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Evaluation
of cowpea aphid-borne mosaic virus (CABMV) coat protein and RNA mediated
resistance strategies in Nicotiana
benthamiana
R. Mundembe
& I. Sithole-Niang
Pathogen-derived resistance to viruses has been very
successful for many virus groups, but has not been widely used to
control viral diseases of importance to farmers in developing countries.
Cowpea aphid-borne mosaic potyvirus (CABMV) is one of the
pathogens that significantly reduces cowpea yields of resource poor
farmers. The goal of this
study was to determine the most effective virus resistance mechanism
using the CABMV coat protein (CP) gene.
The CP gene was amplified using the reverse transcription-polymerase
chain reaction (RT-PCR) technique, incorporating an ATG translation
start codon and the Kozak consensus sequence for optimum expression in
plants, and cloned into a PCR cloning vector pCRII, to result in a
plasmid that was named pCRII-CPk. The CP gene fragment was excised from pCRII-CPk using Bam
HI and Sal I and cloned into a similarly digested expression cassette
vector pCa2Nos, between the promoter and terminator. The plasmid pCRII-CPk was also used as a template to amplify
an untranslatable CP gene with stop codons in all three reading frames (CPstop),
the CP gene in an anti-sense orientation (PC), and the central region of
the CP gene (CPcore). All
three amplification products were cloned into pCa2Nos, between the
promoter and terminator, and the entire insert was ligated into the
unique Hind III site of the
binary plasmid pBI 121, to result in plasmids pBI121-CPk, pBI121-CPstop,
pBI121-PC and pBI121-CPcore. The
constructs were used to transform Agrobacterium tumefaciens which was then co-cultivated with Nicotiana
benthamiana leaf sections. The
regenerated plants were analyzed by PCR and Southern blotting, and the
seeds were germinated on kanamycin and challenged with CAMBV-infected
sap, viral RNA and whole virions.
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