Masters Degrees (Viticulture and Oenology)

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Browsing Masters Degrees (Viticulture and Oenology) by Author "Barkhuizen, Helmien"

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    Mode of action studies of defensin peptides from native South African Brassicaceae species
    (Stellenbosch : Stellenbosch University, 2013-03) Barkhuizen, Helmien; Vivier, Melane A.; Rautenbach, Marina; De Beer, Abre; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Plant defensin peptides have become promising and attractive candidates to be used as antifungal agents in agricultural biotechnology. These peptides have a broad spectrum antifungal activity and play a vital role in the innate immune system of plants. Plant diseases caused by fungi are a major contributor to the decrease in the quality and safety of agricultural products. Due to the dangerous effects and negative environmental impact of pesticides, an effective, safe, natural and durable method to control crop pathogens has therefore become one of the major concerns in modern agriculture. Although these peptides are promising and attractive candidates, their precise mechanism of action is to date still unknown. Several common observations have been made. These include the antagonistic effect of cations on the activity of plant defensins. It is of vital importance to understand the underlying mechanism of the cation-antagonistic effect on the antifungal potency of defensin peptides in order to evaluate the possible contribution to defence reactions against microorganisms in planta. To this end we set out to characterize the effect of cations in the form of biological salts, NaCl, KCl, MgCl2 and CaCl2 on the structural stability and activity in terms of growth inhibition, morphological effects and permeabilization. In order to perform these characterization experiments, a production method resulting in a greater yield and involving simple and rapid purification methods was required. Heliophila coronopifolia peptides have previously been produced in a bacterial system, however the purification methods were tedious resulting in poor yields. Pichia pastoris was selected as production system as several other plant defensins have been successfully produced in this eukaryotic system. Hc-AFP1 and Hc-AFP3 was successfully produced using the Pichia production system and rendered active peptides. Hc-AFP2 and Hc- AFP4 was, however, not produced correctly, due to a post-translational modification event leading to the cyclization of the N-terminal glutamine to generate pyroglutamic acid. This modification negatively influenced the activity of these peptides. An active Hc-AFP2 could be produced by replacing the production buffer with a reduced ionic buffer. The effect of divalent and monovalent cations on the secondary structure of Hc-AFP1 was evaluated by circular dichroism spectroscopy. These cations induced a conformational change in the secondary structure of Hc-AFP1, with NaCl and MgCl2 inducing a more defined secondary structure and KCl and CaCl2 inducing a less defined secondary structure. Monovalent cations caused a slight reduction in the growth inhibition activity of Hc-AFP1 on Botrytis cinerea, however, characteristic hyperbranching and other morphogentic effects were still visible. Divalent cations had a greater antagonistic effect on the activity of Hc-AFP1, completely abolishing the growth inhibitory activity of the peptide, but the induced morphological effects on hyphae remained present. The activity of Hc-AFP1 to permeabilize B. cinerea hyphae was not influenced by the addition of cations, however it was in fact increased to up to 10-fold. However, since the growth inhibition activity of Hc-AFP1 was reduced in the presence of the biological salts indicates that permeabilization is not the sole activity responsible for growth inhibition caused by Hc-AFP1. This peptide probably has an alternative/primary target and more complex MOA. This is the first known report of the investigation of the influence of cations on the structure of plant defensin peptides. It is clear that cations induce a secondary structural conformational change in Hc-AFP1. This may be linked to the antagonism on the activity of this peptide. This study provides significant progress towards the structure-function analysis of plant defensins.