Molecular Mechanism Of Nuclear Inclusion Protein B Regulating The Replication Of Tobacco Vein Banding Mosaic Virus

Potyvirus is the largest genus of plant viruses and consists of more than 200 virus species.Typical among them are potato virus Y(PVY)and tobacco vein banding mosaic virus(TVBMV)which cause huge economic losses to agricultural production in the world.Planting disease-resistant varieties is the most economical and effective way to prevent and control viral diseases,but there is a lack of effective disease-resistant varieties in production.Identifying host factors that interact with plant viruses not only clarify the molecular mechanism of virus infection,but also cultivate virus-resistant varieties through gene editing.In addition,screening attenuated mutants and applying them to cross-protection is also an effective strategy to resist viral diseases.This study analyzed the molecular mechanism of TVBMV nuclear inclusion protein b(NIb)and host factors interacting to regulate virus replication.The main findings are as follows:(1)This study determined the molecular mechanism by which NIb recruited NbRPL1 to regulate TVBMV replication.50 S ribosomal protein large subunit 1 of Nicotiana benthamiana(NbRPL1)interacted with TVBMV replicase NIb.NbRPL1 was located on the chloroplast,and its N-terminal 61 amino acids is a chloroplast transit peptide(c TP),which is cleaved in plants.Moreover,the intact c TP was essential for the NbRPL1 and NbRPL1/ NIb interaction complex to target the chloroplast.The NbRPL1/ NIb interaction complex co-localized with 6K2-induced vesicles,but could not localize to the nucleus and plasmodesmata.In TVBMV infection,NbRPL1 was recruited into the chloroplast-associated TVBMV-induced replication complex(VRC).Silencing of NbRPL1 inhibited the replication and coat protein accumulation of TVBMV,thereby inhibiting the virus systemic infection.Silencing of NbRPL1 did not affect the targeting of replication-related protein 6K2 to chloroplasts,but affected the distribution of NIb in VRC.Overexpression of NbRPL1 promoted virus replication and NIb protein accumulation,but did not affect virus translation.TVBMV infection activated autophagy,and autophagy-related genes,including Beclin1 were up-regulated.The autophagy receptor protein NbBeclin1 also interacted with TVBMV NIb and promoted the degradation of NIb,thereby inhibiting virus replication.Overexpression of NbRPL1 reduced the degradation of NIb protein mediated by NbBeclin1 and promoted the accumulation of viral RNA.NbRPL1 and NbBeclin1 competitively bind to NIb in vitro.TVBMV recruits NbRPL1 to chloroplast-related VRC to stabilize NIb,thereby positively regulating virus replication;while plants recognize and degrade NIb through NbBeclin1,thereby negatively regulating virus replication.(2)This study determined the molecular mechanism by which NIb recruited NbANXD1 to regulate virus infection.We determined that annexin D1 of Nicotiana benthamiana,NbANXD1,interacted with TVBMV replicase NIb through bimolecular fluorescence complementation(Bi FC),yeast two-hybrid(Y2H)and co-immunoprecipitation(Co-IP).Y2 H assay showed that NbANXD1 also interacted with the helicase CI of TVBMV.There were four repeated ANX domains in NbANXD1,and a single ANX domain did not interact with NIb.NbANXD1 interacted with the N-terminus and C-terminus of NIb,but did not interact with the middle region of NIb.In TVBMV-infected cells,NbANXD1 was enriched at VRC,and the NbANXD1/ NIb complex was co-localized with VRC.Silencing of NbANXD1 reduced the systemic infection of TVBMV and PVY.Moreover,ATPase interacted with NbANXD1,silencing of ATPase slowed down the TVBMV infection.In summary,TVBMV hijacks the host factors NbRPL1,NbANXD1 and NbATPase for virus replication and movement,whereas the host plants utilize the NbBeclin1 to resist virus infection.