagged N (N-flag), and FLAG-tagged NS (NS-flag). A. mefaciens cells carrying the NSs-flag, NC-flag, NS-flag, RSV NSvc4 (the positive contr or the GUS expression vector (the negative control) were mixed with A. tumefaciens c carrying the PVX-GFPΔp25 expression vector and A. tumefaciens cells carrying the TB P19 expression vector. The mixed A. tumefaciens cells were individually infiltrated into benthamiana leaves. At 5-7 dpi, the infiltrated leaves were harvested and examined un a confocal microscope and a UV lamp. The result showed that in the presence of NSs-fl the mutant PVX-GFPΔp25 moved into the neighboring cells, similar to that observed leaves co-expressing PVX-GFPΔp25 and RSV NSvc4 ( Figure 6A, B). This indicates t NSs can function as a viral movement protein (MP). However, both N and NS prote

RDaBV NSs Protein Functions as the Viral Movement Protein
To determine which viral protein encoded by RDaBV can function as a viral movement protein in the plant, we produced three expression vectors that can express FLAG-tagged NSs (NSs-flag), FLAG-tagged N (N-flag), and FLAG-tagged NS (NS-flag). A. tumefaciens cells carrying the NSs-flag, NC-flag, NS-flag, RSV NSvc4 (the positive control), or the GUS expression vector (the negative control) were mixed with A. tumefaciens cells carrying the PVX-GFP∆p25 expression vector and A. tumefaciens cells carrying the TBSV P19 expression vector. The mixed A. tumefaciens cells were individually infiltrated into N. benthamiana leaves. At 5-7 dpi, the infiltrated leaves were harvested and examined under a confocal microscope and a UV lamp. The result showed that in the presence of NSs-flag, the mutant PVX-GFP∆p25 moved into the neighboring cells, similar to that observed in leaves co-expressing PVX-GFP∆p25 and RSV NSvc4 ( Figure 6A,B). This indicates that NSs can function as a viral movement protein (MP). However, both N and NS proteins could not complement the cell-to-cell movement of mutant PVX-GFP∆p25 in infiltrated N. benthamiana leaves ( Figure 6A). We collected the infiltrated N. benthamiana leaves and analyzed them with Western blot using a GFP-specific antibody to further confirm this finding. Our results demonstrated that in the presence of NSs-flag, more GFP had accumulated in the leaf tissues, which is similar to that in leaves co-expressing PVX-GFP∆p25 and RSV NSvc4 ( Figure 6C). The Western blot result with the anti-flag antibody showed the expressions of GUS-flag, NSs-flag, N-flag, and NS-flag fusion proteins ( Figure 6C). However, because the expressed NSvc4 did not fuse a flag tag, NSvc4 could not be monitored by the anti-flag antibody ( Figure 6C).  Previous studies have demonstrated that plant virus infections can induce callose depositions around plasmodesmata (PD) to inhibit virus cell-to-cell spread, while viral MPs can reduce callose depositions near PDs [37,38]. To further investigate the function of RDaBV NSs, we transiently expressed NSs-flag or GUS in N. benthamiana leaves. Two days later, the infiltrated leaves were harvested, stained with aniline blue, and examined under a confocal microscope. We found that leaves expressing NSs had fewer aniline blue-stained dots than those expressing GUS ( Figure 7A). We counted the number of fluorescence dots in individual cells and confirmed that the leaves expressing NSs had fewer fluorescence dots compared to the leaves expressing GUS ( Figure 7B). To investigate whether RDaBV NSs can target PD in cell walls, we co-expressed NSs-GFP and TMV MP-RFP fusion proteins in N. benthamiana leaves through agro-infiltration. Two days later, the leaves were treated with a 1 M sucrose solution followed by a confocal microscopy observation. As the TMV MP (red punctate signal) shows the position of PDs in cell walls, our results provide support for the hypothesis that RDaBV NSs can target PDs ( Figure 7C, white arrows).

RDaBV NS Protein Roles as an RNA Silencing Suppressor
A. tumefaciens harboring a vector expressing GFP was mixed with A. tumefaciens harboring a vector expressing RDaBV NS-flag, N-flag, NSs-flag, P19, or GUS. The mixed A. tumefaciens combinations were then individually infiltrated into transgenic 16c N. benthamiana leaves. In this experiment, A. tumefaciens harboring the vector expressing GUS and P19 was used as the negative and positive control. At 6 dpi, the leaves co-expressing GFP and GUS, GFP and N-flag, GFP and NSs-flag, GFP and NS-flag, or GFP and P19 were harvested and photographed under a UV light. We found that GFP silencing was suppressed in the presence of RDaBV NS or P19 ( Figure 8A). On the other hand, GFP silencing occurred in the presence of RDaBV N, NSs, or GUS ( Figure 6A). The Western blot result showed that leaves co-expressing GFP and NS-flag or GFP and P19 accumulated more GFP compared to leaves co-expressing GFP and NSs-flag, GFP and N-flag, or GFP and GUS ( Figure 8B), indicating that RDaBV NS is a viral suppressor of RNA silencing.

