Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Gray leaf spot (GLS) of maize, caused by the fungus Cercospora zeae-maydis, can reduce grain yields by up to 60% and it is now recognized as one of the most significant yield-limiting diseases of maize in many parts of the world. The most sustainable and long-term management strategy for GLS will rely heavily on the development of high-yielding, locally adapted GLS resistant hybrids. Molecular markers could be useful to plant breeders to indirectly select for genes affecting GLS resistance and to identify resistance genes without inoculation and at an early stage of plant development. Only two studies in the USA have examined quantitative trait loci (QTL) association with GLS resistance. The aim of this study was to map GLS resistance genes in a resistant Seed Co LTD, Zimbabwean inbred line. Molecular markers linked to the GLS resistance QTL were identified by using the amplified fragment length polymorphism (AFLP) technique together with bulked segregant analysis. Eleven polymorphic AFLP fragments were identified and converted to sequence-specific PCR (polymerase chain reaction) markers. Eight of the 11 converted AFLP markers were added to the maize marker database of the University of Stellenbosch. Five of the 8 converted AFLP markers were polymorphic between the resistant and the susceptible parent. They were amplified on the DNA of 230 plants of a segregating F2 population and linkage analysis was performed with MAPMAKER/EXP. Two linkage groups consisting of two markers each, with a linkage distance of 10.4 cM (LOD 22.83) and 8.2 cM (LOD 55.41) between the two markers, were identified. QTL mapping with MAPMAKER/QTL confirmed the presence of QTL in both linkage groups. Two publicly available recombinant inbred families (Burr et a/., 1988) were used to localize the converted AFLP markers on the genetic map of maize. The QTL, which were identified with the AFLP markers, were mapped to chromosomes 1 and 5. Another AFLP marker was mapped to chromosome 2 and a further to chromosome 3. To obtain more precise localizations of the QTL on chromosomes 1 and 5, sequence-tagged site markers and microsatellite markers were used. The markers were amplified on the DNA of the 230 plants of the F2 population and linkage analysis was performed with MAPMAKER/EXP. The order of the markers was in agreement with the UMC map of the Maize Genome Database. Interval mapping using MAPMAKERlQTL and composite interval mapping using QTL Cartographer were performed. The QTL on chromosome 1 had a LOD score of 21 and was localized in bin 1.05/06. A variance of 37% was explained by the QTL. Two peaks were visible for the QTL on chromosome 5, one was localized in bin 5.03/04 and the other in bin 5.05/06. Both peaks had a LOD score of 5 and 11% of the variance was explained by the QTL. To test the consistency of the detected QTL, the markers flanking each QTL were amplified on selected plants of two F2 populations planted in consecutive years and regression analysis was performed. Both the QTL on chromosome 1 and the QTL on chromosome 5 were detected in these populations. Furthermore, the presence of a QTL on chromosome 3 was confirmed with these populations. A variance of 8 -10% was explained by the QTL on chromosome 3. In this study, a major GLS resistance QTL was thus mapped on chromosomes 1 and two minor GLS resistance QTL were mapped on chromosomes 3 and 5 using a resistant Seed Co LTD, Zimbabwean inbred line. Markers were identified which could be used in a marker-assisted selection program to select for the GLS resistance QTL. / AFRIKAANSE