Thesis (MSc)--Stellenbosch University, 2002. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has established its importance as a wine yeast and also proven itself as a reliable starter culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly, its metabolic activities result in the production of higher alcohols, fatty acids and esters, which are important flavour and aroma compounds that are essential for consistent and predictable wine quality. There is a growing consumer demand for wine containing lower levels of alcohol and chemical preservatives. Glucose oxidase (GOX) has received considerable research interest regarding its potential application in the wine industry to reduce alcohol levels and as a biocontrol agent. Several physical processes are used for the removal or reduction of alcohol in wine and some of them are sometimes used in combination. These processes tend to involve expensive equipment and can be intensive from a processing point of view. An alternative approach was introduced with the concept of treating grape must with GOX to reduce the glucose content of the must, and therefore produce a wine with a reduced alcohol content after fermentation. Due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialised wine yeast strains possessing a wide range of optimised, improved or novel oenological properties. The first and main objective of this study was to genetically engineer wine yeasts to produce wine with a reduced alcohol content. In order to do this, the structural glucose oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5) containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox- PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of S. cerevisiae (~1278). Results obtained indicated the production of biologically active glucose oxidase and showed that it is secreted into the culture medium. This would mean that the enzyme will convert the glucose to gluconic acid in the medium before the yeast cells are able to metabolise the glucose to ethanol. Microvinifications performed with Chardonnay grapes showed that the laboratory yeast starter cultures transformed with GOX1 were indeed able to reduce the total amount of alcohol in the finished product. The second objective of this study involved the potential application of GOX as a biocontrol agent. Screening was performed for wine spoilage microorganisms, such as acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage microorganisms tested formed different sized inhibition zones, indicating varying degrees of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed under a scanning electron microscope. The total collapse of the bacterial cell wáll could be seen and might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a result of the peroxidation of the membrane lipid, and a strong oxidising effect on the bacterial cell, which is the cause of the destruction of basic molecular structures, such as nucleic acids and cell proteins. In this exciting age of molecular yeast genetics and modern biotechnology, this study could pave the way for the development of wine yeast starter culture strains for the production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs) within the wine industry is a limiting factor at present and credible means must be found to effectively address the concerns of traditionalists within the wine industry and the negative overreaction by some consumer groups. There is a vast potential benefit to the wine consumer and industry alike and the first recombinant wine products therefore should unmistakably demonstrate safe products free of potentially harmful compounds, and have organoleptic, hygienic and economic advantages for both the wine producer and consumer. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na wyne met 'n laer alkoholinhoud en minder preserveermiddels. Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en selfs unieke, wynkundige einskappe bevat. Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278) getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei. Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied (H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese molekulêre strukture, soos die nukleïensure en selproteïene. Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste, asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan. Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese voordele toon te opsigte van beide die wynprodusent en gebruiker.