Subcellular Localization Of Banana Bunchy Top Virus Rep/RepA And Their Promoters Activity Analysis

Banana bunchy top disease caused by Banana bunchy top virus(BBTV)is an important viral disease that threatens the development of banana industry.BBTV is the type member of the genus Babuvirus of the family Nanaviridae.There are six major components in the BBTV genome,and some isolates also contain 2?3 satellite components.The Rep protein,encoded by DNA-R,plays a role in the rolling circle replication of the viral genome by cutting and ligating in the stem-loop structure of the conserved region in each component.The RepA protein encoded by satellite components can only cut and ligate the stem-loop structure region of their own components.A Haikou BBTV isolate containing a S4 satellite DNA(B2)was used for this study.Subcellular localization techniques were used to analyze the localization of Rep and RepA in Nicotiana benthamiana leaf cells and to characterize the promoter function of the UTRs of the Rep and RepA components.The RNA/DNA ratio was analyzed by fluorescent quantitative PCR,and was used to infer the relative activity of the gene promoter(compared to the 35S promoter).Results are summarized as follows:(1)The Rep gene and RepA gene were cloned by PCR from the total DNA of the BBTV Haikou isolates archived in the laboratory.Further analysis revealed that both Rep and RepA proteins contain a conserved viral Rep domain at the N-terminus,which is involved in the cleavage and ligation of the viral genome.Based on the property of this domain,Rep and RepA proteins are predicted to localize in the nucleus and function there.In addition,Rep and RepA proteins contain a conserved P-loop-NTPase domain at their C-terminus,which provides energy in the cutting and ligation of the viral genome.(2)In order to explore the localization of the Rep and RepA proteins,876 bp of Rep gene and 852 bp of RepA gene were obtained by PCR amplification,and cloned into the Ti-plasmid GV1300 upstream of and in frame with the eGFP ORF.After infiltration,the green fluorescence in epidermal cells of N.benthamiana leaves was observed under a confocal microscope.The Rep-GFP and RepA-GFP fusion proteins were found in the nucleus of the epidermal cells of N.benthamiana leaves.The results showed that the BBTV Rep and RepA proteins were localized to the nucleus in N.benthamiana.(3)In order to explore whether the UTRs of BBTV Rep and RepA components initiate DNA transcription,the UTRs were cloned upstream of a GFP reporter gene by overlapping PCR in pCAMBIA3300.The vectors contain 387 bp Rep UTR and 360 bp RepA UTR,respectively.Agrobacterium infiltration method was used to study the promoter activities in the epidermal cells of N.benthamiana leaves.Confocal microscopy showed that the GFP gene linked with the promoter of Rep or RepA gene produced obvious green fluorescence in the nuclei and cytoplasm of the leaves of N.benthamiana,indicating that both UTRs contained promoters that can initiate DNA transcription and allow the expression of the downstream reporter gene(GFP).(4)In order to further determine the relative promoter activities of the Rep and RepA genes,fluorescent quantitative PCR analysis of the GFP gene at the DNA and RNA levels were performed.When the 35 S promoter activity,the RNA/DNA ratio of the GFP gene linked to the Rep gene promoter was 0.345,and the RNA/DNA ratio of the GFP gene linked to the RepA gene promoter was 0.696.The results indicated that the promoter activity of the RepA gene is higher than that of the Rep gene promoter,which is about 70%and 35%of the 35S promoter activity,respectively.This study identified the localization and promoter function of BBTV Rep and RepA in native tobacco cells,laying an important scientific foundation for further study of its mechanism of action.