OPSOMMING: Grys blaarvlek (GBV) van mielies, veroorsaak deur die swam Cercospora zeaemaydis, kan graanopbrengs met tot 60% verlaag en word beskou as een van die vernaamste opbrengs-beperkende siektes wêreldwyd. Die toepaslikste langtermyn stragtegie vir GBV beheer sal wees om plaaslike mieliebasters met hoë opbrengs en GBV weerstand te ontwikkel. Molekulêre merkers kan nuttig deur plantetelers gebruik word om weerstandsgene te selekteer. Seleksie is moontlik in die afwesigheid van inokolum en op 'n vroeë stadium van plant ontwikkeling. Slegs twee vorige studies (in die VSA) het kwantitatiewe-kenmerk-Iokusse (KKL), vir GBVweerstand ondersoek. Die doel van hierdie studie was om die GBV weerstandsgene in 'n weerstandbiedende ingeteelde lyn (Seed Co BPK, Zimbabwe) te karteer. Molekulêre merkers gekoppel aan die GBV weerstands KKL is geïdentifiseer deur gebruik te maak van die geamplifiseerde-fragmentlengte-polimorfisme- (AFLP-) tegniek en gebulkte-segregaat-analise. Elf polimorfiese merkers is geïdentifiseer en omgeskakel na volgorde-spesifieke PKR (polimerase kettingreaksie) merkers. Agt van die elf omgeskakelde AFLP-merkers is by die mieliemerker databasis van die Universiteit van Stellenbosch gevoeg. Vyf van die 8 omgeskakelde AFLP-merkers was polimorfies tussen die bestande en vatbare ouers. Hulle is geamplifiseer op die DNA van 230 plante van 'n segregerende F2-populasie en is gebruik in 'n koppelingstudie met MAPMAKER/EXP. Twee koppelingsgroepe, elk bestaande uit twee merkers, met onderskeidelik koppelingsafstande van 10.4 eM (LOD 22.83) en 8.2 eM (LOD 55.41) tussen die merkers, is geïdentifiseer. KKL-kartering het getoon dat KKL in albei koppelingsgroepe aanwesig is. Twee kommersieël beskikbare, rekombinant-ingeteelde families (Burr et aI., 1988) is gebruik om die omgeskakelde AFLP-merkers op die mielie genetiese kaart te plaas. Die KKL wat met die AFLP-merkers geïdentifiseer is, is gekarteer op chromosome 1 en 5. 'n Verdere AFLP-merker is op chromosoom 2 gekarteer en 'n ander op chromosoom 3. Ten einde die KKL op chromosome 1 en 5 meer akkuraat te karteer, is volgordege- etikeerde en mikrosatelliet merkers gebruik. Die merkers is geamplifiseer op die DNA van die 230 plante van die F2-populasie en koppelings-analises is uitgevoer. Die volgorde van die merkers was dieselfde as die van die UMC-kaart in die Mielie Genoom Databasis. Interval kartering met MAPMAKER/QTL en komposiet interval kartering met QTL Cartographer is uitgevoer. Die KKL op chromosoom 1 het 'n LOD-telling van 21 gehad en is in bin 1.05/06 geplaas. Die KKL was verantwoordelik vir 37% van die variansie. Twee pieke was onderskeibaar vir die KKL op chromosoom 5, een in bin 5.03/04 geleë en die ander in bin 5.05/06. Elke piek het 'n LOD-telling van 5 gehad en die twee KKL was verantwoordelik vir 11% van die variansie. Om die herhaalbaarheid van die effek van die KKL te toets is die merkers naaste aan elke KKL geamplifiseer op geselekteerde plante van twee F2-populasies wat in opeenvolgende jare geplant is. Regressie analise is op die data gedoen. Beide die KKL op chromosoom 1 en die KKL op chromosoom 5 kon in hierdie populasies geïdentifiseer word. Verder kon die aanwesigheid van 'n verdere KKL op chromosoom 3 in hierdie populasies bevestig word. Laasgenoemde KKL was verantwoordelik vir 8-10% van die totale variansie. In hierdie studie is daar dus 'n hoof GBV-weerstands KKL gekarteer op chromosoom 1 en twee kleiner GBV-weerstands KKL gekarteer op chromosome 3 en 5. Merkers is geïdentifiseer wat moontlik in merker-gebaseerdetelingsprogramme gebruik kan word om plante te selekteer wat die GBVweerstands KKL het.

