Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species. / AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.

Thesis (MScAgric)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Grapevine supports one of the oldest industries in South Africa today, and is also of significant international importance. With increasing international trade and the transport of fruit and other grapevine-derived products between borders, it has become increasingly important for South African farmers and viticulturalists to ensure their products conform to strict international market requirements if they are to remain competitive. Such requirements include the correct and accurate identification of berries and wines according to cultivar. In light of this, 26 different wine, table grape and rootstock cultivars, as well as a number of clones from KWV's core germplasm collection were characterised at 16 microsatellite marker loci. Microsatellite markers are known for their high level of informativeness, reliability and reproducibility, and are widely used in the identification and characterisation of plant varieties, population analyses and forensic applications. Unique allelic profiles were obtained for all but two plants, which proved to be identical at all loci considered, and thus 'clones'. These profiles were collated to form a database, containing the DNA fingerprints of each sample at each locus. The relative levels of informativeness of each marker used were also determined, and compared with those found in the literature. Six markers proved to be highly informative, and are promising in the potential application of this technology to other cultivars. The applicability of microsatellite markers to such studies is confirmed; this approach could easily be extended to include any number of cultivars of national and international interest. The results of such an investigation would have important implications for both the farming and commercial industries alike. / AFRIKAANSE OPSOMMING: Wingerd ondersteun een van die oudste industriee in Suid-Afrika vandag, en is ook van groat intemasionale belang. Met die toenemende intemasionale ruilhandel en die vervoer van vrugte en ander wingerd produkte tussen grense, het dit toenemend belangrik geword vir SuidAfrikaanse wingerdboere om te. verseker dat hulle produkte voldoen aan die streng vereistes van die intemasional mark, indien hulle kompeterend wil bly. Hierdie vereistes sluit in die korrekte en akkurate identifisering van druiwe en wyn volgens kultivar. Met hierdie vereistes in ag geneem, is 26 verskillende wyn, tafeldruif en wortelstok kultivars, asook 'n aantal klone van die KWV se kern kiemplasma versameling, gekarakteriseer by 16 mikrosatelliet merker loki. Mikrosatelliet merkers word gekenmerk deur 'n hoe vlak van informatiwiteit, betroubaarheid en herhaalbaarheid en word wydverspreid gebruik in die identifisering en karakterisering van plant varieteite, populasie analises en forensiese toepassings. Unieke alleliese profiele is vir a1 die plante verkry, behalwe vir twee plante wat identiese resultate by alle loki opgelewer het en dus as "klone" beskou kan word. Hierdie profiele is bymekaar gevoeg om 'n databasis te vorm wat die DNA vingerafdrukke van elke monster by elke lokus bevat. Die relatiewe vlak van informatiwiteit van al die merkers is ook bepaal en vergelyk met merkers in die literatuur. Ses van die merkers blyk om hoogs informatief te wees en lyk belowend in die potensiele toepassing van hierdie tegnologie op ander kultivars. Die toepaslikheid van mikrosatelliet merkers op sulke studies is bevestig; hierdie benadering kan maklik aangepas word om enige aantal kultivars van nasionale en intemasionale belang in te sluit. Die resultate van s6 'n ondersoek sal belangrike implikasies inhou vir beide die boerdery en kommersiele industriee.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L. / AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies. / AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the ever-growing world population. Among the many challenges faced by food producers, crop losses and damage caused by fungal plant pathogens is a major problem. The study of fungal pathogens and the interaction between plants and fungi is therefore essential, and has been carried out for many years. Much has been learned in this time, but the full mechanisms of the various modes of fungal attack and plant defence have still not been elucidated. Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first enzymes to be secreted at the start of fungal attack, and many of them are considered to be essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of fungi, including Botrytis cinerea. An important defence mechanism of plants involves the inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act on fungal ePGs. Many different PGIPs from a number of diverse plant species have been described to date. They are known to have differential inhibition capabilities that often result from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains. Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP activity and a strong resistance response against Botrytis, leading to the characterisation of these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic analysis of these lines it was found that they display differential expression of cell wall metabolism genes and biochemical characteristics that might indicate possible cell wall strengthening compared to wild-type tobacco under uninfecting conditions. The V. vinifera cultivars are all very susceptible to fungal attack, whereas other grapevine species, specifically the North American Vitis species, are known for their strong resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have previously been isolated from these more resistant species. The amino acid sequences of the active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera PGIPs separated into 14 subgroups based on their active domain sequences. For this study, one PGIP from each group was selected for functional analysis in tobacco. The selected PGIP-encoding genes were transformed into tobacco by means of Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco populations consisting of three to twelve individually transformed lines of nine different nonvinifera PGIPs were obtained. A subset of the resultant transgenic lines was infected with B. cinerea in two independent whole plant infections over 11-14 days in order to investigate the disease resistance afforded by the various PGIPs towards this fungus. A line from the previously characterised VvPGIP1 population was included as reference; all the infections were contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs displayed very strong disease reduction in comparison to the WT control: after initial primary lesion formation, the spread of fungal infection was contained and halted in these lines, while wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions, indicating very strong resistance phenotypes. The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1 line and the WT control were also used to further evaluate the previous observation that overexpression might lead to changes in expression of cell wall genes. Analysis of the expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic lines tested. This confirmed previous results and have confirmed in all grapevine PGIP overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell wall. From previous work it is known that downregulation of this gene leads to strengthening of the wall. The results obtained in this study showed that the PGIP-specific resistance phenotype seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance towards B. cinerea provides an interesting angle for further investigation into the structural differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen interactions. / AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie. Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme (SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas. Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage (Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1 plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van infeksie. Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies, spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak. Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op baie sterk weerstandsfenotipes. Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van ‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die plantselwand. Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen interaksies.