Genetic manipulation of programmed cell death (PCD) for reduced susceptibility to necrotrophic fungi in narrow-leafed lupin (Lupinus angustifolius)

[Truncated abstract] Necrotrophic fungi have been one of the major constraints to lupin production and genetic and agronomic control of these diseases has so far been limited. Current evidence suggests that the interaction of these pathogens with susceptible plants involves the induction of programmed cell death (PCD), which is referred to as apoptosis in animal systems. Transgenic manipulation of PCD pathways could be a viable strategy for engineering necrotrophic pathogen disease resistance into crop plants. This project investigated the effect of baculovirus anti-apoptotic p35 gene on necrotrophic fungal diseases in narrow-leafed lupin (Lupinus angustifolius). In addition, work was initiated to investigate the underlying basis for low transformation efficiency and reduced transmission of transgene between T0 to T1 in this plant species. The baculovirus anti-apoptotic p35 gene was inserted into lupins using Agrobacterium-mediated transformation. The p35 gene was confirmed to be integrated into the lupin genome, inherited in a regular fashion in T2 to T4 plants and expressed in leaves, stems and roots. The presence of the transgene did not appear to have any effect on visible growth and development of p35 transgenic plants. P35 transgenic lupin lines were challenged with three necrotrophic fungal diseases, anthracnose, pleiochaeta root rot and pleiochaeta brown spot. Reduced susceptibility to all three necrotrophic fungal diseases was observed in some of the 12 p35 transgenic lupin lines tested in glasshouse experiments. These results support the hypothesis that fungal necrotrophs induce PCD in lupin plants as part of the disease syndrome and implicates a role of uncharacterised lupin caspase(s) in the compatible lupin-necrotrophic pathogen interaction. The localisation of a transgene in T0 lupin stems was assessed to inform future efforts to improve lupin transformation efficiency and overcome the problem of ii reduced transmission of transgenes to T1 plants. Cellular localisation of the gus transgene in T0 lupin stem sections showed it was expressed in sectors. ... The transformation efficiency of a gus gene construct was much higher than the observed frequency of transformants in non-p35 germplasm, indicating a possible role of Agrobacterium in inducing PCD and thereby reducing transformation efficiency. These findings should be confirmed and utilised in optimising lupin transformation protocols. This research confirmed previous results with other plant-pathogen interactions, where the p35 construct also inhibited necrotrophic pathogen disease. The use of the p35 gene gave an important opportunity to improve understanding of plantnecrotrophic pathogen interactions in lupin and other crops in general. The findings provide a solid basis for further research into genetic manipulation of necrotrophic pathogen resistance and also, for more efficient lupin transformation. The findings also support the hypothetical presence of functional caspase enzymes in apoptotic pathways of PCD in plants. It is hoped that increasing public acceptance of genetic manipulation will lead to utilisation of these results for the benefit of world society in the future.