Thesis (PhD (Agric)) -- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Chromosome arm 7DL of common wheat carries genes for agronomically important traits such as leaf rust, stem rust, Russian wheat aphid and eye spot resistance. Some of these genes occur on introgressed foreign chromatin, which restricts their utility in breeding. The 7DL genetic maps are poorly resolved, which seriously hampers attempts to manipulate the genes and introgressed regions in breeding. This dissertation represents an attempt to improve our knowledge of the relative map positions of three resistance genes that have significant potential for use in local breeding programmes. The leaf rust resistance gene, Lr19, is located on a Thinopyrum ponticum-derived translocation which occupies a large part of the terminal end of 7DL. The translocation also carries genes for less favourable traits such as yellow flour colour. Attempts have been made to reduce the size of the translocation through allosyndetic pairing induction; the primary aims being to remove deleterious genes and to minimise the amount of foreign chromatin associated with Lr19 so it can be recombined with other useful 7DL genes. Twenty-nine 'Indis'-derived Lr 19 deletion mutants were previously produced by gamma irradiation and a physical map was constructed. In this study, the set of mutant lines were further analysed using 144 Sse8387I/Msei and 32 EcoRI/Msel amplified fragment length polymorphism (AFLP) primer combinations. The previous physical map, which was based on five restriction fragment length polymorphism (RFLP) markers and five structural gene loci, was extended and now includes 95 novel AFLP markers (86 Sse8387I/Msei and 9EcoRI!Msel markers), of which seven map close to Lr 19. Most of the deletions could be ordered according to size and the improved map has already been used to characterise shortened recombinant forms of the Lr 19 translocation. An unsuccessful attempt was made to convert one of the seven markers closest to Lr 19 into a sequence-specific marker. However, an AFLP marker located distally from Lr 19 was successfully converted into a sequence-specific marker in collaboration with other researchers. An attempt was also made to map and tag the Russian wheat aphid (RWA) resistance gene, Dn5. A doubled haploid mapping population consisting of 94 lines was created and typed for Dn5, four microsatellite loci and the endopeptidase locus, Ep-Dl. The Dn5 locus mapped 25.4 cM and 28.6 cM distally from Xg.vm111 and Xg.vm437, respectively, but was not linked to Xgwm428, Xgwm3 7 or Ep-Dl. Tagging of Dn5 was attempted by screening twelve homozygous resistant and seven homozygous susceptible F2 lines from a cross between 'Chinese Spring' and 'PI 294994' with 70 Sse8387IIi\1sei AFLP primer combinations. Only two potentially useful polymorphisms (one in coupling and one in repulsion phase) were identified. Conversion of the coupling phase marker to a sequence-specific marker was not successful. The eyespot resistance gene, Pchl , was derived from Triticum ventricosum and is present in the wheat VPM-1. Close association between Pchl and the endopeptidase Ep-Dlb allele has been reported previously. Pchl/Ep-Dl was tagged by screening ten wheat genotypes (each homozygous for the confirmed presence or absence of Pchl and/or Ep-Dl b) with 36 Sse83 87I/ Msei AFLP primer combinations. Three AFLP markers were closely associated with Pchl I Ep-D 1, one of which was targeted for conversion into a sequence-specific marker. The sequence-specific marker contained a microsatellite core motif and was found to be useful for tagging Pchl!Ep-Dl. A genetic distance of 2 cM was calculated between the novel microsatellite marker and Ep-Dl. The microsatellite marker was also polymorphic for the Lr 19 translocation and it was possible to map it between the Wsp-Dl and Sr25 loci. In this dissertation, mapping and/or tagging of three important resistance genes were achieved. Due to the fact that all markers used in these studies were not polymorphic between all of the targeted regions, it was not possible to fully integrate the data obtained for the three regions. / AFRIKAANSE OPSOMMING: Chromosoom arm 7DL van broodkoring dra gene vir agronomies-belangrike kenrnerke soos blaarroes, stamroes, Russiese koringluis en oogvlek weerstand. Sommige van hierdie gene kom voor in blokke spesie-verhaalde chromatien wat hul bruikbaarheid in teling beperk. Die genetiese kaarte van 7DL is swak ontwikkel en dit maak dit baie moeilik om hierdie gene en spesie-verhaalde streke tydens teling te manipuleer. Hierdie proefskrif verteenwoordig 'n paging om kennis van die relatiewe kaart liggings van drie weerstandsgene, met betekenisvolle potensiaal in plaaslike