| Strawberry vein banding virus（SVBV）,a double-stranded DNA virus belonging to Caulimovirus,is the main virus infecting cultivated strawberry.Previous studies have shown that SVBV P6 protein is a multifunctional protein,which is closely related to the pathogenesis and infection of SVBV.SVBV P6 was used as bait protein to screen the yeast c DNA library of Fragaria vesca infected with SVBV.A protein with zinc finger structure was found to have the highest screening abundance,which was called FvZFP protein.In order to explore the function of FvZFP protein and the function of FvZFP protein in the process of SVBV infection,this study conducted an in-depth study of FvZFP protein.Luciferase complementation imaging assay proved that P6 and FvZFP could interact in vivo.Further studies showed that FvZFP protein had E3 ubiquitin ligase activity,and FvZFP protein could use P6 protein as a ubiquitin ligase substrate and degrade P6 protein by ubiquitin.FvZFP gene was transformed into a transgenic N.benthamiana strain overexpressing FvZFP gene.Transgenic N.benthamiana can inhibit the infection of PGR106-P6 induced by P6 and delay the occurrence of P6-induced symptoms.TRV was used as an expression system to construct TRV-FvZFP,and the FvZFP gene was overexpressed in F.vesca.It was found that the F.vesca lines with overexpression of FvZFP gene could significantly inhibit SVBV infection and delay the occurrence of SVBV symptoms.In addition,FvZFP also has DNA binding function.Electro mobility shift assay demonstrated that FvZFP could bind the SVBV 35S promoter.SVBV 35S-GFP was constructed and co-infiltrated with FvZFP in N.benthamiana.FvZFP could significantly inhibit the activation activity of SVBV 35S and reduce the expression of GFP.This study indicated that F.vesca zinc finger protein could inhibit SVBV infection through ubiquitination pathway at the protein level,and could also affect the activity of viral promoter,which might affect the accumulation of SVBV.This study provides an insight into the replication and infection of SVBV and the disease resistance of the host to SVBV,and lays a theoretical foundation for the study of plant resistance to DNA viruses.1.FvZFP protein has E3 ubiquitin ligase activityUsing LCI interaction system,n LUC-P6 and c LUC-FvZFP were constructed and co-infiltrated in N.benthamiana,which proved that P6 protein and FvZFP protein could interact systematically in vivo.According to previous laboratory studies,protease inhibitor MG132 can inhibit the degradation of P6 protein mediated by FvZFP protein,and this study verified whether FvZFP protein has E3 ubiquitin ligase activity.Prokaryotic expression of FvZFP protein,simulation of ubiquitin system in vitro,found that FvZFP protein can bind Ub molecule.Prokaryotic expression of FvZFP protein and P6 protein,in vitro simulation of the ubiquitin system,FvZFP protein can ubiquitin P6 protein.The results showed that the degradation of P6 protein mediated by FVZFP was achieved by ubiquitination pathway.2.FvZFP transgenic N.benthamiana can delay the occurrence of symptoms induced by P6 proteinFvZFP transgenic N.benthamiana was obtained by leaf plate method,and the function of FvZFP protein was further studied.There was no significant difference in phenotypes between F3 transgenic strains and wild-type strains.PGR106-P6 was constructed,and P6could induce severe mosaic symptoms in the leaves of N.benthamiana.Transgenic N.benthamiana could significantly inhibit the occurrence of mosaic symptoms induced by PGR106-P6.Western blot analysis showed that the expression level of P6 protein in transgenic N.benthamiana was significantly lower than that of wild type,while the expression level of P6 mRNA was consistent.The results showed that FvZFP inhibit P6protein accumulation,but had no effect on P6 gene expression at the RNA level,which proved that FvZFP inhibited P6 protein-induced symptoms via the inhibition of P6 protein accumulation.3.FvZFP overexpressed F.vesca inhibited SVBV infectionThe FvZFP gene of F.vesca was overexpressed by TRV The full-length FvZFP gene was cloned into TRV virus vector,and the FvZFP gene was highly expressed in the F.vesca by the systematic infection characteristics of the virus.Fluorescence quantitative PCR detection showed that FvZFP gene expression could be up-regulated 3-4 times after TRV-FVZFP infection in F.vesca 15,25,35 or 45 days after infection.Overexpression of FvZFP gene can inhibit SVBV infection The overexpression of FvZFP gene in F.vesca obtained by TRV was continued to be used for functional identification.An infectious clone of SVBV was inoculated again 15 days after TRV-FVZFP inoculation.After 32 days,the leaves of the control group showed severe mosaic symptoms.After 38 days,FvZFP overexpressing F.vesca showed milder mosaic symptoms.The nucleic acid and protein detection results showed that the accumulation of DNA and protein of SVBV in the experimental group was significantly lower than that in the control group,suggesting that FvZFP could participate in the infection process of SVBV and inhibit the infection of SVBV in F.vesca.4.FvZFP protein can affect the activity of SVBV 35S promoterP6 protein can bind to the SVBV 35S promoter The P6 protein encoded by cauliflower mosaic virus binds to the 35S promoter of the virus and initiates transcriptional translation of other viral genes.Based on the DNA binding region,the site prediction was carried out in this study,and the SVBV 35S promoter was divided into the first to the fourth segments,which were 1-237bp,238-491bp,492-697bp and 698-951bp,respectively,to meet the requirements of Electro mobility shift assay.The Electro mobility shift assay showed that the SVBV-encoded P6 protein could bind to the SVBV 35S promoter,which was consistent with the results of previous studies.The FvZFP protein can bind to the SVBV 35S promoter Our study demonstrated that P6 protein can interact with FVZFP protein,and P6 protein can bind to the SVBV 35S promoter.According to the above studies,it is speculated that the FvZFP protein may affect the binding of P6 to the SVBV 35S promoter.Electro mobility shift assay showed that FvZFP could also bind to SVBV 35S promoter,suggesting that FvZFP might inhibit the binding of P6 protein to SVBV 35S promoter.FvZFP inhibits the ability of SVBV 35S promoter to transcribe GFP In order to explore the association between FvZFP protein and SVBV 35S promoter,SVBV 35S-GFP was constructed and co-infiltrated with FVZFP in the leaves of N.benthamiana.The expression of GFP was observed by confocal analysis,and it was found that the fluorescence of GFP was strong when SVBV 35S-GFP was infiltrated alone.The fluorescence of GFP decreased significantly after co-infiltration with FvZFP.The results of protein detection were consistent with those of confocal observation. |
