Mechanism For Regulation Of Rice Stripe Virus Cell-to-cell Movement By Plant Remorin Protein

Rice stripe virus(RSV)is the pathogen of rice stripe disease that causing serious yield and quality loss in East Asia.Identification of host factors involved in RSV infection and deciphering the interaction mechanism between RSV and plant host could offer better strategies for controlling rice stripe disease.RSV is transmitted to natural host rice by the small brown plaathopper(Laodelpbax striatellus,SBPH),it can also mechanically infect experimental host plant Nicotiana benthamiana.In this study,we explore the plant responses to RSV infection at early and late infection time in the proteomic level by isobaric tags for relative and absolute quantitation(iTRAQ)technique.Overall,142 proteins were differential expressed by comparison between mock inoculated and RSV infected plants.The NbREM1 protein showing significantly down regulated at early infection stage was selected for analyzing the transcriptional response in time course and NbREM1 mRNA had no significant difference with mock plant and RSV infected plants at all time points,while NbREM1 protein was remarkable down-regulated in early RSV infection.Further research showed that NbREM1 silenced plant was mechanically inoculated with RSV,resulting enhancement of RSV infection on N.benthamiana and causing an increase of virus accumulation.To investigate the biological significance of NbREM1 during RSV infection,we applied Clustered Regularly Interspaced Short Palindromic Repeats-associated protein-9 nuclease system(CRISPR/Cas9)to induce NbREM1 knockout in N.benthamiana.We also obtained NbREM1 overexpression transgenic N.benthamiana lines by expression of NbREM1 cDNA sequence under 35S cauliflower mosaic virus promotor.The inoculation experiments showed that the deletion of NbREM1 accelerated RSV infection and caused an increase of virus accumulation during the early infection while a delayed symptom development and a less accumulation of RSV were observed in systemically infected leaves of overexpressed line.The above results indicate that NbREM1 is a negative regulator of RSV infection.We also found that NbREM1 could regulate callose deposition and plasmodesmata(Pd)permeability,and then affect the ability of RSV NSvc4 to recover the movement of PVX-GFP A p25.Mutagenic analysis showed that C-terminal 33 amino acid(AA)of NbREMl is crucial for its membrane localization of NbREM1.We also found the plasma membrane binding ability of NbREM1 is indispensable for its function on negative regulation of virus cell-to-cell movement.Biotin switch assay demonstrated that NbREM1 can be modified by S-acylation and the cysteine at the locus 206 is the only S-acylation site.S-acylation of NbREM1 was found to be important for NbREM1 protein membrane anchorage,stability and inhibition of virus cell-to-cell movement.Subcellular localization analysis demonstrated that S-acylation deficient NbREM1C206A was retained in the endoplasmic reticulum(ER)and cytoplasm.In addition,semi-in vivo degradation and inhibition measurement assay revealed that S-acylation deficient INbREM1C206A is subjected to autophagy degradation pathway.We found that NSvc4 can interact with NbREMl by the yeast split-ubiquitin assay,co-immunoprecipitation(CO-IP)and bimolecular-fluorescence complementation test(BiFC).RSV infection or expression of NSvc4 alone interferes the S-acylation of NbREM1 and induce lose membrane anchorage and degradation through autophagy pathway.While the NSvc4 mutant(NSvc4 a 106-125)that lost ER localization cannot bind with NbREM1,and is unable to inhibit S-acylation of NbREM1,indicating that NSvc4 interferes NbREMl S-acylation depending on protein interaction and its ER localization.As the primary host of RSV,the similar interaction analysis in rice plant was conducted.We also found that OsREM 1.4 is a negative regulator of RSV infection and OsREM1.4 can be S-acylated.In addition,S-acylation deficient OsREM1.4C200A is retained in the ER and subjected to autophagy degradation pathway.Furthermore,the S-acylation of OsREM1.4 is significantly inhibited in the presence of NSvc4.In this study,we deciphered the mechanism of remorin acting as a negative regulater of RSV cell-to-cell movement and unveiled a new strategy used by a plant virus to overcome plant defense:NbREM1 could regulate callose deposition and Pd permeability,and then affect the ability of RSV cell-to-cell movement.As counteract,RSV encodes viral NSvc4 protein that can interfere the S-acylation of remorin,inducing remorin weakened membrane anchorage and finally is subjected to degradation through autophagy pathway,facilitating virus infection.