Characterization of transgenic grapevine ectopically expressing plant defensin peptides

Barkhuizen, Helmien (2019-12)

Thesis (PhDAgric)--Stellenbosch University, 2019.


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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