Discussion
It is well known that the number of known plant-infecting viruses accounts for a small fraction of the total number of plant-infecting viruses in nature. In our field survey in 2019, rice plants showing dwarfing, reduced tillering, and blighted grain symptoms, which are similar to that shown by virus-infected rice plants, were found in several rice fields in Zhejiang province, China. Because analyses of the collected rice samples for known virus infections through RT-PCR failed, we speculated that these rice samples

Discussion
It is well known that the number of known plant-infecting viruses accounts for a small fraction of the total number of plant-infecting viruses in nature. In our field survey in 2019, rice plants showing dwarfing, reduced tillering, and blighted grain symptoms, which are similar to that shown by virus-infected rice plants, were found in several rice fields in Zhejiang province, China. Because analyses of the collected rice samples for known virus infections through RT-PCR failed, we speculated that these rice samples could be infected with unidentified virus(es). To test this speculation, we analyzed these rice samples using RNA-seq. The RNA-seq and RT-PCR results showed that these dwarf rice plants contained an ambisense-segmented viral RNA genome, similar to that reported for viruses in the order Bunyavirales. Based on the phylogenetic relationship, we tentatively named this new virus the rice dwarf-associated bunya-like virus (RDaBV).
The organization of the RDaBV genome shares similar characteristics with that of viruses in the family Discoviridae in the order Bunyavirales. The RDaBV genome consists of three RNA segments: L, M, and S. The genome organization of RDaBV was determined according to the existence of conserved motifs and previously reported features of bunyaviruses [1][2][3][4]. The L segment encodes an RdRp, and the M segment encodes a nonstructural protein (NS). The RDaBV S segment encodes a nucleocapsid and another nonstructural protein (NSs). The termini of three RDaBV RNA segments have the same complementary conserved sequence CACACAAAG, a feature shared by other bunyaviruses, including melon chlorotic spot virus and rice grassy stunt virus [35,36].
Though some plant viruses are diffused by contact and seeds [39], the majority of plant viruses are transmitted by vectors, including insects, mites, nematodes, or fungi [40]. Of these vector-transmitted plant viruses, most are transmitted via insect vectors in fields [41]. In this study, we tried to transmit RDaBV to rice plants using insect vectors, including the small brown planthopper (Laodelphax striatellus), brown planthopper (Nilaparvata lugens), white-backed planthopper (Sogatella furcifera), and Recilia dorsalis, but all failed (data not shown). A phylogenetic analysis using RDaBV RdRp showed that this novel rice virus belonged to the newly created family Discoviridae and was evolutionarily close to Plasmopara viticola lesions-associated mycobunyavirales-like virus 8 [25]. The hosts of all six reported viruses in the family Discoviridae are from the phylum Oomycota or Ascomycota. Given that we did not find the insect vector of RDaBV, we suspect that the novel virus could be transmitted by fungi but could also be transmitted by other insects.
The encoding of MPs is one of the most important characteristics of plant viruses. The plasmodesmata (PD) is the critical macromolecule transport corridor in plant cell walls. Because of its size exclusion limit (SEL), PD is a bottleneck during plant virus infections [42]. Many studies have demonstrated that plant virus infections can induce callose accumulation around PDs, leading to a restriction of virus intercellular trafficking. On the other hand, plant virus-encoded MPs have been shown to interact with ankyrin repeat-containing proteins (ANK) to reduce callose accumulation, relax callose sphincters, and increase the SEL of PDs [37,38]. In this study, we identified the NSs protein as the MP of RDaBV by the movement complementation and callose staining experiment, which demonstrates that RDaBV is a plant-infected virus. At the same time, during the survey of rice viral diseases in 2021, we collected 30 rice plants showing dwarfing symptoms in paddy fields in the Zhejiang province of China and monitored RDaBV and the known rice viruses in these samples by RT-PCR. We found that four rice plants showing dwarfing symptoms were only infected with RDaBV, and the infection rate of RDaBV reached 13.3% ( Figure S2).
We also found that the NS protein can act as a viral RNA silencing suppressor. RNA silencing (RNAi) is a common defense strategy used by plants to resist viral invasions. This anti-viral strategy can be interfered with by viral suppressors of RNA silencing (VSRs) encoded by plant viruses via inhibiting dsRNA recognition, interfering with RNA-induced silencing complex (RISC) assembly, directly binding to proteins crucial to the RNA silencing pathway, and interfering with silent signal transmission [43][44][45][46]. However, in this study, we do not know which step of plant RNA silencing was suppressed by the RDaBV NS protein.
In summary, we identified a novel rice-infecting bunya-like virus, RDaBV, belonging to the family Discoviridae of the order Bunyavirales in Zhejiang province, China. We determined that the RDaBV NSs protein is a viral movement protein and its NS protein is a viral RNA silencing suppressor. Since vectors play important roles in rice virus spread in fields, further studies are needed to determine how RDaBV is transmitted in rice fields. Furthermore, the pathogenicity of RDaBV must be verified using Koch's postulate. Additionally, its evolutionary history and impact on rice production must be further studied. Nonetheless, the findings presented in this study enrich our understanding of bunyaviruses and rice viruses and can contribute to more effective strategies for controlling this rice viral disease.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14061183/s1, Table S1: Primers used in this study; Table S2: Viruses used for the nucleotide and amino acid identity analyses; Table S3: Viruses used for phylogenetic analysis; Table S4: The BLASTx search using the L segment of RDaBV query; Table S5: The BLASTx search using the M segment of RDaBV query; Table S6: The BLASTp search using the N protein of RdaBV query; Figure