Functional Analysis Of NbP3IPH In Turnip Mosaic Virus Infection In N. Benthamiana

Turnip Mosaic Virus(TuMV),a member of the genus Potyvirus,are mostly transmitted non-persistently by aphids,causing significant damage and loss in a variety of crops such as Brassica oleracea and cabbage.TuMV is a single-molecule single-stranded RNA(ss RNA)virus that encodes a polyprotein,and this polyprotein can be hydrolyzed into 10 types of viral proteins,including P1,P3,6K1,6K2,HC-Pro,NIa-Vpg,NIa-Pro,NIb,CI,CP,and P3N-PIPO proteins in the P3 coding region.Membrane protein 6K2 has been confirmed to be related to the replication vesicles of the virus,which can favor the virus infection by affecting the replication and movement of the virus.This study investigated the relationship between an unknown functional protein,NbP3 IPH,and TuMV in Nicotiana benthamiana,and analyzed its function and role in resisting TuMV infection.First,we analyzed the sequence and localization of NbP3 IPH,and found that NbP3 IPH is localized in the nucleus,cell membrane and chloroplast.Using prediction software,we predicted that the first 32 amino acids of the sequence are chloroplast signal peptides;and we found that expression level of NbP3 IPH was reduced after TuMV infection.The tobacco rattle virus(TRV)-induced gene silencing(VIGS)system was used to investigate the effect of NbP3 IPH silencing on TuMV infection.We found that the expression level of NbP3 IPH was reduced by60% in NbP3IPH-silenced plants and there is no significant change in plant phenotype.After the inoculation of TuMV,the viral symptom presented mild in wild plants compared with NbP3IPH-silenced plants,which indicated that NbP3 IPH silencing is beneficial for TuMV infection.Moreover,Western blot of viral protein confirmed that TuMV accumulated less on NbP3 IPH over-expressed side,which indicated that over-expression of NbP3 IPH helped plants to better resist TuMV infection.These results suggest that NbP3 IPH plays an important role in the process of resisting TuMV infection.Bimolecular Fluorescence complementation(BiFC),Yeast two hybrid(Y2H),Co-Immunoprecipitation(Co-IP)confirmed the interaction between NbP3 IPH and TuMV 6K2.Moreover,we found that the expression level of 6K2 was reduced after its co-expression with NbP3 IPH.Three inhibitor chemicals for autophagy(3-MA,E64 d and Pep A)were applied to identify the pathway to degrade 6K2 when NbP3 IPH and 6K2 were expressed together.And NbP3 IPH can activate autophagosome production.The interaction between NbP3IPH?6K2 and ATG5?ATG8f was verifiedusing BiFC and Y2 H technologies.Therefore,we suggested that the degradation of6K2 protein induced by NbP3 IPH depend on the autophagy pathway.In summary,our study clarified the mechanism of an unknown functional protein NbP3 IPH in resisting TuMV infection.NbP3 IPH interacts with 6K2 to mediate the degradation of autophagy pathway and participate in the plant's resistance to TuMV infection.This study has great value for deeper understanding of complicated relationship between virus and host interaction,viral pathogenicity,and plant antiviral pathway.