The Mechanism For The Regulation Of Rice Stripe Virus Infection By The Host's Interacting Factor Of Movement Protein And The Unfolded Protein Response

Rice stripe disease caused by rice stripe virus(RSV)is an important virus disease on rice in China,which has caused serious damage to rice production in China and even in Asia.It is important to study the interactions between RSV and host plants as well as to investigate the pathogenic mechanism for the prevention and control of rice stripe disease.As we know,plant cells are encapsulated by strong cell walls(CW),resulting it difficult for viruses to move between cell and cell.To complete systemic infection,plant viruses usually encode one or more movement proteins(MP)to help themselves cross the plasmodesmata(PD)of plant cells to enter and infect the next neighboring cell.In previous studies,the NSvc4 protein,encoded by the complementary strand of the RNA4 of RSV,is identified to be the movement protein.The unfolded protein response(UPR)is a series of intracellular processes in response to the unfolded proteins accumulated in the endoplasmic reticulum(ER),including activation of cellular autophagic degradation pathway and upregulation of the expression of some molecular chaperone proteins.Plant virus infection can activate host UPR,and most studies elucidated that UPR facilitates plant virus infection.However,previous studies mainly focused on plant positive-strand RNA viruses,while the role of host UPR in negativestrand RNA virus(e.g.,RSV)infection are still unclear.This study mainly focuses on the role of the UPR in RSV infection and proposes a new model in which UPR regulates RSV infection by fine-turning the protein accumulation level of virus-encoded MP.First,we confirmed that RSV infection could activate the UPR of Nicotiana benthamiana cells.Then,we found that the accumulation of the autophagy-related protein ATG8-II in N.benthamiana increased after RSV infection,and the number of autophagosomes in the cells was counted to increase after infection,demonstrating that RSV infection activates the autophagic pathway in N.benthamiana cells.The 4-phenyl butyric acid(4-PBA)is an inhibitor of endoplasmic reticulum stress(ER stress)and UPR,and 4-PBA treatment could inhibit autophagy activation caused by RSV infection,indicating that RSV could activate the cellular autophagy pathway through UPR.Our further study found that either cellular autophagy pathway inhibitor treatment or silencing of autophagy-related genes could inhibit the degradation of the movement protein NSvc4.It indicates that NSvc4 is degraded through the cellular autophagy pathway.Tunicamycin(TM)treatment of N.benthamiana leaves could artificially trigger UPR and activate autophagy.Strangely,however,TM treatment of N.benthamiana leaves could not promote the degradation of NSvc4.This finding indicates that there may be some factors in the host that regulate the stability of NSvc4 and inhibit its autophagic degradation.To search for potential host factors regulating the stability of NSvc4,we identified a molecular chaperone protein containing the J domain in the results of a previous yeast library screening for interacting proteins between NSvc4 and N.benthamiana.Bioinformatics analysis revealed that the protein is a type-I J-domain protein,belonging to the Nb MIP1 protein family.We have demonstrated that NSvc4 can interact with Nb MIP1 family proteins in vivo by bimolecular fluorescence complementation(Bi FC)and co-immunoprecipitation(Co-IP).Also,RSV infection was found to up-regulate the expression of Nb MIP1 family through RT-qPCR assay,with Nb MIP1.4 being the most highly up-regulated and the main member responding to RSV infection.The UPR triggered by TM treatment resulted in the up-regulation of Nb MIP1 expression,while4-PBA treatment inhibited the up-regulation of Nb MIP1 family expression caused by RSV infection.This suggests that RSV infection also up-regulates the expression of Nb MIP1 via UPR.Next,we confirmed that Nb MIP1 protein could regulate the protein stability of NSvc4.We found that the stability of NSvc4 was enhanced by overexpressing Nb MIP1.4b in N.benthamiana,while silencing Nb MIP1 family promoted the degradation of NSvc4 via the autophagy pathway.Through laser confocal microscopy,we observed that overexpression of Nb MIP1.4b reduced the autophagosome-localized NSvc4-e GFP.This indicates that Nb MIP1 may inhibit the autophagic degradation of NSvc4 by blocking the transportation of NSvc4 into the autophagosome,but the mechanism remains to be investigated further.TM treatment promoted the degradation of NSvc4 upon silencing Nb MIP1 family genes in N.benthamiana,suggesting that UPR-activated autophagy could lead to the degradation of NSvc4,and Nb MIP1 is the host factor that regulates stability of NSvc4 and inhibits NSvc4 from autophagic degradation.Also,we found that the mutant Nb MIP1.4b(?HPD),which was unable to interact with heat shock protein 70(Hsp70),still stabilized NSvc4,indicating that Nb MIP1 inhibits NSvc4 from autophagic degradation in an Hsp70-independent manner.Although there have been some reports describing the Hsp70-independent J-domain proteins,to our knowledge,this has never been found in the plant-pathogen interaction before.Finally,we proved that Nb MIP1 and its homologous gene Os Dj A5 in rice could positively regulate RSV infection.Silencing of Nb MIP1 in N.benthamiana through TRV-VIGS inhibited RSV infection and restricted the cell-to-cell movement of RSV.Os Dj A5,the homologue of Nb MIP1 in rice,interacted with NSvc4 and was upregulated after RSV infection.The symptoms of RSV infection and viral accumulation level in rice Osdja5 knockout mutant have also been significantly reduced.In summary,this study illustrates a new model of UPR regulates RSV infecion: on the one hand,RSV activates cellular autophagy and promotes the degradation of the movement NSvc4 through UPR;on the other hand,UPR upregulates the expression of Nb MIP1/Os Dj A5 and inhibits the autophagic degradation of NSvc4.This model also exemplifies the intense arms race between hosts and plant viruses.