| Autophagy is a highly conserved evolutionary degradation pathway that transports proteins or damaged organelles in animal and plant cells to vacuoles or lysosomes for degradation in the form of autophagic vesicles formed by double membranes,so as to achieve intracellular material circulation and maintain the stability of the intracellular environment in response to biological and abiotic stresses.Recent studies have shown that autophagy plays important roles in the process of virus infection in plants.Autophagy can participate in the defense against virus infection in plants,or collaborates with viruses to facilitate their infections.Previous studies in our laboratory have shown that autophagy can be induced by rice stripe virus(RSV)infection and plays an important role in the defense against RSV infection by degrading RSV P3 protein.However,the mechanism of autophagy activation by RSV infection remains unclear.In this study,we found that the previously identified eukaryotic initiation factor 4A(eIF4A),which was a target gene regulated by RSV-derived viral small interference RNA(vsiRNA),can negatively regulate autophagy during RSV infection.The main results are as follows:1.Previous studies have found that eIF4 A gene was targeted and cleaved by RSV-derived vsiRNA-4A,which resulted in impaired mRNA expression of eIF4 A and to further understand the role of eIF4 A gene in the process of RSV infection,we used tobacco rattle virus(TRV)-mediated gene silencing(virus-induced gene silencing,VIGS).The results showed that,RSV infection symptoms were relieved on NbeIF4A-silenced plants and the accumulation of virus was significantly reduced compared with the control,indicating that RSV infection was inhibited on NbeIF4A-silenced plants.In order to understand the possible mechanisms of impaired RSV infection in NbeIF4A-silenced plants,we analyzed that transcription level of key genes in plant immune pathways by RT-q PCR,including salicylic acid(SA),jasmonic acid(JA),reactive oxygen species(ROS),26 S proteasome,RNA silencing,and autophagy pathways.Our results revealed that the transcription level of autophagy genes ATGs(NbATG2,NbATG3,NbATG5,NbATG6,NbATG7 and Nb PI3K)were significantly induced.Considering that autophagy has been reported to play a defensive role in RSV infection,thus,we hypothesized that autophagy pathway might play a role during RSV infection in NbeIF4A-silenced plants.We further found that the number of autophagosomes was significantly increased on NbeIF4A-silenced plants.Consistently,protein degradation of RSV P3(suppressor silencing)was also significantly enhanced in NbeIF4A-silenced plants.In addition,the replicase protein NIb of turnip mosaic virus(Tu MV)was also degraded in NbeIF4A-silenced plants.These results suggest that autophagy pathway was activated in NbeIF4A-silenced plants.To further prove that autophagy plays a role in RSV infection on NbeIF4A-silenced plants,we silenced the key autophagy gene NbATG3 or NbATG5,together with NbeIF4 A,and monitored RSV infection in these plants.We found that co-silencing of these genes significantly inhibited the resistance of NbeIF4A-silenced plants to RSV infection.These results demonstrate that the impaired RSV infection in NbeIF4A-silenced plants is dependent on the autophagy pathway.2.To further clarify the molecular mechanism of eIF4 A in autophagy regulation,we analyzed the protein-protein interaction between eIF4 A and important proteins of autophagy by Yeast two-hybrid(Y2H),Bimolecular fluorescence complementation(BiFC),Co-immunoprecipitation(Co-IP)and Luciferase complementation imaging(LCI).Our results showed that NbeIF4 A interacted with the autophagy key protein NbATG5.Moreover,the 71-241 amino acid in NbeIF4A(71-241)is responsible for its interaction with NbATG5.Transiently expression of NbATG5 can activate autophagy,while overexpression of NbeIF4 A can inhibit NbATG5-mediated or MV-induced autophagy activation,which relies on the NbATG5-NbeIF4 A interaction.Further studies showed that the interaction between NbeIF4 A and NbATG5 interfered with the protein-protein interaction strength between NbATG5 and NtATG12,thereby preventing the formation of ATG5-ATG2 complex.3.Since previous studies have shown that NbeIF4 A is targeted by RSV vsiRNA and leads to down-regulation of its mRNA,we analyze that the ability of vsiRNA expression to activate autophagy.Here,we found that the expression of vsiRNA-4A can indeed activate autophagy,however,the overexpression of NbeIF4 A can inhibit the autophagy activated by vsiRNA-4A,indicating that the production of RSV vsiRNA can activate autophagy through down-regulating the expression of NbeIF4 A.In this study,we also found that the rice(Oryza sativa)eIF4A(OseIF4A)and NbeIF4 A shared 74.6% and 81% identities in nucleotide and aa level,respectively.Moreover,showing that the interaction OseIF4 A and OsATG5.The activity of autophagy induced by OsATG5 expression was suppressed by OseIF4 A.The result shows OseIF4 A and NbeIF4 A have similar functions in autophagy regulation.In summary,this research reveals the molecular mechanism of eIF4 A serving as a negative regulator of autophagy,in RSV infection.Our results reveal eIF4 A mRNA is targeted by RSV vsiRNA for cleavage,thereby blocking its inhibition of ATG5 and activating autophagy.This study provides a new theory in the role of interactions between RSV and autophagy in viral infection,and lays a foundation for the development of effective virus prevention and control strategies. |
