Silencing Mechanism And Infecting Identification Of China Isolate Of Strawberry Vein Banding Virus

Strawberry vein banding virus(SVBV) P6 protein encoded by ORF VI has been proved to be a silencing suppressor, can inhibit local RNA silencing on Nicotiana benthamiana 16 c line which induced by single strand GFP, while the mechanism is not very clear so far. In this paper, we investigate the mechanism how P6 inhibits gene silencing. It is identified that P6 can not inhibit gene silencing induced by double strand GFP(ds GFP), and also can not locally revert GFP gene on silenced N. benthamiana 16 c line. We clone the complete genome of SVBV isolate of Shenyang(SVBV-SY) and SVBV isolate of Beijing(SVBV-BJ), construct infectious clone of SVBV-SY and SVBV-US, and reveal their infection difference of the phenotype on woodland strawberry(Fragaria vesca). Meanwhile, we construct SVBV infectious clone with P6 gene frameshift mutated, validate that P6 is a essential component for infecting.Subsequently, SVBV virus vector is constructed, and successfully infect woodland strawberry, providing a useful tool to research gene function of virus and strawberry.1. SVBV P6 cannot inhibit RNA silencing induced by ds GFP Agrobacterium tumefaciens which contain recombinant plasmid p CAM-P6,p CAM-GFP and p CAM-ds GFP, respectively, mix together and co-infiltrate N.benthamiana. Not any manifest green fluorescence can be observed on infiltrated sites of leaves, under UV light 3 days post-infliltration(dpi). It is demonstrated that SVBV P6 cannot not inhibit RNA silencing induced by ds GFP.2. SVBV P6 cannot locally retrieve RNA silencing of GFP gene on silenced N.benthamiana 16 c line A. tumefaciens which contain recombinant plasmid p CAM-GFP infiltrate N.benthamiana 16 c line plant. Green fluorescence of the plant totally vanish, and turn red thoroughly, under UV light 30 dpi. The GFPgene is totally silenced.A. tumefaciens containing recombinant plasmid p CAM-P6 infiltrate the GFP silenced N. benthamiana 16 c. Not any green fluorescence can be observed on infiltrated sites of leaves, under UV light 3 dpi. A. tumefaciens containing recombinant plasmid p CAM-P6 and p CAM-GFP, respectively, co-infiltrated the GFP silenced N. benthamiana 16 c. Green fluorescence also cannot be observed on co-infiltrated sites, under UV light 3 dpi. It is demonstrated that SVBV P6 cannot not locally retrieve RNA silencing of GFP on silenced N. benthamiana 16 c line.3. Complete genome cloning of SVBV-SY and SVBV-BJ Strawberry samples which be infected with SVBV, are collected, and their total DNA is prepared to use. SVBV-SY and SVBV-BJ genome fragments is amplified by PCR,respectively. Complete genome clone, p SVBV-SY and p SVBV-BJ, are constructed by linking fragments together. SVBV-SY contain 7, 864 base pair(bp), and Gen Bank accession number is KP311681. SVBV-BJ contain 7, 863 bp, and Gen Bank accession number is KR080547.The alignment and analysis of the genomic sequences, It is found that SVBV-SY shares a high identification(97.8%) with that of SVBV-BJ, and also share relative high similarity(85.7%~94.2%) with that of other two isolates of SVBV, while share low similarity(43.5%~45.9%) with that of other members of genus Caulimovirus.Subsequently, nucleotide sequences alignment of ORFs of SVBV-SY and SVBV-BJ with other SVBV isolates, the results show that the ORF II comprise the highest variation, the similarity is 74.3%~75.1%. And ORF V share the minor difference, the similarity is88.8%~97.6%.4. Construct infectious clone and validate infectivity of SVBV-SY and SVBV-US By using complete genome clone p SVBV-E3, SVBV-US infectious clone p BIN-1.25SVBV-US is constructed successfully, transform into A. Tumefaciens GV3101,and inoculate F. vesca, F. × ananassa, N. tabacum, N. benthamiana, N. tabacum var.Samsun NN and N. glutinosa to validate its infectivity. The F. vesca menifest vein banding symptom on leaves 8 weeks post-inoculation. But no symptom could be observed on F. × ananassa and 4 species of Nicotiana plants, at the same time. It is indicated that infectious clone of SVBV-US can infect F. vesca and F. × ananassa with high-performance, but it can not infect 4 species of Nicotiana plants, through PCR detection.By using complete genome clone p SVBV-SY, SVBV-SY infectious clone p BIN-1.25SVBV-SY is constructed successfully, transform into A. Tumefaciens GV3101,and inoculate F. vesca and F. × ananassa to validate its infectivity. F. vesca manifest leaves twist, early blossom, and plant debilitating symptoms, 8 weeks post-inoculation.And no obvious symptom could be observed on F. × ananassa at the same time. It is indicated that infectious clone SVBV-SY can infect F. vesca efficiently, but it can not infect F. × ananassa through PCR detection.5. Construct and validate of SVBV P6 frameshift mutated infectious clone Frameshift mutation of SVBV P6, and the recombinant plasmid p SVBV(?P6), SVBV genome clone with mutation, was constructed. Subsequently, P6 frameshift mutated infectious clone of SVBV was successful constructed, the recombinant plasmid p BIN-1.25SVBV(?P6). And no symptom could be observed on the F. vesca, which inoculated with mutated infectious clone of SVBV-US after 8 weeks. SVBV cp gene cannot be detected by PCR method. It is proved that P6 frameshift mutation lead to the loss of infectivity of SVBV.6. Construction of SVBV vector The recombinant plasmid p SVBV-MCS was constructed through replacing the ORF II fragment of p SVBV-E3 with multiple cloning sites. Subsequently, SVBV vector p BIN-1.2SVBV-MCS was successful constructed and inoculated on F. vesca. No obvious symptom could be observed on the F. vesca 8 weeks post- inoculation, which inoculated with SVBV vector. While SVBV cp gene can be detected from these samples by PCR method. Part of the Fv Su gene of F. vesca was inserted into p BIN-1.2SVBV-MCS and inoculated F. vesca. The F. vesca plant also did not present vein banding symptom 8weeks post-inoculation. However, cp gene and the inserted Fv Su gene can be detected in the new emerged leaves by PCR method. It is demonstrated that SVBV vector could infect F. vesca.