tee! programme, te verbreed. Die blaarroes weerstandsgeen, Lr 19, kom voor op 'n Thinopyrum ponticum-verhaalde translokasie wat 'n groot terminale gedeelte van 7DL beslaan. Die translokasie dra ook gene vir minder gewensde kenrnerke soos gee! meelkleur. Pogings is aangewend om die translokasie deur homoeoloe parings-induksie te verkort. Die doe! was om nadelige gene te verplaas en die hoeveelheid vreemde chromatien geassosieer met Lr 19 te minimiseer sodat dit met ander nuttige gene op 7DL gerekombineer kan word. Nege-en-twintig 'Indis'-verhaalde Lr 19 delesie mutante is vroeer met gamma bestraling geproduseer en gebruik om 'n fisiese kaart op te stel. Teenswoordig is die stel mutante verder ontleed met behulp van 144 Sse8387I!Msei en 32 EcoRII Msel amplifikasie-fragment-lengte-polimorfisme (AFLP) inleier kombinasies. Die bestaande fisiese kaart, wat gebaseer was op vyf restriksie-fragment-lengte-polimorfisme (RFLP) merkers en vyf strukturele geen loki, is uitgebrei en sluit nou 95 unieke AFLP merkers (86 Sse8387I/Msel en 9EcoRI/Msel merkers) in, waarvan sewe naby aan Lr19 karteer. Die meeste van die delesies kon op grond van hulle grootte gegroepeer word en die verbeterde fisiese kaart is alreeds gebruik om verkorte rekombinante vorms van die Lr 19 translokasie te karakteriseer. 'n Onsuksesvolle paging is aangewend om een van die sewe merkers naaste aan Lr 19 om te skakel na 'n volgorde-spesifieke merker. 'n AFLP merker wat distaal van Lr 19 karteer is egter wel suksesvol in samewerking met ander navorsers omgeskakel na 'n volgordespesifieke merker. 'n Paging is ook aangewend om die Russiese koringluis (RKL) weerstandsgeen, Dn5, te karteer en merkers gekoppel aan die geen te identifiseer. 'n Verdubbelde-haplo!ede karteringspopulasie van 94 lyne is geskep en getipeer vir Dn5, vier mikrosatelliet loki en die endopeptidase lokus, Ep-D1. Die Dn5 lokus karteer 25.4 cM en 28.6 cM distaal van Xgwml11 en Xgwm437, respektiewelik, maar was me gekoppel met Xgwm428, Xgwm37 of Ep-D1 me. Twaalf homosigoties weerstandbiedende en sewe homosigoties vatbare F2 lyne uit die kruising: 'Chinese Spring' I 'PI 294994' is met 70 Sse8387VMsel AFLP inleier kombinasies getoets in 'n poging om merkers vir Dn5 te identifiseer. Slegs twee moontlik bruikbare polimorfismes (een in koppelings- en een in repulsie fase ), is ge'identifiseer. Omskakeling van die koppelingsfase merker na 'n volgorde-spesifieke merker was onsuksesvol. Die oogvlek weerstandsgeen, Pch1, is uit Triticum ventricosum oorgedra en kom voor in die koringlyn, VPM-1. Noue koppeling van Pch1 en die endopeptidase alleel, Ep-D1 b, is vantevore gerapporteer. Merkers is vir P chl I Ep-D 1 gevind deur tien koring genoti pes ( elkeen homosigoties vir die bevestigde teenwoordigheid of afwesigheid van Pch1 en/of Ep-D1 b) te toets met 36 Sse83871/kfsel AFLP inleier kombinasies. Drie AFLP merkers is gevind wat nou koppel met Pchl!Ep-D1 , waarvan een gekies is vir omskakeling na 'n volgorde-spesifieke merker. Die volgorde-spesifieke merker het 'n mikrosatelliet kernmotief bevat en was nuttig as merker vir Pch1/Ep-D1. 'n Genetiese afstand van 2 cM is tussen die unieke mikrosatelliet merker en Ep-D1 bereken. Die mikrosatelliet merker was ook polimorfies vir die Lr 19 translokasie en dit is tussen die Wsp-D1 en Sr25 loki gekarteer. Kartering en/of identifikasie van merkers vir drie belangrike weerstandsgene was suksesvol in hierdie studie. Omdat al die merkers wat gebruik is, nie polimorf was tussen al die streke van belang nie, was dit nie moontlik om die data vir elk van die drie streke ten volle te integreer nie.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco. / AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plant-pathogen interactions have been intensively investigated in the last decade. This major drive towards understanding the fundamental aspects involved in plant disease resistance is propelled by the obvious agricultural and economical benefits that are intrinsically linked to disease and stress resistant plants. It is, therefore, not surprising that fundamental research in this area is not just restricted to model organisms, such as Arabidopsis and tobacco, but also extends to more traditional crop plants, such as maize, bean, soybean, apples, grapevine etc. In grapevine for instance, several genes involved in disease resistance have been isolated. One of these genes, encoding for a polygalacturonase inhibiting protein (PGIP), has been studied extensively. PGIPs are cell wall bound, contain leucine rich repeats (LRR) and are found in all dicotyledonous plants so far examined. In most cases, pgip genes occur in small multigene families and expression is often tissue specific and developmentally regulated. Up-regulation