Doctoral Degrees (Viticulture and Oenology)

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Browsing Doctoral Degrees (Viticulture and Oenology) by Subject "Antimicrobial peptides"

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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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    Isolation and characterization of antifungal peptides from plants
    (Stellenbosch : University of Stellenbosch, 2008-03) De Beer, Abre; Vivier, Melane A.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Over the last decade research has shown the importance of small antimicrobial peptides in the innate immunity of plants. These peptides do not only play a critical role in the multilayered defense systems of plants, but have proven valuable in the engineering of disease resistant food crops towards the ultimate aim of reducing the dependency on chemical fungicides. As the lists of isolated and characterized peptides grew, it became clear that other biological activities, in addition to the antimicrobial capacity, could be linked to some of these peptides; these alternative activities could have important applications in the field of medicine. This has made the defensin encoding genes prime targets for the agricultural and medical biotechnology sectors. To this end we set out to evaluate South African flora for the presence of plant defensin sequences and to isolate plant defensin genes that might be useful in biotechnology applications. Moreover, by isolating and characterizing these novel peptides, also in an in planta environment and in interaction with fungal pathogens, important knowledge will be gained of the biological role and importance of the peptides in the plant body. The plant host targets were South Africa Brassicaceae species including indigenous species, as well as Vitis vinifera, as the most important fruit crop in the world and since no defensins have been isolated from this economically important crop plant. The Brassicaceae family has been shown to be abundant in defensin peptides and several of the best characterized peptides with potent activity have been isolated from this family. Based on initial activity screens conducted on selected South African Brassicaceae spp. we concluded that these spp. contain promising antifungal peptide activities, warranting further efforts to isolate the genes and encoding peptides and to characterize them further. The preliminary activity screens used a peptide-enrichment isolation strategy that favored the isolation of basic, heat-stable peptides; these properties are characteristic features of plant antimicrobial peptides. These peptide fractions showed strong antifungal activities against the test organisms. A PCR-amplification strategy was subsequently designed and implemented, leading to the isolation of 14 novel defensin peptide encoding genes from four South African Brassicaceae spp., including the indigenous South African species Heliophila coronopifolia. Amino acid sequence analysis of these peptides revealed that they are diverse in amino acid composition and share only 42% homology at amino acid level. This divergence in amino acid composition is important for the identification of new biological activities within closely related plant defensins. Single amino acid changes have been contributed with the divergent biological activities observed in closely related plant defensin peptides. Phylogenetic analysis conducted on the deduced amino acid sequences revealed that all the new defensins share a close relationship to other Brassicaceae members of the plant defensin superfamily and was furthest removed from the defensins isolated from the families Solanaceae and Poaceae. Classification analysis of these peptides showed that they belong to subgroup A3 of the defensin superfamily. A putative defensin sequence was also isolated from V. vinifera cultivar, Pinotage, and termed Vv-AMP1. Genetic characterization showed that only a single gene copy of this peptide is present within the V. vinifera genome, situated on chromosome 1. Genetic characterization of this peptide encoding gene within the Vitis genus showed that this gene has stayed conserved throughout the divergent evolution of the Vitis genus. Expression studies of Vv-AMP1 revealed that this gene is expressed in a tissue specific and developmentally regulated manner, being only expressed in grape berries and only at the onset of vèraison. Induction of Vv-AMP1 in grapevine leaf material could never be achieved through the external application of hormones, osmotic stress, wounding, or pathogen infection by Botrytis cinerea. Deduced amino acid analysis showed that Vv-AMP1 encoded for a 77 amino acid peptide consisting of a 30 amino acid signal peptide and a 47 amino acid mature peptide, with putative antifungal activity. The Vv-AMP1 peptide grouped with the subclass B type defensins, which have been documented to have both antifungal and antibacterial activities. The Vv-AMP1 signal peptide directed the green fluorescent protein (GFP) reporter gene to the apoplastic regions in cells with high levels of accumulation in the vascular tissue and the guard cells of the stomata. Recombinant Vv-AMP1 peptide was successfully purified from a bacterial host and shown to have a size of 5.495 kDa. Recombinant Vv-AMP1 showed strong antifungal activity at low concentrations against a broad spectrum of fungal pathogens, which included Verticillium dahliae (IC50 of 1.8 μg mL-1) and the necrotrophic pathogen Botrytis cinerea (IC50 of 12-13 μg mL-1). Antifungal activity of Vv-AMP1 did not induce morphological changes in fungal hyphae, but its activity was associated with induced membrane permeabilization in treated hyphae. Vv-AMP1 was successfully introduced into Nicotiana tabacum as confirmed by Southern blot analysis and 20 individual lines were generated. Genetic characterization confirmed the integration and expression of the gene in the heterologous tobacco environment. The peptide was under control of its native signal sequence which has been shown to direct its product to the apoplastic regions of cells. The transgenic lines were analyzed to determine the presence and activity of the grapevine defensin peptide. Western blot analyses of partially purified plant extracts detected a signal of the expected size in both the untransformed control and the transgenic lines. Comprehensive analysis of EST databases identified three highly homologous sequences from tobacco that probably caused the background signal in the control. These crude protein extracts were able to inhibit the growth of V. dahliae in vitro when tested in a microtiter plate assay, but the inhibition could not be conclusively linked to the presence of the transgenic peptide, since non-expressing transgenic lines, included as controls, also showed inhibition. Similar results were obtained with infection studies, clearly showing that despite successful integration and expression of the transgene, the peptides was either not functional in the heterologous environment, or perhaps unstable under the particular regulatory conditions. This peptide belongs to a subclass of peptides known for associated activities that might activate tight control by plant hosts if threshold levels are reached. These aspects need further investigation, specifically since it is in stark contrast to previous results obtained with defensins from a different subclass. This study has also yielded significant other related resources that would be instrumental for further possible biotechnology exploitation of some of the novel peptides, but also to provide genetic constructs and plant material that would be invaluable to address fundamentally important questions such as the regulation and mode of action of defensin peptides, specifically in interaction with pathogen hosts. The novel peptides have been transformed to various hosts, including grapevine and these transgenic populations are available to facilitate the next rounds of research into this extremely promising group of antifungal peptides.