Development Of The Serological Detections For Rice Stripe Virus And Preliminary Analysis Of The Biological Functions Of Viral NS3 Protein

Rice stripe virus(RSV)is a representative member of Tenuivirus.The virus is transmitted by Laodelphax striatellus Fallen in a persistent manner.Rice stripe disease caused by RSV is one of the most important diseases in rice production that causes serious economic losses in agricultural production region distributed in East Asia.The molecular mechanism of the interaction network between the virus and plants was studied,and the serological detection system of RSV was established for the prevention and control measures of rice stripe disease.In this study,NSvc3,NS2,and NS3 proteins of RSV were expressed and purified using a prokaryotic expression system.The purified recombinant protein was subjected to immunize New Zealand white rabbits and the specific antiserum were obtained.After antibodies purification by ammonium sulfate-extraction,the titer was evaluated and subsequently applied in developed serological detections.All the testing results showed that the prepared antibodies could be used for the detection of RSV-infected rice and RSV-carrying planthopper.Previous studies showed that NS3 protein functions as a RNA silencing suppressor and plays a key role in the pathogenesis.To deeper understand the pathogenic mechanism of NS3 in rice,two potential NS3-interacted proteins were screened out through bimolecular fluorescence complementation(BIFC)in Nicotiana sylvestris,which were named Ascorbate Peroxidase 3(APX3)and Light-induced protein(LIP).Based on the exisiting sequences of the NS3-interacted proteins,the full-length opening reading frames(ORFs)sequences of the corresponding candidate gene were cloned in Nicotiana benthamiana,which were named as NbAPX3 and NbLIP.Then,the direct intercactions between NbAPX3/NbLIP and NS3 were comfirmed through yeast two-hybrid assay(Y2H),pull-down assay,and co-immunoprecipitation analyses(Co-IP)in vivo and vitro.Transient co-expression experiment mediated by Agrobacterium tumefaciens infiltration showed that interactions of NbAPX3-NS3 and NbLIP-NS3 seriously attenuated the RNA silencing suppression activity of NS3.Taken together,these results demonstrated that the interactions of NbAPX3-NS3 and NbLIP-NS3 could weaken the pathogenecity of RSV in N.benthamiana.To clarify the biological functions of the NS3-interacted proteins(NbAPX3 and NbLIP)to viral replication and movement,then the GFP-tagged tobacco mosaic virus(TMV-GFP)was selected as the testing virus.Down-regulate the expression of NbAPX3 and NbLIP by tobacco rattle virus mediated virus-induced gene silencing system(TRV-VIGS)in N.benthamiana,the replication of TMV-GFP were both suppressed;while overexpression of the NbAPX3 could increase the viral accumulation level in upper leaves,and overexpression of the NbLIP enhanced the replication of TMV-GFP.In sum,NbAPX3 and NbLIP proteins can interact directly with NS3 proteins and the NS3-interacted proteins play an important role in enhancing the replication and accumulation of TMV.All these results also implied that NbAPX3 and NbLIP may play the significant role of in the pathogenesis of RSV infection.The profound mechanism of the NS3 interacting proteins played in RSV life cycle was still obscure and needs further exploration.