| Summary: | Thesis (MSc)--University of Stellenbosch, 2011. === ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants
became increasingly apparent over the past decade. Antimicrobial peptides are unique
and diverse molecules that are found in many tissue types in a variety of invertebrate,
plant and animal species. Many of these peptides, such as plant defensins, have been
found to be ubiquitous throughout the plant kingdom and have been isolated from
flowers, leaves, roots, seeds, seedlings, pods, tubers and bark.
The growing relevance of antimicrobial peptides (including plant defensins) in
research can be largely attributed to their broad-spectrum antifungal activity. This
makes them promising potential targets, both as therapeutic agents and for their use in
crop protection and disease resistance. The continuing discovery of novel antimicrobial
peptides has advanced the development of strategies to overexpress these genes in
plants to attempt to enhance the plant’s natural ability to resist pathogenic attack.
The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized
and was shown to be tissue specific and developmentally regulated, being expressed
only in berries at the onset of berry ripening. The peptide showed strong antifungal
activity against a number of plant pathogenic fungi in vitro. In this study, the biological
role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis
vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens.
As a first step, recombinant production of Vv-AMP1 using an existing bacterial
expression system was evaluated and the heterologous production of the Vv-AMP1
peptide improved. Specific optimizations targeting both production and purification of
the peptide showed to improve the yield of Vv-AMP1. Steps in the production process
targeted for improvement included induction conditions of peptide production by the
bacterial culture as well as a number of purification steps, such as lysate preparation,
binding conditions, column washing, elution conditions and thrombin protease cleavage.
The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from
1.6 L of overnight culture. While production was markedly improved, the resultant
purified Vv-AMP1 proved biologically inactive and structurally unstable. This is
uncharacteristic of the peptide, suggesting that an important aspect necessary for
peptide activity, such as folding or the presence of specific co-factors might not be
supported in this non-host prokaryotic production system.
The study also entailed the characterization and evaluation of the Vv-AMP1
peptide against a panel of grapevine-specific pathogens that are culturable to
sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1
showed strong inhibitory activity against all pathogens tested, inhibiting the growth of
Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8
μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved
particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively.
Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a
severe inhibition on fungal germination and growth. The peptide did not induce
morphological changes in fungal hyphae but compromises the fungal membranes,
supporting the theory that the peptide induces membrane permeabilization.
Functional analysis of a transgenic V. vinifera (cv. Sultana) population
overexpressing Vv-AMP1 was included in this study to provide the opportunity to study
the in planta role of the peptide in its native host. The genetic characterization of the
putative population included confirming gene integration and copy number through PCR
and Southern blot analysis as well as gene expression through northern blot analysis. A
confirmed transgenic population was evaluated for improved disease resistance against
Botrytis cinerea as a first test organism in an attempt to link the overexpression of the
Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average
lesion size and further statistical analysis concluded that the transgenic population
showed a definite, albeit slight, improved resistance when compared to the
untransformed control lines.
In conclusion, the study determined that Vv-AMP1 had a strong antifungal action
against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be
established between the overexpression of Vv-AMP1 and a mild resistance phenotype
within its native host plant. The characterized transgenic population is important for
further work to evaluate the in planta activity of the peptide against more grapevine
pathogens such as the stem pathogens that were proven sensitive and specifically
those that cannot be cultured and are obligate pathogens, such as the downy and
powdery mildews. === AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante
het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is
unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van
invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv.
“plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds
geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas.
Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”)
in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit.
Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese
middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende
ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van
strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike
vermoeë om patogeniese aanval teen te staan te verbeter.
Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer
as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die
aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale
aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie
word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy
natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van
wingerd-spesifieke patogene.
As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n
bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1
peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die
produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1
verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit
beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n
aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom
wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale
suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L
oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig
verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is
buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir
antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore,
moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem.
Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen
'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend
in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk
inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia
seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en
9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders
sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese
analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op
swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in
swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie
bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer.
Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die
Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in
planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese
karakterisering van die vermeende transgeniese bevolking het die bevestiging van
beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel
as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking
is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur
infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n
weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer.
Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die
gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe)
verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne.
Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke
patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel
tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die
transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard
belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer
teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het
teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte
patogene soos dons- en poeierskimmel.
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