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is able to sense and respond to changes in its immediate environment. Information regarding the nutritional status of the extracellular environment is sensed by membrane receptor systems and relayed through signalling pathways to the nuclear interior, affecting the transcription of specific genes., Transcription factors, which function downstream of these signal transduction pathways, have to be transported into the nucleus after synthesis in the cytoplasm in order to regulate transcriptional events. Transport into the nucleus occurs in a tightly regulated manner at the nuclear pore complex, which is located in the nuclear membrane, and requires the recognition of transport signal sequences, which are present in the proteins that are to be transported. Signalling pathways control the nuclear accessibility of transcriptional regulators by modifying their respective signal sequences. In response to a limited availability of carbon or nitrogen, cells are able to change their morphology from a unicellular ovoid form to elongated cells attached to each other. This morphological change is associated with daughter cells that remain attached to their respective mother cells following unipolar budding, thus forming filamentous structures referred to as pseudohyphae. The regulation of the development of pseudohyphae is correlated with other physiological processes, such as starch degradation and the invasion of agar-containing media. Mss11p performs a central role in the regulation of the genes required for these processes and it has been shown to specifically regulate the expression of FL011, which encodes a cell surface protein critical for pseudohyphal development, and STA2, which encodes an extracellular glucoamylase functioning in the degradation of starch. The aim of this study was to characterise the functioning of Mss11p. Overexpression analysis indicates that Mss11p functions as an inducer of invasive growth, cell elongation and flocculation. Furthermore, MSS11 deletion improves biomass formation and suppresses the growth defect of yeast from a L:1278b genetic background transformed with the RAS2val19 allele on non-fermentable carbon sources. Biochemical analysis shows that Mss11p is a nuclear protein of approximately 97 kDa in apparent size that is maintained at relatively low levels in yeast. Finally, the data suggest a model in which Mss11p functions as a mediator of the transcriptional regulation of various genes. / AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae is in staat om veranderinge in sy onmiddelike omgewing waar te neem en daarop te reageer. Inligting betreffende die beskikbaarheid van voedingstowwe in die omgewing word vanaf membraan reseptorsisteme deur middel van seintransduksiekaskades na die nukleus herlei, waar die transkripsie van spesifieke gene beïnvloed word. Transkripsie faktore wat stroom af van hierdie seintransduksie funksioneer, moet na die nukleus vervoer word na vervaardiging in die sitoplasma, om sodoende transkripsionele gebeurtenisse te reguleer. Die vervoer van faktore na die binnekant van die nukleus vind onder streng regulering plaas by die nukleêre porie kompleks, wat in die nukleêre membraan gesitueer is. Vervoer vind plaas deur middel van die herkenning van nukleêre lokaliseringsekwense wat in die proteïene wat vervoer word, teenwoordig is. Seintransduksiekaskades beheer die beskikbaarheid van proteïene tot die nukleus deur hulonderskeidelike nukleêre lokaliseringsekwense te modifiseer. Selle is in staat om hul morfologie te verander van 'n eensellige eliptiese vorm tot verlengde selle wat aan mekaar geheg bly in reaksie op die beperkende beskikbaarheid van koolstof of stikstof bronne. Hierdie morfologiese verandering word geassosieer met dogterselle wat ná monopolêre botselvorming aan hul moederselle geheg bly, en dus filamentagtige strukture vorm wat pseudohifes genoem word. Die regulering van die ontwikkeling van pseudohifes word gekorreleer met ander fisiologiese prosesse, soos styselafbraak en die penetrerende groei van selle op agar-bevattende media. Mss11p vervul 'n sentrale rol in die regulering van gene wat vir hierdie prosesse benodig word en reguleer die uitdrukking van FL011, wat kodeer vir 'n selwandproteïen wat krities is vir die ontwikkeling van pseudohifes, en STA2, wat kodeer vir 'n ekstrasellulêre glukoamilase wat vir die afbraak van stysel benodig word. Die doel van hierdie studie was om Mss11p-funksie te karakteriseer. Deur middel van oorproduksie is Mss11p as die induseerder van penetrerende groei, selverlenging en flokkulasie geïdentifiseer. Verder is bevind dat MSS11-delesie lei tot verhoogde biomassa formasie, en dat dieselfde delesie lei tot 'n oorkoming van 'n groeidefek van gis van die 2:1278b genetiese agtergrond wat met die RAS2val19aleel op nie-fermenteerbare koolstofbronne getransformeer is. Biochemiese analise dui daarop dat Mss11p 'n nukluêre proteïen is van ongeveer 97 kDa in oënskynlike grootte, wat teen lae vlakke in gis onderhou word. Die data stel 'n model voor waarin Mss11p as bemiddelaar optree vir die transkripsionele regulering van verskeie gene.