of PGIP-encoding genes typically occurs upon pathogen infection, treatment with elicitors, salicylic acid (SA), jasmonic acid (JA), cold treatment and wounding. Differential regulation and specificity have been shown to occur between members of the same multigene family. Differential regulation even extends to the utilization of separate pathways to induce pgip genes from the same family in response to a single stress stimulus. PGIPs interact with cell wall macerating polygalacturonases (PGs) that are secreted by pathogenic fungi during the infection process. The antifungal action of PGIPs is thought to depend on a dual action. The physical interaction of PGIP with PGs has an inhibitionary effect, resulting in (i) a slower fungal infection rate and (ii) the prolonged existence of long chain oligogalacturonides (OGs). These oligosaccharides are able to elicit a general plant defense response, enabling the plant to further retard or curb the spread of infection. The main objective of this study was to investigate the regulatory aspects underlying PGIP expression in grapevine. Unlike most characterized PGIP encoding genes from other dicotyledonous plant species, no evidence to support the existence of a V. vinifera PGIP multigene family could be found from either genetic or biochemical analyses. Recently, a genomic DNA fragment from Vitis vinifera cv Pinotage was pathogen interactions with regards to the fundamental processes underlying defense gene regulation. / AFRIKAANSE OPSOMMING: Die ooglopende voordele wat, vanuit 'n landboukundige én ekonomiese oogpunt, uit siekte- en stresbestande plante spruit, het gedurende die laaste dekade aanleiding gegee tot die ontwikkeling van plantpatogeen-interaksies as "n baie belangrike studieveld. Dit was dus ook te verwagte dat fundamentele navorsing in hierdie area nie net beperk gebly het tot modelorganismes soos Arabidopsis en tabak (ook natuurlik van landboukundige belang) nie, maar ook na meer tradisionele landbougewasse soos mielies, boontjies, sojaboontjies, appels, druiwe, ens. oorgevloei het. Verskeie siekteweerstands-verwante gene is byvoorbeeld al vanuit wingerd geïsoleer. Een só "n geen wat vir "n poligalakturonase-inhiberende proteïen (PGIP) kodeer, vorm deel van hierdie groep gene. Die funksie en regulering van PGIP's is baie goed bestudeer. Hierdie proteïene word normaalweg in die selwande van die meeste dikotiele plante aangetref. Leusienryke herhalings is algemeen in PGIP's en hierdie tipe van herhalings is kenmerkend van proteïene betrokke by proteïen-proteïen-interaksies. Verder word pgip-gene gewoonlik in klein multigeenfamilies aangetref, waar in die meeste gevalle die uitdrukking weefselspesifiek en die regulering spesifiek ten opsigte van die ontwikkelingsfase is. Verskeie faktore kan tot die induksie van pgip-gene lei, soos onder andere patogeen-infeksie, elisitoor-, salisiensuur-, jasmoonsuur- en kouebehandeling, asook verwonding. Differensiële regulering word in baie gevalle tussen lede van dieselfde multigeenfamilie aangetref. Hierdie differensiële regulering kan selfs bemiddel word deur onafhanklike reguleringsweë in reaksie op dieselfde induksiestimulus. PGIP's is in staat om te reageer met poligalakturonases (PGs), wat selwande afbreek en wat gedurende die infeksieproses deur swamme of fungi afgeskei word. Die effek van hierdie interaksie is tweeledig: (i) Die fisiese interaksie tussen PGIP en PG moduleer die aktiwiteit van die PG deur die ensiemaksie te inhibeer, en (ii) PGinhibisie lei tot die verhoogde stabiliteit van langketting-oligogalakturonades, molekules wat daartoe in staat is om die weerstandsrespons van plante te ontlok. Die inhibisie van die patogeen-PG's, tesame met die geïnduseerde weerstandrespons, stel die plant dan in staat om verdere infeksie te vertraag of te verhoed. Die doel van hierdie studie was om die onderliggende aspekte van PGIPregulering in wingerd te bestudeer. In teenstelling met die meeste plantspesies waar pgip-gene in klein multigeenfamilies aangetref word, is daar nie 'n pgip-multigeenfamilie in wingerd nie. Veelvuldige kopieë van In enkele pgip-geen word egter in die wingerdgenoom aangetref. Daar is onlangs in ons laboratorium In genoom-DNAfragment vanaf Vitis vinifera cv Pinotage geïsoleer wat die oopleesraam en 5'-stroomopsekwense van In PGIP-enkoderende geen (Vvpgip1) bevat. In hierdie studie is die uitdrukkingspatroon van Vvpgip1 ten opsigte van weefselspesifisiteit, korrelontwikkelingsfase, asook die effek van verskeie omgewings en patogeenverwante stres-stimuli ontleed. Die regulatoriese meganismes van Vvpgip1 bevat spesifieke in planta-ontwikkelingsfaseseine wat verder deur spesifieke faktore, insluitende omgewings- en patogeenstres, gereguleer