Thesis (Master of Applied Sciences in Chemistry)--Cape Peninsula University of Technology, 2018. / Antimicrobial Peptides (AMPs) are short peptides of about 45 - 54 amino acids that exhibit antibacterial and antifungal activities. Plant defensin is a type of AMP in plants which belong to a family of cationic peptides with a characteristic 3D folding pattern held in place by four disulfide bridges. AMPs especially defensins have been identified to have a huge biotechnological potential and are being patented for many applications. The aim of this work was to clone an antimicrobial peptide from Vigna subterranea and characterise it with bioinformatics analysis. 4 sets of primers were synthesized according to the sequences of conserved regions in AMPs i.e. defensins from legumes like Vigna unguiculata, Vigna radiata, Cicer arietinum and Cajanus cajan, amongst others, which have defensins with only a few sequence differences. The primers were designated VsDef P1 to P4. Using Vigna subterranea total genomic DNA as a template, fragments of expected sizes were successfully amplified and cloned into the pDRIVE vector and used to transform Escherichia coli JM109 cells in each case. Representative clones were sequenced and analysed using BLAST from National Center for Biotechnology Information. However, only the VIG clone was shown to be a bona fide defensin (over 90% identity, E-value of 1ex102, 99% query coverage of the nucleotide sequence, compared to Vigna unguiculata defensin). Based on this high sequence identity, a new pair of primers VsDef P5 was designed based on the Vigna unguiculata defensin sequence to specifically amplify the complete Vigna subterranea defensin gene, hereafter called VsDef1. Attempts to clone VsDef1 were however unsuccessful, and evidence of clone deletion and insert re-arrangement of insert DNA was observed. Direct sequencing of the PCR product demonstrated that it was indeed the complete VsDef1 pre-protein, composed of 433 nucleotides. In silico translation and analysis showed that VsDef1 has an intron at position 105 − 259 of the nucleotide sequences and encodes for a 78 amino acid peptide. Phylogenetic analysis revealed to be similar to the sequence of the defensins for Vigna unguiculata (96%), Vigna radiata (95%), Vigna angularis (95%) and Phaseolus vulgaris (93%) on the NCBI database. The three - dimensional structure of the peptide was modelled with SWISS-MODEL expasy and the structure was found to include one α- and three β domains, similar to those of other defensins. The failure to identify VsDef1 clone in a V. subterranea library and the failure to recover its cDNA clone are consistent with the hypothesised toxicity of VsDef1 to Escherichia coli. It is suggested that a different host, such as yeast, should be used in the future. The VsDef1 mRNA levels in germinating V. subterranea seeds was however successfully investigated using real-time reverse transcription quantitative PCR. VsDef1 mRNA is present in both the testa and embryo of dry seed and will persist through the early stages of seedling growth. This demonstrates the importance of VsDef1 in fighting off infection during germination in order to ensure successful germination. It is therefore essential to characterise more antimicrobial peptides from V. subterranea. The diversity of AMPs and their patterns of expressed genes will enable understanding of complex regulatory networks, which will likely enable identifying of genes involved in diseases and new biological processes.

Plant Defensins type 1 (PDF1) sont principalement décrites pour leur rôle dans l'immunité innée en réponse à des attaques pathogènes via l'activation de la voie de signalisation de l'éthylène (Et) et de l'acide jasmonique (JA). Les défensines PDF1 du genre Arabidopsis sont également impliquées dans la tolérance cellulaire au zinc chez la levure. In planta, de nombreux résultats mettent en évidence une corrélation entre la forte accumulation des transcrits AhPDF1 et leur contribution dans la tolérance à un excès de zinc. Dans cette étude, l'analyse du transcriptome (qRT-PCR) révèle que les paralogues PDF1s, aussi bien chez A. thaliana que chez A. halleri sont très peu voire pas du tout sensibles au zinc. Toutefois, il y a une spécialisation des PDF1s en réponse à l'activation de la voie de l'acide jasmonique dans le genre Arabidopsis. De plus, la contribution fonctionnelle des membres de la famille PDF1s dans la tolérance au zinc a été caractérisée chez A. thaliana à l'aide d'une approche génétique combinant des mutants KO après insertion d'un ADN-T et la technologie de miRNA artificiel. L'étude de ces mutants souligne par ailleurs la diversité fonctionnelle au sein de la famille des défensines AtPDF1s qui ne confèrent pas toutes la tolérance au zinc. En effet, une diversité de déterminants moléculaires des PDF1s a été mise en évidence lors de cette étude. La forte accumulation des PDF1s n'est pas l'unique paramètre requis pour la tolérance au zinc. Il faut également considérer la spécificité de tissu où s'expriment ces PDF1s. A ces considérations s'ajoutent aussi des régulations post-transcriptionnelles et post-traductionnelles. L'étude de ces modifications est envisagée afin de comprendre la contribution des différentes défensines PDF1s dans la tolérance au zinc. / Plant Defensin type 1 (PDF1s) are mainly recognized for their response to pathogen attack via ethylene (Et)/jasmonate (JA) signaling activation pathway. However, PDF1s originating from Arabidopsis genus also showed their capacity to induce cellular zinc tolerance up on expression in yeast. In planta, a group of observation highlighted the correlation of AhPDF1 high transcript accumulation for their contribution to zinc tolerance. Here, transcriptomic analysis (qRT-PCR) revealed that in both A. thaliana and A. halleri species, PDF1 paralogues were barely or not at all responsive to zinc. Nevertheless, there is a species specialization of PDF1s in response to activation of JA-signaling in Arabidopsis genus. In addition, in A. thaliana, the functional contribution of PDF1 members in zinc tolerance was investigated through genetic approach. Examining combination of T-DNA insertion knockout mutant and artificial miRNA, these studies were first direct demonstration of the functional involvement of AtPDF1s in zinc tolerance. These also highlighted the functional diversity among AtPDF1s because not all of them could play a role in zinc tolerance. Indeed, a diversity of PDF1 molecular determinants for zinc tolerance in plants was underlined. Remarkably, PDF1 high transcript is not the only important parameter for zinc tolerance and PDF1 tissue specificity could be an important factor to consider. Moreover, post-transcriptional and post-translational regulation might occur. Studies on these modifications are now the further questions in order to understand the contribution of the different PDF1s to zinc tolerance.

