Mechanisms Of Ubiquitination Of SVBV P6 Mediated With ZFP Of Woodland Strawberry(Fragaria Vesca)

Interaction between Virus pathogenic factors and host factors affects on virus pathogenicity is always an hotspot in molecular biology of viruses.It was demonstrated in our research that protein P6 of SVBV is a RNA silencing suppressor and a symptom determinant which closely related to the virus pathogenicity.But so far,there haven't seen any research about the interactions between SVBV P6 with strawberry proteins.The yeast cDNA library of the Fragaria vesca infected with SVBV was constructed and protein P6 was used as a bait to screen out a host factor Zinc finger protein ZFP which interacts with P6.Throughing yeast two-hybrid assay,bimolecular fluorescence complementation assay and Co-immunoprecipitation assay,we demonstrated that SVBV P6 interacted with ZFP.we proved that ZFP can affect the P6 protein aggregation,expression and influences the symptom of PVX mediated by P6 in N.Benthamiana.Proteasome inhibitor MG132 can inhibits P6 protein degradation mediates by ZFP.This study lays a theoretical foundation in revealing the molecular mechanism of the interaction of SVBV P6 with strawberry ZFP and the defense and counter-defense mechanism between host plant and DNA virus.1 Verifying the interaction between P6 and ZFP proteinVerifying the interaction between P6 and ZFP by yeast two-hybrid assayPlasmids p GAD-ZFP+pGBK-P6 and pG AD-P6+pGBK-ZFP were cotransformed into yeast.The experiment results show that plasmids pG AD-ZFP+pGBK-P6 and p GAD-P6+pGBK-ZFP were cotransformed into yeast cells which has a capability of growing on Select medium plate.Indicating that P6 could interacts with ZFP.Verifying the interaction between P6 and ZFP by bimolecular fluorescenc e complementation A.tumefaciens cells containing the constructs pCV-nYFP-P6+pCV-cYFP-ZFP and pCV-nYFP-ZFP+pCV-cYFP-P6 were infiltrated into N.B enthamiana.The results show that only fluorescence can be seen in N.bentham ianaleaves epidermal cells with A.tumefaciens cells containing plasminds pCV-n YFP-P6+pCV-cYFP-ZFP and pCV-nYFP-ZFP+pCV-cYFP-P6,indicating that P6 could interacts with ZFP in plant cells.Verifying the interaction between P6 and ZFP by Co-immunoprecipitation assay A.tumefaciens cells containing the constructs pCM2300-P6-GFP and pCM2300-ZFP-mCherry were infiltrated into N.Benthamiana.After the Co-IP assay, western blot could detected P6 and ZFP,indicating that P6 could interacts with ZFP in plant cells.2 Fragaria.vesca ZFP gene sequence comparison and phylogenetic tree an alysisSequence similarity comparison of Fragaria.vesca ZFP gene and other plants of ZFP gene were logged in GenBank.Results showed that there was high similarity of Fragaria.Vesca ZFP amino acid sequence between Plum blossom,Pyrus bretschneideri and Malus pumila and the ratio is 88.6% to 88.8%,and low similarity with other plants and the ratio is 76.7% to 76.7%.The phylogenetic tree of Fragaria.Vesca ZFP protein amino acid sequence and other plants species was constructed.The result shows that Fragaria.Vesc a ZFP amino acid sequence and other Rosaceae plants like P.mume,P.Bretsc hneideri,and M.Domestica together to a cluster.The Fragaria.Vesca ZFP am ino acid sequence was closest relative to other Rosaceae plants.3 Differentially expressed of ZFP mRNA in Fragaria.Vesca infected by SVBVFragaria.Vesca was infitrated with SVBV and EHA105 repectively,25 days later.Real-time qPCR analysis the exprssion differences of ZFP between infected by SVBV and EHA105.The results shows that SVBV can up regulates the exprssion of ZFP mRNA.4 Intracellular subcellular localizations of ZFP and co-localization with P6A.tumefaciens cells containing the constructs pCM2300-P6-GFP and pCM2300-ZFP-mCherry were infiltrated into N.Benthamiana.The results indicates that ZFP can locals in cytoplasm and nucleus,P6 co-localization with ZFP in the nucleus and the boundary of the cell.5 Fragaria.Vesca ZFP mediates P6 degration and ubiquitination in plant.ZFP protein affects P6 protein aggregation We use agrobacterium-mediated transient infiltration experiments,A.tumefaciens cells containing the plasmids p CAM2300-P6-GFP and p CM1307-ZFP-3*flag with OD value of the ratio of 1:2 were infiltrated into N.Benthamiana.And the A.tumefaciens carry the pCAM2300-P6-GFP and vector with the same ratio of OD as a negetive control,3 days later,confocal observation of P6 protein aggregation in plant cells.Results shows that whe co-infiltration with P6 and ZFP will significantly decreases the size and the number of P6 aggregation.So we come to the conclusion that ZFP can affects the P6 proteins aggregation in plants.ZFP can decrease the expression of P6 in plant A.tumefaciens cells containing the plasmids pCM1307-ZFP-3*flag and 2300-P6-GFP were infitrated into growing well N.Benthamiana,3 days later,the P6-GFP fluorescence was observed under uv lamp,it shows that the fluorescence of co-infitrated with pCM1307-ZFP-3*flag and 2300-P6-GFP were weaker than P6 with the vector,and we suggest that ZFP could affects the exprssion of P6.Further,we test the protein exprssion of P6 and extracts the protein of infitrated area,western blot assay shows that the expression of P6 was decreased by ZFP.Moreover,we detected the mRNA level of P6,the results shows that the level of P6 wasn't decreased.Verification the proteasome inhibitor can inhibits P6 protein degradation in vivo A.tumefaciens cells containing the plasmids pCAM2300-P6-GFP and pCM1307-ZFP-3*flag were infitrated into N.Benthamiana,after 48 hr,we use MgCL2 solution dilutes the ubiquitin/26 S proteasome inhibitor MG132 and CHX(cycloheximide)into 100 uM,then we infitated the dilution into the plants have been infitrated with P6 and ZFP.Negative control was used DMSO instead of MG132,16 hr after infiltrated,protein extracted by Shenggong RIPAII,western blot assay detected the exprssion of P6.The results shows that the MG132 can inhibits the ZFP protein mediates the degradation of P6.Detection of MG132 effect on the stability of P6 protein by semi-in vi vo assay We use agrobacterium-mediated transient infiltration experiments,A.tumefaciens cells containing the plasmids pCAM2300-P6-GFP and pCM1307-ZF P-3*flag,pCAM2300-P6-GFP sample was extracted in plant cell,P6 protein le vels were analyzed with anti-GFP antibody at different times.Compared with t he DMSO treatment,MG132 treatment of P6 protein degradation was slower t han DMSO.The results show that 26 S proteasome inhibitor MG132 can inhibit the degradation of P6 protein and the P6 protein degradation through the 26 S proteasome pathway.6 ZFP influences the symptom of PVX mediated by P6 on N.BenthamianaA.tumefaciens cells containing the plasmids pGR106-ZFP,pGR106-P6,pGR106,were infitrated into N.Benthamiana with certain proportion and two mixed bacteria liquid.Symptoms of comparative analysis of differences after 10 days,when pGR106-P6 combined with pGR106 empty infiltration,its symptoms similar to P6 mediated,but when pGR106-ZFP combined with pGR106-P6 infiltration,plants showing symptoms weaker than the symptoms P6.The results show that the ZFP influences the symptom of PVX mediated by P6 on N.Benthamiana.