Interactions Between Genes Of Southern Rice Black-streaked Dwarf Virus(SRBSDV) And White-backed Planthopper (WBPH)

Plant viruses are naturally transmitted by vectors, including insects, mites, nematodes and fungi, in which approximately80%of plant viruses are insect-borne. Southern rice black streaked dwarf diseases, cuased by Southern rice black streaked dwarf virus (SRBSDV), was first reported in Yangjiang city, Guangdong Province, China. The virus was initially considered an isolate of Rice black streaked dwarf virus (RBSDV). Until2008, it has been identified to be a novel species of rice viruses and tentatively named Southern rice black streaked dwarf virus (SRBSDV) or Rice black streaked dwarf virus2(RBSDV-2), which was a putative member of second group within genus fijivirus, family reoviridae. SRBSDV is transmitted by the white-backed planthopper (WBPH), Sogatella furcifera Horvath, in a persistent manner, but not transmitted by eggs, seeds, andmechanical mode.In order to screen interactions between the genes of WBPH and SRBSDV, the WBPH cDNA expression library was first constructed into the yeast plasmids using Gateway technique in this study. Quality assay of the cDNAlibrary titer total colony-forming units5.09×107CFU, the average insert size was between0.75and2kb and the recombinant percentage of the cDNA library was very high. These results consistently indicated that the cDNA library can be used for screening interactions by the Yeast Two Hybrid system. Based on the published sequences of SRBSDV-GD and-HN isolates, a series of primers were designed and used to construct the recombinant plasmids inserted with the encoding regions of SRBSDV S1-S4in Yeast Two Hybrid (YTH) system. Toxical assay showed that all the proteins encoded on the SRBSDV S1-S4have no significant toxic effects on the growth of yeast strains. Auto-activation tests showed that only P3protein of SRBSDV could autonomously activate the expression of reporter genes in yeast, indicating SRBSDV P3had transcriptional activation effect.Then the P8protein, a core structural protein of SRBSDV, was used a bait for screening the expression cDNA library of the white-backed planthopper (WBPH), Sogatella furcifera Horvath, by yeast mating method. A total of543positive colonies were selected on SD/-Ade/-His/-Leu/-Trp/X-a-Gal medium. Sequence analysis showed insertions of these prey plasmids derived from5genes of WBPH, includingcarboxylest-erase of Sogatella furcifera (SfCarE),Sogatella furcifera cytochrome oxidase subunit I gene (SfCO I), and other3genes with unknown function. There is strong interaction betweenSfCarE and SRBSDV P8in yeast cells. Bimolecular fluorescence complement-ation(BiFC)assay showed the interaction also occurred in plant cells and the reconstituted YFP fluorescence was mainly distributed in the cytoplasm. Subcellular localization assay showed that SfCarE was distributed in the cytoplasm and SRBSDV P8was detected in both the cytoplasm and the nucleus, supporting the interaction between them in cytoplasm.In addition, to understand the interactions of viral proteins, SRBSDV P6, a multifunctional protein, was used as bait and a SRBSDV cDNA library as prey in a yeast two hybrid (YTH) assay by yeast mating method. Total3pairs of interactions, including P6/P5-1, P6/P6, and P6/P9-1, were selected. Here we further studied on the interactions of P6/P6and P6/P9-1. Co-transformed yeast assays showed that there was a stronger interaction of P6/P6than that of P6/P9-1. These interactions were confirmed by bimolecular fluorescence complement (BiFC) assay in plant cells. The reconstituted YFP fluorescence was randomly distributed in the cytoplasm and aggregate into granular structures, which appeared similar to the viroplasm-like structures (VLS). No YFP fluorescence was observed in the control cells. YTH analyses using truncated mutants showed that all the4truncated mutants of P6could not interact with P6, indicating that both N-and C-terminal fragments were necessary for its self-interaction. YTH analyses using truncated mutants also indicated that both N-and C-terminal fragments of P9-1were necessary for its interaction with P6, and the N-terminal region (aa1-93) P6was crucial for its interaction with P9-1. Immunogold labeling showed that the immunogold particles were specifically labeled in amorphous Viroplasm with higher electron-density in infected cell of rice plants, indicating that P6is a component of the Viroplasm and supporting P6was involved in viroplasm formation by interacting with another component, P9-1, of viroplasm in infected plants. These results provided foundation for further study on the self-interactions among the viral proteins and interactions between SRBSDV and its vector, white-backed planthopper (WBPH), Sogatella furcifera Horvath.