Construction Of SVBV-AH Infectious Clone And The Mechanism Of Interaction Between RD21 Of Woodland Strawberry And SVBV P6

Strawberry vein banding virus(SVBV)is a member of the genus Caulimovirus infecting Fragaria species.In this study,the full-length genome of SVBV isolated from Anhui Province,construct infectious clone of SVBV-AH,and reveal the phenotype of the clone infection on woodland strawberry(Fragaria vesca).P6 protein encoded by SVBV has been proved to be a silencing suppressor,transactivator and 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 Fragaria vesca infected with SVBV was constructed,SVBV P6 was used as a bait to screen out a host factor cysteine proteinase RD21.Using yeast two-hybrid assay(Y2H),we demonstrated that SVBV P6 interacted with RD21 in vitro.we proved that RD21 can affect the P6 protein expression in N.Benthamiana.The results provide a basis for further understanding the molecular mechanism of interaction between SVBV P6 protein and strawberry RD21 and the defense and anti-defense mechanisms between host plants and viruses.1 Complete genome cloning of SVBV-AHTotal DNA extracted from strawberry samples which be infected with SVBV from AnHui Changfeng.SVBV-AH fragment was separatly amplified by using PCR,and the full length pSVBV-AH genome was obtained by linking fragments together.The genomic sequence of SVBV-AH is 7862 base pair(bp),and GenBank accession number is KX787430.The alignment and analysis of the genomic sequences,SVBV-AH was highly similar to SVBV-BJ and SVBV-SY genome sequence,reaching 97.7% and 98.5%,respectively.The sequence similarity with SVBV-US was 85.6%,while The full-length genome sequence of SVBV-AH share low similarity with that of other members of genus Caulimovirus,reaching only 43.4%~44.7%.Subsequently,comparing the nucleotide sequences of SVBV-AH and SVBV-US,we found that the difference of ORF II sequence between SVBV-AH and SVBV-US was highest variation,and the similarity was only 69.6%.And ORF V share the minor difference,the similarity was 86.5%,indicating that variation of SVBV in the process of adapting to strawberry due to geographical isolation.2 Construct infectious clone and validate infectivity of SVBV-AHBy using complete genome clone SVBV-AH,infectious clone pBIN-1.25 AHSVBV issuccessfully constructed,transform into A.Tumefaciens GV3101,and inoculate F.vesca to validate its infectivity.After 6 weeks,it was found that the F.vesca inoculated with SVBV-AH infection clone showed obvious vein banding symptom,while no symptom could be observed on the cultivated strawberry inoculated with pBINPLUS.Southern blot analysis showed that SVBV-AH clone could effectively infect forest strawberry.3 Verifying the interaction between P6 and RD21 by yeast two-hybrid assayPlasmids pGBK-P6 +pGAD-RD21 and pGBK-P6+pGAD-RD21 were cotransformed into yeast.The transformants were incubated on SD/-Trp/-Leu mediumand SD/-Trp/-Leu/-His/-Ade/-Glu medium.The experiment results show that plasmids pGAD-RD21+pGBK-P6 and pGAD-P6+pGBK-RD21 were cotransformed into yeast cells which has a capability of growing on Select medium plate.Indicating that P6 could interacts with RD21.4 Fragaria.vesca RD21 gene sequence comparison and phylogenetic tree an alysisSequence similarity comparison of Fragaria.vesca RD21 gene and other plants of RD21 gene were logged in GenBank.Results showed that there was high similarity of Fragaria Vesca RD21 nucleotide sequences between Prunus mume,Malus x domestica,Prunus avium,Pyrus x bretschneideri and Prunus persica and the ratio is 83.5% to 84.8%,and low similarity with other plants and the ratio is 67.4% to 76.5%.The phylogenetic tree of Fragaria.Vesca RD21 nucleotide sequenceand other plants species was constructed.The result shows that Fragaria.Vesca RD21 amino acid sequence and other Rosaceae plants like P.mume,M.domestica,P.avium,P.bretschneideri,and P.persica together to a cluster.Fragaria.Vesca RD21 amino acid sequence was closest relative to other Rosaceae plants.5 Differentially expressed of RD21 mRNA in Fragaria.Vesca infected by SVBVFragaria.Vesca was infitrated with SVBV and GV3101 repectively,35 days later.Real-time qPCR analysis the exprssion differences of RD21 between infected by SVBV and GV3101.The results shows that SVBV can up regulates the exprssion of RD21 mRNA.6 Intracellular subcellular localizations of RD21 and co-localization with P6A.tumefaciens cells containing the constructs pCM2300-RD21-GFP and pCM2300-P6-RFP were infiltrated into N.Benthamiana.The results indicates that GFP can locals in cytoplasm and nucleus boundaries,P6 co-localization with RD21 in the boundary of the cell.7 Cysteine Protease RD21 can inhibit the expression of P6 in plantA.tumefaciens cells containing the plasmids pGR106-P6-GFP+pGR106 and pGR106-P6-GFP+pGR106-RD21 were separately co-infitrated into growing well N.Benthamiana,4 days later,the P6-GFP fluorescence was observed under uv lamp,it shows that the fluorescence of co-infitrated with pGR106-P6-GFP + pGR106-RD21 were weaker than pGR106-P6-GFP+pGR106,and we suggest that RD21 could affects the exprssion of P6.Further,we test the protein exprssion of P6 and extracts the protein of infitrated area,western blot and northern blot assay shows that the expression of P6 was decreased by RD21.