Thesis (MScAgric)--Stellenbosch University, 1946. / No Abstract Available

Thesis (MScAgric)--Stellenbosch University, 1951. / No Abstract Available

Thesis (MScAgric)--Stellenbosch University, 1953. / No Abstract Available

Thesis (MScAgric)--Stellenbosch University, 1963. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MScAgric)--Stellenbosch University, 1977. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

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.

Thesis (MScAgric)--Stellenbosch University, 1975. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine. / AFRIKAANSE OPSOMMING: Die nuutste strategieë wat deel vorm van die beskerming van plant gewasse teen mikrobiese patogene het hul oorsprong in die inspanning van die natuurlike, hoogs gekompliseerde verdedigingsmeganismes van die plant deur middel van genetiese enginieurswese ten einde die gebruik van chemiese plaagdoders te verlaag. Hierdie benadering maak staat op ‘n in-diepte begrip van plant-patogeen interaksies om verstandige strategieë vir plantverbetering te kan ontwikkel. Van hierdie hoogs-gespesialiseerde verdedigingsmeganismses in die plant se arsenaal teen patogeen aanvalle sluit die de novo produksie van proteinagtige antimikrobiese peptiede (AMPs) in as deel van die plant se ingebore immuunstelsel. Hierdie AMPs is klein, sisteïen-ryke peptiede soos die plant “defensins” en is bekend vir hul breë-spektrum antifungiese aktiwiteit. Hierdie plant defensinpeptiede word aangetref in meeste, indien nie alle plant spesies nie en die defensin koderende gene word oor-verteenwoordig in plant genome. Meeste van hierdie defensins is gewoonlik die produkte van enkele gene wat die plant in staat stel om hierdie molekules relatief spoedig en met minimale energie verbruik in die plant te vorm. Hierdie faktore dra by tot die vestiging van AMPs as uitstekende kandidate vir genetiese ingenieursstrategieë as deel van die strewe na alternatiewe gewasbeskermingsmeganismes. Die eerste antimikrobiese peptied wat geïdentifiseer en geïsoleer is uit wingerd, Vv-AMP1, word beheer deur die ontwikkelingsstadium en word eksklusief uitgedruk in korrels vanaf die aanvang van rypwording. Rekombinant-geproduseerde Vv-AMP1 het sterk antifungiese aktiwiteit getoon teen ‘n wye reeks plantpatogeniese swamme teen merkwaardige lae peptied konsentrasies in vitro, alhoewel geen in planta verdedigingsfenotipe tot dusver gekoppel kon word aan hierdie peptied nie. In hierdie studie was die antifungiese aktiwiteit van Vv-AMP1 wat ooruitgedruk is in sy natuurlike gasheerplant (Vitis vinifera) ge-evalueer teen wingerd-spesifieke nekrotrofiese- en biotrofiese swamme. Eerstens is ‘n afgeharde geneties-gekarakteriseerde transgeniese V. vinifera (cv. Sultana) populasie wat Vv-AMP1 ooruitdruk gegenereer en morfologies gekarakteriseer. Om die in planta funksionaliteit van Vv-AMP1 ooruitgedruk in wingerd te evalueer is hierdie bevestigde transgeniese populasie blootgestel aan antifungiese toetse met die nekrotrofiese swam, B. cinerea en die biotrofiese swam, Erysiphe necator. Vir die doel om infeksiestudies uit te voer met ‘n biotrofiese swam is ‘n metode geoptimiseer vir die kweek en infeksies met E. necator wat gelei het tot ‘n herhaalbare patosisteem vir hierdie swam op wingerd. Blaarstudies, volgens die pas-verbeterde metode vir E. necator infeksies het ‘n geprogrammeerde seldood-geassosieërde weerstand, gekoppel aan die ooruitdrukking van Vv-AMP1 onthul, wat vergelyk kan word met dié van die hoogs-weerstandige wingerdspesie, Muscadinia rotundifolia. Hierteenoor het heel-plant infeksie studies met B. cinerea onthul dat Vv-AMP1 ooruitdrukking geen verhoogde weerstand teen dié nekrotrofiese swam aan V. vinifera bied nie. ‘n In silico analise van die transkripsie van defensin-agtige (DEFL) gene wat vroeër in wingerd geïdentifiseer is, is by hierdie studie ingesluit. Hierdie analise het vermeende gesamentlike uitdrukking van hierdie DEFL gene en ander gene in die wingerd genoom onthul wat aangedryf word deur weefsel- of kultivar-spesifieke regulering of die plant se reaksie tot biotiese en abiotiese stress stimuli. Ten slotte, hierdie resultate het bygedra tot ons kennis in verband met Vv-AMP1 en het ‘n in planta verdedigingsfenotipe vir hierdie defensin in wingerd onthul. In silico analiese van die DEFL gene in wingerd het verder toestande onthul wat die uitdrukking van hierdie gene aandryf wat ons toelaat om aannames te maak ten opsigte van die moontlike biologiese funksies van DEFL peptiede in wingerd en ondersteun die opstel en toets van hipoteses vir die rol en megansimes van aksie van die wingerd defensin familie.