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
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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.