Study On The OTU-like Protein Encoded By Rice Stripe Virus

Rice stripe disease is caused by rice stripe virus, which is transmitted by small brown planthopper, Laodelphax striatellus Fallen. The disease has occurred at many rice-grown areas in several countries in Southeast Asia since its discovery in Japan in the late19th century. RSV is a member of genus Tenuivirus and contains four genomic segments of linear RNAs (RNA1-RNA4), in which each of RNA2-4contains two open reading frames (ORFs) while RNA1just contains one ORF, encoding a polypeptide (PC1) with2919amino acids (MW337kDa). Sequence analysis showed that the polypeptide was characteristic of RNA dependant RNA polymerase (RdRp) domain, and thus thought to be a viral RdRp. In addition to the RdRp domain, the polypeptide harboured an ovarian tumor-like (OTU-like) domain at its N-terminus. OTU-like proteins are one type of deubquitinating enzyme family, which could play an important role in interaction between viruses and hosts by deubquitination. Previously studies in our laboratory showed that the N-terminal OTU-like protein was involved in maturation of viral RdRp and intereactions between the virus and its hosts. Here the encoding region of OTU-like protein was divided into four fragments, which were separately subcloned into the prokaryotic expression vector pET-32a and transformed into E. coli strain BL21(DE3). Prokaryotic expression of the fusion proteins were induced by IPTG and purified for preparation of antiserum against the proteins by immunizing mice. Western blot assay showed that the antiserum were specific for the corresponding fusion proteins. In the meantime, the encoding region of OTU-like protein was cloned into the yeast vector pGBKT7to generate the recombinant plasmid pGBK-OTU. An auto-activation test showed that the protein could not autonomously activate the expression of reporter genes in Yeast Two Hybrid (YTH) system. Thus the protein was used as bait for screening the expression cDNA library of rice plants by mating. Total3genes were identified to interact with the OTU-like protein in yeast. Bimolecular fluorescence complementation (BiFC) assay showed that there was strong interaction between the OTU-like protein and rice LHCP in plant cells. Down-regulation of LHCP expression by the method of virus-induced gene silencing (VIGS) could induce an obvious chlorotic symptom. These results provided foundation for further study on the function of the OTU-like protein.