Browsing by Author "Barkhuizen, Helmien"

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    Characterization of transgenic grapevine ectopically expressing plant defensin peptides
    (Stellenbosch : Stellenbosch University, 2019-12) Barkhuizen, Helmien; Vivier, Melane A.; Rautenbach, Marina; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is one of the most important and widely grown food crops in the world. The cultivation and commercial production of this crop has, however, became highly dependent on the use of pesticides. One of the strategies to limit the use of chemicals is to harness the species natural defence mechanisms. This strategy requires the understanding of these plant defence mechanisms. Among the highly specialized defence mechanism of plants is the production of specific antimicrobial peptides, called plant defensins. These peptides are small, basic, positively charged and cysteine-rich with a potent broad range of antimicrobial activity. The plant defensins form a vital part of the innate immune system of plants and are widely distributed throughout the plant kingdom. Several plant defensins have been isolated and predominantly characterized for their in vitro antifungal activity. However, other biological activities, such as heavy metal tolerance, ion channel blocking, α-amylase and protease inhibition and modulators of growth and development have also been attributed to these peptides. Some well-studied defensins have been described in literature in terms of their three-dimentional structure, antimicrobial in vitro functions/activities, their mode(s) of action, as well as their applications. Limited information, however, is available on their broader potential impacts on plant growth and development and more specific, non-defence related stress-mitigating functions within their host plants. Furthermore, little information exists on grapevine plant defensins. Although 79 defensin-like genes (DEFL) have been identified in the reference genome, only four grapevine plant defensins have been isolated and characterised to date for potential antifungal activities. The goal of this study was therefore to evaluate potential in planta/in vivo functions of plant defensins in grapevine. In this study functional characterisation studies were performed on plant defensins overexpressed in grapevine. Genotypical screens were conducted on uncharacterised transgenic populations of two V. vinifera cultivars (Sultana and Red Globe), overexpressing three different defensin peptides (Heliophilia coronopifolia antifungal peptides 1 and 4 (Hc-AFP1, Hc-AFP4) and Raphanus sativus antifungal peptide 2 (Rs-AFP2)) to assess transgene integration and expression. These analyses revealed unique transgenic lines for the transgenic populations expressing the plant defensins Hc-AFP1 and Rs-AFP2, with the majority of these lines expressing the transgene. Although the presence of the transgene was confirmed for the transgenic V. vinifera (cv. Sultana) Hc-AFP4 and V. vinifera (cv. Red Globe) Hc-AFP4 lines, they did not exhibit any transgene expression and was not included in the growth, or biotic and abiotic stress phenotypical analyses. A previously characterised population of Vvi-AMP1 overexpressed in Sultana was also included in this study. Two in silico approaches were used to contextualise the functional characterisation studies. The first was to compare the different peptides in terms of their sequence similarities, as well as deduced structural features. The Rs-AFP2 peptide’s crystal structure has been resolved and the structure-function in silico analyses made use of the data available for this peptide. The Hc-AFP1, Hc-AFP4 and Rs-AFP2 peptides showed more similarities in sequence and structure compared to the Vvi-AMP1 defensin peptide. The majority of the sequence and structural differences between Hc-AFP1, Hc-AFP4 and Rs-AFP2 and the Vvi-AMP1 peptide resided in the conserved λ-core motif that is known to be important in determining the antifungal activities of plant defensin peptides. The second in silico approach was to use existing gene expression data in grapevine to evaluate where (in which organs and tissues) and under which (stressful) conditions grapevine defensin genes show differential expression patterns. Using the Corvina gene atlas, it was shown that under normal non-stressed conditions, DEFL genes were expressed in all grapevine organs and tissues at various developmental stages. Furthermore, using available microarray data, it was found that some defensins responded to biotic stress such as B. cinerea infection, although the Vvi-AMPs did not respond. Similarly, strong upregulation was found in response to Planococcus ficus (mealybug) infestation and in response to abiotic stress such as locally applied heat stress and leaf dehydration, with Vvi-AMP1 showing the strongest upregulation to the latter. Guided by the results of the in silico gene expression analysis, the in vivo functions of plant defensin peptides in grapevine were evaluated by analysis of the transgenic grapevines, overexpressing plant defensin peptides Hc-AFP1, Rs-AFP2 and Vvi-AMP1 in terms of growth, as well as biotic and abiotic stress. From these phenotypical observations it was evident that genotypical background (Sultana versus Red Globe) had a strong effect on several of the phenotypes observed. Furthermore, some of the observed phenotypes were peptide-specific, whereas in most other instances all peptides caused the same type of response to a particular stress, but with varying strength of the response, or some differences in mechanism. In terms of growth, the transgenic populations only showed mild phenotypes and no overt stunting or abnormalities were observed. Some plant lines, however, showed slower growth and root inhibition in vivo and these observed growth alterations were possibly a result of higher metabolic load on the plants due to the overexpression of the peptides, an aspect that deserves further study. The transgenic populations were evaluated for their in vivo functions towards biotic stress through evaluating their defence phenotypes against two fungal pathogens, namely the necrotrophic fungus, Botrytis cinerea and the biotrophic fungus Erysiphe necator, as well as the insect pest, Planococcus ficus (mealybug). None of the transgenic plant lines displayed a resistant defence phenotype towards B. cinerea, whereas all the transgenic lines showed enhanced resistance towards the biotrophic powdery mildew fungus through an increased penetration resistance mechanism. Some plant lines also displayed programmed cell death (PCD) associated resistance, especially the transgenic plants that contained the Rs-AFP2 construct. PCD is associated with the mechanism of action of the Rs-AFP2 peptide. All tested plant lines also showed promising results towards the soft scale insect P. ficus, all reducing the infestation significantly, making this the first report of an in planta anti-insect activity of plant defensins Hc-AFP1, Rs-AFP2 and Vvi-AMP1. The transgenic populations were also evaluated for their in vivo functions towards abiotic stress by subjecting plants to an active drying experiment and evaluating their intrinsic water use efficiencies (WUE). The majority of the plant lines all demonstrated an increase in intrinsic WUE, but the Red Globe Rs-AFP2 plant line showed a decrease in intrinsic WUE, which can at least partly possibly be linked to the reduced growth parameters demonstrated for this plant line, specifically the reduction in root growth. In conclusion, this study contributed to the current understanding of how plant defensins function in vivo, confirming growth impacts, antifungal activities, anti-insect activity and a role in water stress management. Furthermore, we gained a vital insight into the in vivo functions of grapevine plant defensins.
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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.