Research For The Interaction Between RSV NSvc4 And Two Rice Proteins

In the recent years rice stripe disease caused by Rice stripe virus (RSV) has broken out in china and caused great decreases to rice yields. Main proteins encoded by RSV include coat protein (CP), pathogenesis-related protein (SP) and a putative movement protein (NSvc4 ) .For avoidance of host monitoring and propagating itself, there may be a chain of compatible interactions between viral proteins and a set of host factors . Employing yeast two hybrid system, several rice proteins were identified to interact with NSvc4. The main aim of this study was to confirm their interactions.Firstly, possible application of co-immunoprecipitation in identifing rice proteins that interact with viral proteins was exploited. On the one hand, cell suspensions were prepaired and total proteins were extracted after inoculation of RSV. The total proteins were immunoprecipited by antibodies to CP and SP respectively. On the othe hand, rice seedlings inoculated with RSV was used for total proteins extraction and the total proteins were used for subsequent co-immunoprecipitation experiments. In all, total proteins from rice cell suspension cultures and seedlings were used to screen possible proteins interacting with CP or SP of RSV.Recently, using CP,SP and NSvc4 as the baits, a number of rice proteins were identified to interact with them. In this study, two proteins, namely NB8 and NB9, were selected to confirm their interactions with NSvc4.Total RNA was extracted from RSV-infected rice leaves with Trizol and the full-length ORF of the NB8 and NB9 were amplified by RT-PCR with primer pairs designed according to rice cDNA sequences. The specific fragments were cloned into pMD18-T respectively. Construct containing NB8 ORF was digested and fragment was ligated into linearized pGADT7 vector. Construct containing NB9 ORF was digested and fragment was ligated into linearized pGADT7-CP vector. The recombinant plasmids were designated as pGAD-NB8 and pGAD-NB9 respectively. The yeast cells co-transformed with pGBK-NSvc4/pGAD-NB8, pGBK-NSvc4 /pGAD-NB9, and pGBKT7-53/pGADT7-T were able to grow on the selective media.To test the interaction further, co-immunoprecipitation experiments were used to determin whether such interactions occur in plant cells. The fusion gene c-Myc-NSvc4 was amplified by PCR using pGBK-NSvc4 as template and cloned into pMD18-T. Construct containing c-Myc-NSvc4 was digested and ligated into a cauliflower mosaic virus 35S-based pEGAD transient-expression vector, and the recombinant plasmid was designated as pEGAD-Myc-NSvc4. The c-Myc-NSvc4 fragment was inserted into linearized pKYLX71 :35S2 vector and recombinant plasmid was designated as pKYLX-Myc-NSvc4. Alse, the recombinant plasmids were designated as pEGAD-HA-NB9, pKYLX-HA-NB9, pKYLX-HA-NB8 respectively, the c-Myc-epitope-tagged NSvc4 co-immunoprecipitated with the HA-epitope-tagged NB8 and HA-epitope-tagged NB9 after Agrobacterium- mediated transient expression in Nicotiana benthamiana. This interaction was confirmed with the reciprocal experiments, in which HA-epitope-tagged NB8 and HA-epitope-tagged NB9 co-immunoprecipitated with the c-Myc-epitope-tagged NSvc4, respectively. These results provided evidence that RSV NSvc4 interacts with NB8 and NB9 in plant cells, while there is no interaction of the two rice proteins with RSV CP and SRFinally, expression levels of NB8 and NB9 in RSV inoculated and non-inoculated rice plants were detected using Real-time PCR.No significant changes were observed for mRNA expression of NB8, while that of NB9 was slightly decreased after RSV inoculation.