Functional Analysis Of CP And TGB Proteins Of Apple Stem Pitting Virus In Viral Movement And Pathogenicity

Apple stem pitting virus is the type species of the genus Foveavirus in the family Betaflexiviridae.The virus commonly infects apple and pear.In some susceptible apple and pear plants,ASPV infection can induce vein yellowing,red mottling and necrotic spots on leaves,stony pits on fruits,and stem xylem pits.The co-infection of the virus with other viruses seriously affects the growth of apple and pear plants.In this study,we investigated the function of coat protein(CP)and triple gene block proteins(TGBps1-3)encoded by ASPV in viral replication,movement,and pathogenesis.The main research results are as follows:1.The split-ubiquitin-based membrane yeast two-hybrid and the bimolecular fluorescence complementation assays revealed that homologous interactions occurred within each of proteins TGBp1,TGBp2,TGBp3,and CP,and potential heterologous interactions were observed between TGBp2 and each of the three proteins TGBp1,TGBp3 and CP.Subcellular localization analysis showed that TGBp2 altered the location of TGBp1 and CP in Nicotiana benthamiana leaf cells,and TGBp2,TGBp1 and CP co-located in the perinuclear aggregates,whereas TGBp3 recruited the TGBp2/TGBp1/CP complex to its induced mobile circular vesicles.Within ASPV-infected N.benthamiana leaf cells,each of TGBp1,TGBp2,and CP localized to circular vesicles,indicating that TGBp3 encoded by viral genomic RNA recruited these three proteins into circular vesicles.The treatments with chemical inhibitors demonstrated that the formation and intracellular transportation of circular vesicles depended on ER and microfilaments.TGBp1,TGBp2,and CP have the ability to increase the size exclusion limit(SEL)of PD,TGBp1 and CP themselves can pass through PD,and TGBp1 can help TGBp2 pass through PD.TGBp3 does not have the ability to regulate SEL of PD or intercellular movement.2.Results showed that TGBp3-e YFP altered the m Cherry-HDEL-labeled ER structures in N.benthamiana leaf cells,and formed ER granules with circular envelop.Further treatment with a chemical thapsigargin showed that circular vesicles induced by TGBp3 completely relied on cell ER structures.Results also showed that the TGBp3 transmembrane domain was a critical region for the protein to remodel the ER and induce vesicles formation.Transmission electron microscopy(TEM)revealed the presence of vesicles with diameters of 200-500 nm inside N.benthamiana leaf cells transiently expressing TGBp3 and infected with ASPV.Electron dense vesicles and multivesicular bodies also presented within N.benthamiana cells infected with ASPV and viroplasm-like structures at the perinuclear and pericellular were observed.When the ds RNA tagged proteins were coexpressed with TGBp3-m Cherry in ASPV-infected N.benthamiana leaves,it was found that TGBp3-m Cherry partially colocalizated with ds RNA tagged proteins in perinuclear and pericellular aggregates.Confocal and TEM observations revealed that TGBp3 expression and ASPV infection altered perinuclear membrane structures in N.benthamiana cells.These results suggested that TGBp3 induced vesicles were associated with viral replication.3.Results showed TGBp3-e YFP labeled vesicles colocalized with the COPII coat protein Sec24,indicating that the generation of TGBp3-vesicles was dependent on the N.benthamiana COPII mechanism.Transcriptome combined with RT-q PCR analysis revealed that the expression level of the COPII-associated factor Nb UNC93 was significantly upregulated in N.benthamiana leaves transiently expressing TGBp3.Bi FC and luciferase complementation assays showed that TGBp3 interacted with Nb UNC93.In N.benthamiana plants with downregulated the expression level of Nb UNC93,the number of TGBp3-vesicle and the accumulation of ASPV were significantly reduced,indicating that the replication and transportation of ASPV in plants depend on the packaging of viral components to COPII vesicles by Nb UNC93.4.The ASPV encoded CP induced ROS accumulation in both pear and N.benthamiana leaves.By using yeast two hybrid,a CP interacting immune related factor TLP was identified from a pear c DNA library.Transcriptome analysis showed that CP upregulated the expression of Nb TLP.The accumulation level of ASPV in N.benthamiana plants with a down regulated Nb TLP level was significantly increased,indicating that Nb TLP negatively regulated the infection of ASPV and could be a potential antiviral factor.It was found that the interaction intensity of CP from the two ASPV isolates with Nb TLP was negatively correlated with the degree of HR response in N.benthamiana leaves,implying that the interaction of the viral CP with TLP interfered the antiviral function of the latter protein.This study revealed the mechanism of ASPV-encoded CP and TGBps in the virus replication and movement,and identified a host factor Nb TLP,which negatively regulated the ASPV infection.The obtained results provide important information for dissecting the infection mechanism of ASPV.