Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds through specific adaptive cellular programmes, in particular by modifying gene expression. Many adaptive changes affect the physico-chemical properties of the cell wall, and several mechanisms that specifically affect the expression levels of genes that encode for cell wall components have been described previously. Cell wall modification directly impacts on general cell wall properties and cell-cell and cell-surface interactions. Many of these properties have been directly linked to families of cell wall proteins referred to as adhesins. In particular members of the Flocculation (FLO) gene family have been shown to play a crucial role in adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion, plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears to control flocculation. The regulation of FLO11 expression is well documented and is mainly controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A (cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to activate transcription. In this study we further explore additional gene targets of Flo8p and Mss11p, as well as their regulation and their impact on cell wall characteristics and associated adhesion phenotypes. Our analysis shows that Mss11p is also required for FLO1 expression, and functions together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene expression analysis further reveals that altered Mss11p levels leads to the change in the expression of various cell membrane and cell wall genes, notably AQY2 and members of the DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes display an almost exclusive dependence on FLO gene expression. We also demonstrate that these phenotypes require Flo10p and are thus dependent on the specific balance of Flo proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of FLO10 shares signalling components with FLO11, but that the two genes are differentially regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for Mss11p but rather for the MAPK component Ste12p. Whole genome expression analysis were also performed on strains with altered levels of Flo8p which were compared with the above mentioned transcriptome data set. This analysis shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also have significant unique gene targets. The combination of transcriptome data with current information concerning transcription factor (TF) interaction networks reveals the importance of network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we constructed a TF interaction model in which Flo8p acts as the predominantly activating TF component, whereas Mss11p function as a target hub TF, possibly as a mediator- or polymerase II holo-enzyme component. Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and show that by adjusting FLO11 expression in this strain we are able to significantly change this phenotypic behaviour. / AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae neem veranderinge in sy omgewing waar en reageer daarop deur middel van spesifieke sellulêre programme, in die besonder deur geenuitdrukking aan te pas. Verskeie aanpasbare veranderinge beïnvloed die fisieke, asook chemiese eienskappe van die selwand, en talle meganismes is al beskryf wat die uitdrukkingsvlakke beïnvloed van gene wat vir selwandkomponente kodeer. Die modifikasie van die selwand het ’n direkte impak op selwand-eienskappe, asook die sel-sel- en sel-oppervlak-interaksies. Verskeie van hierdie eienskappe word direk gekoppel aan die selwandproteïenfamilies, wat ook as adhesie-faktore bekend staan. Veral lede van die Flokkulasie (FLO) -geenfamilie het ’n noodsaaklike funksie in adhesie-fenotipes. Flo11p speel ’n rol in verskeie fenotipes, wat insluit die indringende groei van agar, plastiekaanhegting en die vorming van pseudohifes, “flor“ en “matte“, terwyl Flo1p flokkulasie beheer. Die regulering van FLO11-uitdrukking is deeglik gedokumenteerd en dit word hoofsaaklik gereguleer deur die mitogeen-geaktiveerde proteïenkinase (MAPK) en sikliese AMP-proteïenkinase A (cAMP-PKA) seintransduksiekaskades. Genetiese analises toon dat Mss11p stroom-af en sentraal tot hierdie kaskades funksioneer, en dit aktiveer transkripsie deur interaksie met die cAMP-PKA-komponent, Flop8. In hierdie studie word ’n ondersoek gedoen na addisionele teikengene van Flo8p en Mss11p, en hoe hierdie gene gereguleer word, asook hul impak op selwandeienskappe en geassosieerde adhesie-fenotipes. Ons analises toon dat Mss11p ook benodig word vir die ekspressie van FLO1 en dat dit, tesame met Flo8p, beheer uit oefen oor verskeie Flo-afhanklike fenotipes. Genoomwye geenekspressie-analises wys verder daarop dat veranderde Mss11p-vlakke lei tot die aanpassing van die ekspressie van verskeie selmembraan- en selwandgene, naamlik AQY2 asook lede van die DAN- en TIR-geenfamilies. Verdere genetiese analise dui daarop dat adhesie-fenotipes byna eksklusief afhanklik is van FLO-geenekspressie. Daar is verder getoon dat hierdie fenotipes ook Flo10p benodig en dus afhanklik is van die spesifieke balans van Floproteïene in die selwand. Die analise van seintransduksiemutante demonstreer dat FLO10 en FLO11 seintransduksie-komponente deel, maar dat hierdie gene verskillend gereguleer word. Anders as FLO11, toon FLO10 nie ’n absolute noodsaaklikheid vir Mss11p nie, maar eerder vir die MAPK-komponent, Ste12p. Totale genoomekspressie-analises is ook gedoen op gisrasse met aangepaste vlakke van Flo8p en dis vergelyk met bogenoemde transkripsiedatastel. Hierdie analise wys dat Flo8p and Mss11p die FLO-gene, asook AQY2 en TIR3, koreguleer, maar ook beduidende unieke teikengene het. Die kombinasie van transkripsiedata met huidig beskikbare informasie betreffende transkripsiefaktor (TF) -interaksienetwerke dui op die relevansie van netwerkinteraksie tussen Cin5p, Flo8p, Mga1p en Mss11p. Hiervan is daar ’n model opgestel waarin Flo8p in die meeste gevalle as die aktiverende TF-komponent optree, terwyl Mss11p as TF-teiken dien, moontlik as ’n mediator- of polimerase II holoënsiemkomponent. Laatens word daar vir die eerste keer verslag gedoen van ”mat”-vorming deur ’n industriële wyngisras en toon ons verder dat hierdie fenotipe beduidend verander word deur middel van die aanpassing van FLO11-uitdrukking.

Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. Although integration of the MJ-EXG1 expression cassette was confirmed, no mRNA levels could be detected with northern blot or RT-PCR analysis of the MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.

The past few decades have witnessed a revolution in recombinant DNA and nucleic acid sequencing technologies. Recently however, technologies capable of massively high-throughout, genome-wide data collection, combined with computational and statistical tools for data mining, integration and modeling have enabled the construction of predictive networks that capture cellular regulatory states, paving the way for ‘Systems biology’. Consequently, protein interactions can be captured in the context of a cellular interaction network and emergent ‘system’ properties arrived at, that may not have been possible by conventional biology. The ability to generate data from multiple, non-redundant experimental sources is one of the important facets to systems biology. Towards this end, we have established a novel platform called ‘spotted cell microarrays’ for conducting image-based genetic screens. We have subsequently used spotted cell microarrays for studying multidimensional phenotypes in yeast under different regulatory states. In particular, we studied the response to mating pheromone using a cell microarray comprised of the yeast non-essential deletion library and analyzed morphology changes to identify novel genes that were involved in mating. An important aspect of the mating response pathway is large-scale spatiotemporal changes to the proteome, an aspect of proteomics, still largely obscure. In our next study, we used an imaging screen and a computational approach to predict and validate the complement of proteins that polarize and change localization towards the mating projection tip. By adopting such hybrid approaches, we have been able to, not only study proteins involved in specific pathways, but also their behavior in a systemic context, leading to a broader comprehension of cell function. Lastly, we have performed a novel metabolic starvation-based screen using the GFP-tagged collection to study proteome dynamics in response to nutrient limitation and are currently in the process of rationalizing our observations through follow-up experiments. We believe this study to have implications in evolutionarily conserved cellular mechanisms such as protein turnover, quiescence and aging. Our technique has therefore been applied towards addressing several interesting aspects of yeast cellular physiology and behavior and is now being extended to mammalian cells. / text