Thesis (MSc)--Stellenbosch University, 2013. / 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. / AFRIKAANSE OPSOMMING: Plantdefensinpeptiede word beskou as belowende en aantreklike kandidate vir gebruik as swammiddles in agribiotegnologie. Hierdie peptiede beskik oor breë spektrum antifungiese aktiwiteit en speel ‘n essensiële rol in die ingebore immuunsisteem van plante. Plant siektes wat deur swamme veroorsaak word dra betekenisvol by tot die afname in die kwaliteit en veiligheid van landbouprodukte. As gevolg van die skadelike effekte en negatiewe omgewingsimpak van plaagdoders, het effektiewe, veilige, natuurlike en duursame metodes om gewaspatogene te beheer, van die belangrikste vraagstukke van moderne landbou geword. Alhoewel hierdie peptiede belowende en aantreklike kandidate is vir die toepassing, is hulle presiese meganisme van aksie tot vandag toe steeds onbekend. Verskeie algemene waarnemings is egter al gemaak. Dit sluit die antagonistiese effek van katione op die aktiwiteit van plantdefensinpeptiede in. Dit is kernbelangrik om die onderliggende meganisme van die katioon-antagonistiese effek op die antifungiese effektiwiteit te verstaan om die moontlike bydrae van die peptiede tot die verdedigingsreaksies teen mikro-organismes in planta te evalueer. Met die doel voor oë het ons gemik om die effek van katione, spesifiek in die vorm van die biologiese soute NaCl, KCl, MgCl2 en CaCl2, op die strukturele stabiliteit en aktiwiteit in terme van groei inhibisie, morfologiese effekte en permeabilisasie te karakteriseer. Om uiteindelik hierdie karakterisasie eksperimente uit te voer was dit nodig om ‘n metode met ‘n groter opbrengs en wat vinnige suiwering van die peptied ondersteun, te optimiseer. Heliophila coronopifolia peptiede was voorheen in ‘n bakteriese sisteem geproduseer, maar die suiweringsmetodes was tydsaam en het gelei tot ‘n swak opbrengs. Pichia pastoris is dus geselekteer as die produksie sisteem aangesien verskeie ander plantdefensinpeptiede al suksesvol geproduseer is in hierdie eukariotiese sisteem. Hc-AFP1 and Hc-AFP3 is suksesvol vervaardig in die Pichia sisteem en het aktiewiteit getoon. Hc-AFP2 and Hc-AFP4 kon egter nie korrek vervaardig word nie as gevolg van ‘n na-vertalingsverandering wat gelei het tot die siklisering van die N-terminale glutamien, om piroglutamiensuur te lewer. Hierdie verandering het die aktiwiteit van die peptied negatief beinvloed. ‘n Aktiewe Hc-AFP2 kon wel vervaardig word deur die produksiebuffer te vervang met ‘n lae-ionise buffer. Die effek van divalente en monovalente katione op die sekondêre struktuur van Hc-AFP1 is ge-evalueer deur van sirkulêre dikroisme spektroskopie gebruik te maak. Hierdie katione het ‘n vouingsverandering in die sekondêre struktuur van Hc-AFP1 geïnduseer, NaCl and MgCl2 het ‘n meer gedefinieërde sekondêre struktuur induseer, terwyl KCl and CaCl2 ‘n minder gedefinieërde sekondêre struktuur geinduseer het. Monovalente katione het ‘n effense vermindering in die groei-inhibisie aktiwiteit van Hc-AFP1 op Botrytis cinerea veroorsaak, alhoewel kenmerkende hife-oorvertakking en ander morfologiese effekte nogsteeds sigbaar was. Divalente katione het ‘n sterker antagonistiese effek gehad op die aktiwiteit van Hc-AFP1, waar dit totaal en al die groei-inhibisie aktiwiteit van die peptied vernietig het, alhoewel die geïnduseerde morfologiese effekte op die hiffes steeds sigbaar was . Die aktiwiteit van Hc-AFP1 om B. cinerea hyphae te permeabiliseer is nie negatief beinvloed deur die byvoeging van katione nie, tewens dit het die aktiwiteit tot 10-voudig verhoog. Aangesien die groei-inhibisie aktiwiteit van Hc-AFP1 nie verminder is in die teenwoordigheid van die biologiese soute nie, dui dit aan dat permeabilisasie nie die enigste aktiwiteit is wat die groei inhibisie veroorsaak het nie. Die peptied het dus moontlik ‘n alternatiewe of primêre teiken en ‘n meer komplekse meganisme van aksie. Dit is die eerste verslag wat die invloed van katione op die struktuur van plantdefensinpeptiede ondersoek het. Dit is duidelik dat katione ‘n sekondêre strukturele vouingsverandering in Hc-AFP1 induseer. Hierdie verandering mag dalk bydra tot die antagonistiese uitwerking op die aktiwiteit van hierdie peptied. Hierdie studie het betekensisvolle vordering gemaak met die analise van die struktuur-funksie interaksie van plantdefensinpeptiede. / The National Research Foundation (NRF), Institute of Wine Biotechnology (IWBT), THRIP and Winetech for financial assistance.