The Expression And Funcation Research Of Segment S5of Rice Black-streaked Dwarf Virus(RBSDV)

Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. RBSDV is transmitted in nature by the small brown planthopper(Laodelphax striatellus) and replicates in cells of both host plants and insect vectors. Naturally-infected rice plants are usually stunted, with dark leaves. Waxy white to black galls along the veins on the underside of leaf blades and on the outer surface of sheaths and culms are also common symptoms. All known Fijivirus contain10linear genomic segments of double-stranded RNA (dsRNA), ranging in size from approximately1.4to4.5kb and named S1-10according to their migration in polyacrylamide gel electrophoresis (PAGE). As reported previously, S5has a major ORF (p5a), and unusually, a second, out-of-phase and partially overlapping ORF (p5b) was identified from sequence analysis. The expression and function of these proteins remain unknown.We now report the use of specific antibodies against each of the segment5encoded proteins, showing that both are expressed in rice plants. In western blots, these antibodies reacted with proteins of about106and27kDa, respectively, as predicted by sequence analysis. In immunoelectron microscopy, antibodies to p5a reacted with viroplasms, indicating that p5a is a component of viroplasms, but no labeling was observed with p5b antisera. Northern blot assays showed that the genome segment S5was transcribed as a single mRNA with no subgenomic RNA. These results show that S5is functionally bicistronic in infected plants.In a yeast two-hybrid (YTH) assay in which RBSDV p6, a putatively multifunctional protein, was used as bait, there was a strong interaction between the p6and p5a proteins. The interaction was confirmed by bimolecular fluorescencecomplement (BiFC) assay in plant cells. RBSDV p5a formed granules positioned at the cell periphery in Nicotiana benthamiana leaves; p6was present in both the cytoplasm and the nucleus and formed punctate bodies associated with the cell periphery. Immunogold labeling showed that both p6and p5a localized within viroplasms in infected cells of rice plants. These results suggest that the interaction between p5a and p6of RBSDV may be involved in the formation of viroplasms.7candidate host factors were acquired by screening a rice cDNA library using RBSDV p5a as bait protein. According to the functional annotation of homologous sequences and reports about the protein function, the role of the interaction between p5a and host factors in the pathogenic process of RBSDV and the process of host response to virus infection was predicted. These data will facilitate future studies on the molecular interactions between RBSDV and Oryza sativa.Experiments using YTH demonstrated that p5b and OsRCA-2interact strongly. p5b subcellular localization analyses showed that p5b was located in chloroplasts, supporting its interaction with OsRCA-2. The results of a Co-IP assay also showed that p5b and OsRCA-2can interact in living plant cells. Plants in which NbRCA-2was silenced had dwarfing symptoms positively related to the extent of NbRCA-2silencing. Interestingly, we found that p5b did not locate to the chloroplast when NbRCA-2was silenced. N. benthamiana showed dwarf symptoms after infection by TRV:p5b and the degree of dwarfing was positively related to levels of p5b expression in the plant. We therefore conclude that p5b is a pathogenesis-related protein of RBSDV.