Interaction Of P7-2Encoded By Rice Black-streaked Dwarf Virus And Host Rice Proteins

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus, infects maize and rice plants and causes significant yield losses of rice and maize crops in Asia. RBSDV is transmitted in nature by the small brown planthopper(Laodelphax striatellus) and replicates in cells of both host plants and insect vectors. Plants with RBSDV infection normally show dwarf growth and production of white tumors along the vein on the back of the leaves and on leaf sheaths. RBSDV has a genome of10dsRNAs (S1～S10) encased in icosahedral double-shelled particles and encodes at least10primary translation products, which are named from their respective genome segments. Among these proteins, six (P1, P2, P3, P4, P8and P10) are structural components of the viral particle, and another six (P5, P6, P7-1, P7-2, P9-1, and P9-2) are non-structural proteins. Among of those non-structural proteins, P7-1had been identified to form the cytoplasmic tubular-like structures that serve as a conduit for virion movement between cells in the insect vector. The ORF1of S9encodes an important non-structural protein, which can form the viroplasm in cells. Moreover, P5and P6are recently study show that they are associated with biological activity of viroplasm. P7-2is expressed in low amounts and their functions are still unknown.In this study, taking advantage of molecular cloning techniques, we first obtained the cDNA of P7-2coding region from segment7. The fragment of P7-2was cloned into prokaryotic expression vector pMAL-C2x. The resulting recombinant plasmid pMAL-P7-2was expressed in E.coli BL21(DE3) strain. The further purified fusion protein MBP-P7-2was prepared as a bait protein and total proteins of leaves of RBSDV infected rice plants were extracted under non-denaturing conditions as the prey proteins. When Pull-down samples were analyzed by SDS-PAGE and silver staining, four protein bands were detected in the MBP-P7-2samples but not in the MBP control. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was further used to identify those proteins. According to the sequence alignment in Rice Genome Annotation Project, two of them were transcription-associated protein, one was aminotransferase protein, and the last one was putative chaperonin60beta precursor. The expression levels of transcription-associated protein and putative chaperonin60beta precursor was higher in infected rice comparing with healthy rice using real-time fluorescence quantitative PCR. The results indicated that the expression aminotransferase protein was suppressed by RBSDV infection. Therefore, we suggested that P7-2may interfere host transcription, and it could reduce the resistance of the host plant by repressing the expression of aminotransferase protein in host plant.In addition, we explore the subcellular localization of P7-2in plant cells using protoplast transformation system. P7-2was fused to eGFP and expressed in Arabidopsis protoplast. Confocal laser scanning microscopy (CLSM) observation revealed that P7-2-GFP fusion protein distributed in both nucleus and cytoplasm. The DNA transcription process was mainly in nucleus. Therefore, the research of subcellular localization of P7-2further confirmed the speculation that this protein may has the ability of transcriptional activation. Moreover, establishing and improving the Arabidopsis protoplast transformation system laid a foundation for further study on the subcellular localization of P7-2in rice protoplast.