Study On Interaction Between Rice Black-streaked Dwarf Virus (RBSDV) P5-1 And OsCSN5B,A Factor Of Host Rice Plant

Rice black-streaked dwarf virus disease is caused by Rice black-streaked dwarf virus (RBSDV). RBSDV is recognized as a member of the genus Fijivirus, family Reoviridae and transmitted by the small brown planthopper (Laodelphax striatellus) in a persistent manner. RBSDV-infected rice plants are always stunted with much more tillering, 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. RBSDV genome is made up of 10 double-stranded RNA, which are named S1 to S10, respectively. S5 has two ORFs, a major ORF (p5-1) and unusually a second ORF (p5-2). Although it is predicted that p5-1 protein is involved in the formation of viroplasms, the real role of p5-1 between interaction with host factors remains unclear.RBSDV p5-1 was used as a bait to screen a rice cDNA library in a yeast two-hybrid, from which a host factor named OsCSN5B was acquired. CSN5, the most special subunit of COP9 signaling complex(CSN), is closely related with plants ubiquitin protein degradation and plants antiviral immunity. In this study both the results from yeast two-hybrid assay(YTHS) and Co-immunoprecipitation(Co-Ip)) can show us a strong interaction between p5-1 and OsCSN5B. The region (amino acids 71-150) of OsCSN5B was found to be crucial for its interaction with p5-1. Analyses of subcellular localization showed that p5-1 accumulated in cytoplasm and OsCSN5B accumulated in both nucleus and cytoplasm. Co-localization of p5-1 and OsCSN5B would change the amount of p5-1 punctate bodies. The expression of OsCSN5B would reduce the amount of punctate bodies which p5-1 and p6 formed together, which indirectly demonstrated the relative realtionship of p5-1 and viroplasms.In addtion,the study showed us that OsCSN5B overexpression sustained ToMv replication in N. benthamiana while p5-1 overexpression facilitated ToMv replication in N. benthamiana. Recombinant protein OsCSN5B was prepared and purified with prokaryotic expression systems, after which Western blotting tests showed that antiserum against OsCSN5B protein could react strongly with OsCSN5B protein. Acorrding to QPCR and Western blotting, the expression of OsCSN5B gene at level of mRNA and protein went down after RBSDV infected healthy rice plants. OsCSN5B positive transgenic rice plants was obtained using CRISPR/Cas9 technology, which was quite signficant to further study the interaction between OsCSN5B with p5-1.