The Interactions Between Functional Proteins Of Rice Stripe Virus And Chloroplast Proteins Of Rice

Recently, the epidemics of Rice stripe virus in some areas have caused great losses to agricultural production. Much work has been done on the traditional and molecular biology of RSV. But the functions of RSV encoded proteins, mechanisms underlying its pathogenicity and interactions between RSV and hosts remain obscure.Yeast two hybrid experiments using several RSV encoded proteins as baits have been cuducted in our lab and several putative RSV interacting proteins were identified. In this study, NB2 and SB16, two chloroplast proteins that interacted with NSvc4 and SP of RSV respectively, were selected. Changes of the expression of NB2 after RSV infection were determined using Real Time PCR. Additionaly, based on the finding that CP of RSV interact with itself, further experiments were carried out to map the domains involved in the self-interaction.Firstly, to verify the interactions between the two chloroplast proteins and specific RSV encoded proteins, the entire ORFs of the two proteins were amplified by RT-PCR and ligated into the pGADT7 vector, obtaining recombinants pGAD-NB2 and pGAD-SB16, respectively. The two recombinants were then cotransformed to yeast AH109 with biat plasmids pGBK-NSvc4, pGBK-SP and pGBK-CP respectively. Clononies were selected on a serias of auxotrophic mediums. The results indicated that NB2 could interact with NSvc4, but not with CP or SP; whereas SB16 could interact with neither NSvc4 nor CP and SP.Secondly, co-immunopricidation experiments were conducted to investigate the interactions in plant cells. HA epitoped NB2 and SB16 fusion genes were obtained and inserted into the plant expression vector pEGAD. The recombinants pEGAD-NB2 and pEGAD-SB16 were cotransformed with pEGAD-NSvc4 and pEGAD-SP, pEGAD-CP (epitoped with c-Myc) into Agrobacterium strain EHA105, respectively. The Agrobacterium strains carring genes of interest were infiltrated into leaves of Nicotiana benthamiana according to different combinations. The total proteins were then extracted from infiltrated leaves and immunopricidated with antisurms to c-Myc or HA respectively. Protein samples were then separated by SDS-PAGE and detected using antibodies to HA and c-Myc, respectively. The results confirmed that NB2, but not SB16 interact with RSV NSvc4.Considering the facts that many previous studies have demonstrated altered expressions of virus interacting host proteins during viral infection, Real Time PCR were employed to detect the expression changes of NB2 during RSV infection. The results revealed that NB2 showed increased expression in response to RSV infection.Finaly, regions involved in the self-association of RSV CP were investigated using yeast two hybrid experiments. It was found that the N-terminal 81 amino acids of RSV CP was critical for the interaction.Collectively, these results confirmed the innteraction between a functional protein of RSV and rice chloroplast protein NB2. Sequence analysis revealed that the NB2 encoded a chloroplast protein. Such an interaction is consistent with typical symptoms associated with RSV infection. But this seems to contradict our present understanding of RSV NSvc4, which has been shown to express only at the very early stages of RSV infection. Thus, either that previously studies are inaccurate, i.e. expression of Nsvc4 can last for a long time or at least can be very strong and stable at sites where active replication of RSV occurs, or that interaction between RSV NSvc4 and NB2 is not involved in pathogenesis. It was demonstrated recently that NSvc4 was a movement protein of RSV. So NB2 could be involved in viral movement. But the molecular mechenisms underlying this involvement are unknown at present. Real time PCR was conducted and it was found that expression of NB2 increased upon RSV infection. There are extensive comunications between nucleus and plastids, which allows the coordination of the expressions of the nuclear genomes with the overall state of chloroplast function. So the upregulation of NB2 during RSV interaction could be the results of feedback regulation. Interactions between RSV proteins and NB2 could divert a high percentage of NB2 from its normal location, resulting in dysfunction of rice chloroplasts. Increasing expression of NB2 could be a mechanism of the host to compensate for this dysfunction. Additionaly, yeast two hybrid experiments in this study indicated that the N-terminal 81 amino acids were involved in the self-association of RSV CP. Further investigation of the self- interaction of CP and mapping the key amino acids should greatly advance our understanding of RSV.Results of this study would provide us with clues to investigate the functions of RSV encoded proteins, the mechenisms of RSV pathogenesis and host responses to RSV infection. They would also provide new alternatives to develop RSV resistant rice plants.