word. In lyn hiermee is mRNS-transkripte van Vvpgip1 tot wortel- en korrelweefsels beperk, terwyl die mRNS-vlakke ook tussen verskillende korrelontwikkelingsfases wissel. Kumulatiewe uitdrukking kon waargeneem word in veráison-korrels in reaksie op verwonding en osmotiese stres. Die weefselspesifieke uitdrukkingspatroon tipies van wingerd-PGIP is in blare opgehef in reaksie op Botrytis cinerea-infeksie, verwonding, osmotiese stres, ouksien (indoolasynsuur) en salisiensuur. PGIP-uitdrukking word ook onderdruk deur In staurosporien-sensitiewe proteïenkinase, wat In goeie aanduiding is van die betrokkenheid van proteïenfosforilasie in die seintransduksiekaskade wat tot PGIPuitdrukking aanleiding gee. Die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare kan ook nageboots word in tabak wat met die Vvpgip1-geen en -promotor getransformeer is. PG-inhibisie-eksperimente met membraan-geassosieerde proteïenekstrakte van geïnduseerde wingerdblare het ook dieselfde profiel getoon as dié van PGIP wat deur die Vvpgip1-geen geënkodeer is. Die uitdrukkingsprofiel van PGIP in die transgeniese tabakplante het ook bewys dat die promotor van die Vvpgip1-geen vir die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare verantwoordelik is. In silica-analise van die promotorarea dui op die teenwoordigheid van verskeie cis-werkende elemente. Die kern promotor en transkripsie-aanvangsgedeelte is gevolglik eksperimenteel bepaal. Verder het uitdrukkingseksperimente met promotorfragmente verskeie dele van die promotor geïdentifiseer wat by stimulis-geassosieerde uitdrukking betrokke is. Posisioneel is hierdie fragmente in goeie konteks met die voorspelde cis-werkende elemente en kan dus die basis vorm vir verdere studies oor Vvpgip-regulering. Met hierdie studie word die eerste data verskaf waar die regulering van PGIP deur omgewingsverwante faktore verbind kan word met onwikkelingspesifieke toestande in die plant. Verder verskaf die resultate verdere bewyse vir die rol van PGIP in plant-patogeen-interaksies en lewer spesifieke bydraes tot die onderliggende prosesse wat by die regulering van siekteweerstandverwante gene betrokke is.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1957 R68 / Master of Science

Ph.D. / Current models regarding plant : pathogen interactions assume that recognition of pathogen-associated molecular pattern (PAMP) molecules can occur through pattern recognition receptors (PRRs) on the surface of plant cells. Lipopolysaccharides (LPS) embedded in the cell wall of Gram-negative bacteria can trigger defence responses or prime the plant in order to respond more rapidly, following perception of bacterial pathogens. Limited data has been reported on signal transduction and the nature of the LPS receptors in plants since no receptors have been identified yet. Parallels have been shown to exist between self-incompatibility and pathogen recognition with regard to self / non-self recognition. The two processes were reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility were discussed herein. The role of S-domain receptor-like kinases (RLKs) in defence mechanisms has previously not been widely recognized or explored. It was reasoned that S-domain RLKs could be utilized to function as resistance (R) genes or as pattern recognition receptors in perception of PAMPs of a non-protein nature. It has been found that genes encoding receptors may be up-regulated in response to perception of its ligand. A putative receptor-like kinase was previously reported to be induced by LPS. This 153 bp differentially expressed transcript, HAP3-15 (GenBank accession number DR109311), might be an expressed sequence tag (EST) for a gene encoding a receptor for LPS. The experimental characterisation of this EST was reported herein. Gene-walking, reverse transcriptase polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), cloning, sequencing and bio-informatic analyses were used to identify the full gene. These results revealed that it encoded a receptor-like protein kinase with an extracellular S-domain recognition motif. The 2842 bp genomic sequence obtained, showed that the sequence had a defined promoter region and six major domains. The first five domains were encoded by the first exon. These domains included a B-lectin / agglutinin domain, an S-locus glycoprotein domain, an EGF-like repeat, a PAN domain, a transmembrane region and part of the 6th domain. The 6th domain was a kinase domain consisting of eleven sub-domains interspersed by three introns. The gene was therefore designated as the N. tabacum S-domain Receptor-like kinase (NS-RLK).

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Typescript (photocopy). / Digitized by Kansas Correctional Industries