Characterisation of Cowpea aphid-borne mosaic virus (CABMV) and evaluation of pathogen derived resistance to the virus.
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Cowpea is a major source of dietary protein in most African traditional diets, and the crop partially replenishes the soil nitrogen content as a result of its association with nitrogen-fixing Rhizobium spp, and is used as fodder. However, cowpea suffers from a wide range of production constraints such as viral diseases. Cowpea aphid-borne mosaic potyvirus (CABMV) is one of the most important viral pathogens of cowpea in major cowpea growing regions of the world and significantly reduces cowpea yields of resource poor farmers. In an effort to characterise a CABMV isolate from Zimbabwe, designated CABMV-Z3, using classical virology techniques the host range of CAMBV-Z3 was tested. The symptoms produced on susceptible Nicotiana bethamiana, N. clevelandii, Vigna unguiculata, Chenopodium amaranticolor and Ch. quinoa plants were described. Nicotiana glutinosa, N. tabacum, N. rustica, Phaseolus vulgaris and Cucumeris sativus were shown to be non-hosts of the virus. The CABMV-Z3 was purified for the first time and polyclonal antiserum was produced against the virus. Full-length cDNA clones of CABMV-Z3 were made by long-range reverse transcription-polymerase chain reaction (RT-PCR). Another aspect of the study was to determine the most effective virus resistance mechanism using the CABMV-Z3 coat protein (CP) gene, in tobacco as a model host since no reliable cowpea transformation protocol was available at the time. The 1.2 kb 3’ terminal region of CABMV-Z3 was amplified using RT-PCR technique and cloned into a PCR cloning vector pGEM-T, and named pGEM-CPRep. This clone was used as template in a PCR to amplify a CP gene optimised for expression in plants (CPk), an untranslatable CP gene with stop codons in all three reading frames (CPstop), an anti-sense CP (PC), and the central region of the CP gene (CPcore). All four amplification products were provided with promoters and terminators from expression cassette vector pCa2Nos, and ligated into the unique Hind III site of the binary plasmid pBI121, to result in plasmids pBI121-CPk, pBI121-CPstop, pBI121-PC and pBI121-CPcore. The constructs were used in Agrobacterium-mediated transformation of Nicotiana benthamiana leaf sections following the co-cultivation method. Regenerated plants were analyzed by PCR and Southern hybidization. R1 seedlings were assayed for kanamycin resistance and for presence of CP, and challenged with CAMBV-infected sap. Lines showing delayed symptom development, tolerance and recovery were identified but no line showing immunity was identified. This was significant resistance, since it affords protection to the plants during the crucial early stages of development and exerts little evolutionary pressure on the virus to evolve new strategies. The ultimate goal of the project was to produce cowpea lines that are resistant to CABMV. However, there was no efficient, reliable and reproducible cowpea transformation and regeneration system. A cowpea transformation protocol was developed and evaluated, utilizing the binary constructs specified above. The binary constructs were electrophoresed into the apical meristems of developing cowpea seedlings under various conditions including different voltage and current settings as well as pretreatment of seedlings with acid or plant growth regulators. Preliminary screening was done using GUS assays, PCR and Southern analysis, and the plants allowed to set seed. The T1 seeds were germinated in-vitro on MS plates. The results show that DNA delivery to meristematic cells was successful, leading to transgenic sectors of the plants. However, the chimeric nature of the plants poses a problem, as there is need for a reliable non-destructive mechanism to track the transgenic branches of the chimeras. This transformation method has the potential to avert some of the concerns often raised against transgenic plants since it does not necessarily require selectable marker and reporter genes, and avoids the expensive and laborious tissue culture step.