Identification of Nanoviruses in banana from Pakistan and possible control through RNAi

Abstract

Banana Bunchy Top Virus (BBTV) is a member of genus Babuvirus of the family Nanoviridae, ssDNA virus transmitted by Pentalonia nigronervosa. Family Nanoviridae is divided into two genera: Nanovirus and Babuvirus. Nanovirus includes FBNYV, MDV, SCSV, while the genus Babuvirus include BBTV. In Pakistan, banana production is under severe loss due to BBTV. In the absence of natural resistance, the use of genetically engineered resistance is an attractive option. The main objective of this study was to develop resistance in banana against banana bunchy top virus through RNAi and the identification of unknown components of BBTV by a new technique called Rolling Circle Amplification (RCA). Rolling circle amplification (RCA) is a novel technique for the amplification of circular DNAs. This technique has been widely used for the amplification of geminiviruses but its use for the characterization of nanoviruses has not been reported. The identification of unknown component is also necessary to find out whether any additional component is associated with infectious unit or not. An analysis of the genetic diversity of BBTV was made by this valuable technique across Tando Jam, Sindh, Pakistan, to characterize components of banana bunchy top virus. The RCA product was digested with several restriction enzymes and was resolved in agarose gel. The resulting RFLP pattern resembled those expected for BBTV. In order to confirm the RFLP analysis, the DNA was probed with cloned components of BBTV. The probes for components DNA-S, DNA-N and DNA-M correctly hybridized to their respective fragment. We further cloned two components of BBTV to verify results. The cloned components were highly homologous to South Pacific group of BBTV as reported from Pakistan. The results of present studies confirmed that RCA technology can be used for characterization of nanoviruses. The technique is of great value to nanovirus research since the components that make up this group are still being discovered. This diversity (low) is also helpful in generating resistance against viruses. So, RNAi construct was made against MRep of BBTV to engineer resistance against BBTV. This construct was transiently checked in banana male flower bud. The buds agro-infiltrated with EHA105 gave better expression as compared to GV3101. Expression of BBTV genes from PVX and under 35S promoter was also observed. Expression of MRep and MP under PVX resulted in necrosis and cell death at the site of inoculation and severe leaf curling and necrosis in newly emerging vii leaves in MP. Clink, NSP and CP produced mild symptoms of leaf curling and mosaic, while CP produced necrotic response in inoculated leaves. When all these genes were expressed under 35S promoter in N. benthamiana 16c line, MP and Clink stabilized GFP specific mRNA and reduced GFP specific siRNA. MRep, NSP and CP did not show accumulation of GFP specific mRNA. These results identified that MP and Clink are supressors of silencing. The ability of MP to induce severe necrosis in inoculated and systemic leaves and RNA silencing suppressors indicates that MP is a major pathogenecity determinant in BBTV genome. Promoter regions of BBTV components may have application for heterologous transgene expression. Promoter regions of BBTV components were cloned in expression vector and checked it in N. benthamiana plants. Out of five components of BBTV, DNA-S, DNA-C and DNA-R did not show any GUS expression in N. benthamiana, while DNA-N showed some level of expression. The deletion of 200bp from 5’ end of DNA-N increased the promoter activity but was still low as compared to CaMV, 35S.