The use of maize streak virus (MSV) replication-associated protein mutants in the development of MSV-resistant plants

Doctoral Thesis


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University of Cape Town

Maize streak virus (MSV) is the type member of the Mastrevirus genus of the Geminiviridae. As the causal agent of maize streak disease (MSD), MSV is the most significant pathogen of maize in Africa, resulting in crop yield losses of up to 100%. Transmitted by leafhoppers (Cicadulina spp.), MSV is indigenous to Africa and neighbouring Indian Ocean Islands. Despite maize being a crucial staple food crop in Africa, the average maize yield per hectare in Africa is the lowest in the world, leading to food shortages and famine. A major contributing factor to these low yields is MSD. To genetically engineer MSV-resistant maize using the pathogen-derived resistance (PDR) strategy, the viral replication-associated (Rep) protein gene was targeted, whose multifunctional products Rep and RepA are the only viral proteins essential for replication. Rep constructs had previously been made containing deleterious mutations in several conserved amino acid motifs. In this study, these mutants and the wild type Rep gene were truncated to remove key motifs involved in viral replication. A quantitative PCR assay was developed to determine the effects of the mutant and truncated Reps on viral replication in black Mexican sweetcorn (BMS) suspension cells. The MSVsensitive grass Digitaria sanguinalis was then transformed with Rep constructs that inhibited MSV replication in BMS, and transgenic lines were tested for virus resistance. Several plants of a D. sanguinalis line transgenic for a mutated full-length Rep gene showed excellent resistance (immunity) to MSV, but the transgene had negative effects on aspects of plant growth and development. Transformation with a mutated/truncated Rep gene resulted in healthy fertile transgenic D. sanguinalis plants, many of which showed good MSV resistance. Fertile maize (Hi-II) T 1 transgenic plants expressing the truncated/mutated Rep gene have been obtained, the offspring of which will be tested for resistance to MSV. Considering the success in achieving MSV-resistant D. sanguinalis, there is good reason to believe that the transgenic maize will too be resistant to MSV.

Bibliography: pages 170-194.