Autophagy Functions As An Antiviral Mechanism Against Tomato Spotted Wilt Virus In Host Plants

Autophagy is an evolutionary conserved intracellular degradation pathway,by which cytoplasmic constituents,including proteins and dysfunctional organelles,delivered to lysosomes or vacuoles for degradation.In plants,autophagy is increasingly recognized for its central importance in development,reproduction,metabolism,hormone signaling,cell death,senescence,and responses to abiotic and biotic stresses.Autophagy plays important roles in regulating immunity-related cell death,antiviral and promoting viral pathogenesis during plant-virus interactions,but the role of autophagy on Tomato spotted wilt virus is unclear.Tomato spotted wilt virus(TSWV)is an important member of Tospovirus and belongs to the Bunyaviridae family.Among the five genera of Buniaviridae,Tospovirus is the only genus that infects plants,and its representative species TSWV is a plant polysegment negative sense strand virus.Among the five genera of Buniaviridae,the TSWV genus is the only one that infects plants,causing serious harm to agricultural production worldwide.In order to discuss the significant roles of autophagy in the defense and counter-defense arm race between host plants and TSWV,this paper analyzed the high-throughput transcriptome of Arabidopsis thaliana infected with TSWV and found that a large number of autophagy-related(ATG)genes appeared up-regulated after virus infection.Quantitative real-time reverse-transcription PCR(qRT-PCR)detected that the expression levels of autophagy-related genes AtATG5 and AtATG7 were significantly up-regulated after TSWV infection.After TSWV infected Arabidopsis thaliana,a large increase in the number of autophagosomes was observed under the microscope.Overexpressed proteins of TSWV activates autophagy,and the number of autophagosomes produced by overexpressing NSs protein is the highest.Therefore,the infection of TSWV activates the autophagic response of the host plant.Virus-induced gene silencing showed that autophagy-related genes NbATG5 and NbATG7 reduced the resistance of plants to TSWV.The autophagy activated by the host plant has an antiviral effect on TSWV.Furthermore,through bimolecular fluorescence complementation(BiFC)and Co-immunoprecipitation(Co-IP)analysis,it was found that the core component of autophagy AtATG6 can interact with NSs protein in vivo,and overexpression of AtATG6 can inhibit the gene silencing suppressor activity of NSs protein.Through subcellular localization studies,it was observed under a laser confocal microscope that the complex of AtATG6 and NSs could be localized on autophagosomes.Western blot analysis showed that AtATG6 can degrade NSs.In addition,AtATG6 can degrade N.Through BiFC and Co-IP analysis,AtATG6 also interacts with the mobile protein NSm.Using the TSWV movement mini-replicons reverse genetics system,overexpression of AtATG6 can affect the intercellular movement of the virus.These results indicate that the autophagy gene AtATG6 plays an important antiviral regulatory mechanism during TSWV infection in plants.The results of the study revealed the antiviral regulatory mechanism of autophagy in the interaction between plants and multipart negative-strand RNA viruses,and improved the understanding of the relationship between negative-sense RNA viruses and plant autophagy.