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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80365 |
| Date | 03 1900 |
| Creators | Barkhuizen, Helmien |
| Contributors | Vivier, Melane A., Rautenbach, Marina, De Beer, Abre, Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 125 p. : ill. |
| Rights | Stellenbosch University |
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