Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Global food security is hampered by a variety of insects/pest and plant diseases. In wheat, the Russian wheat aphid (RWA) is a significant pest problem in many areas of the world. Wheat has developed defensive mechanisms against the RWA over time which are activated upon feeding. One such mechanism is the hypersensitive response (HR) which is effective against phloem-feeding insects i.e. D. noxia (Diuraphis noxia, Kurdjumov, RWA). In this study, two genes associated with the hypersensitive response i.e. ascorbate peroxidase (APX) and glutathione S transferase (GSTF6b) were investigated to elucidate their function in the defensive mechanism of wheat using a reverse genetic approach i.e. particle bombardment. This study has succeeded in the established of a tissue culture and transformation system which generated three genetically modified wheat plants with decreased resistance to RWA feeding due to gene silencing. The establishment of this system enabled to test the association of defensive related genes in wheat to RWA resistance. Expression analysis performed on obtained transgenics before and after RWA infestation reavealed that the silenced plants were more susceptible to RWA feeding. Chlorosis was observed in the Gamtoos-S-APX transgenic plant which is an indicator of oxidative damage to the photosynthetic machinery of the plant. Decreased GSTF6b transcripts was found in the transgenic Gamtoos-S-GSTF6b and transgenic Gamtoos-R-GSTF6b transgenic plants but no visible symptoms of infestation was observed in these two plants. Resistance breeding could be strengthened by developing broad spectrum resistance plants by incorporating wheat defensive related genes with known function into the breeding programs. The use of this transformation system will allow rapid identification and introduction of agronomically important genes by upregulating these genes to enhance bread wheat against aphid infestation.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance. / AFRIKAANSE OPSOMMING: Die Russiese-koringluis (RWA) is ‘n pes wat ‘n belangrike ekonomiese invloed op koring opbrengste het en infestasie kan tot grootskaalse skade en oes verlies lei. Verskeie studies, onder andere komplimentêre DNA amplifiseerde fragment polimorfismes (cDNA-AFLPs), onderdrukkende onderskeidende hibridisaie (SSH) en mikro-reekse wat voorheen op transkriptomiese vlak gedoen is, het moontlike gene wat by RWA weerstand betrokke is, geïdentifiseer. Alhoewel hierdie gene reeds geidentifiseer was, hulle rol is nogtans onbekend. Dié gene moet nog getoets word, duur funksionele genetiese benaderingste maak. In hierdie studie is ‘n gars streep mosaïek virus vektor (BSMV) gebruik om kandidaat-gene van belang in ‘n Dn1-weerstandige geen-bevattende kultivar (TugelaDN) te onderdruk. Ondrukking van gene het deur middel van virus geïnduseerde geen onderdrukking (VIGS) plaasgevind. In hierdie studie is die hipotese gestel dat die gene betrokke by die hipersensitiewe reaksie (HR) ‘n invloed op plantweerstand kan hê en is dus geteiken vir geen-onderdrukking-studies. Hierdie gene het die volgende ingesluit: glutatioon-S-transferase (GST), superoksied dismutase Cu/Zn (SOD) en askorbien peroksidase (APX). Egter, omdat luisinfestasie ook tot verwonding aanleiding gee, is die onderdrukte gene ook onder alleenlik verwondingstoestande getoets. Luis vrugbaarheid is gebruik as indikator van betrokkenheid omdat meer vatbare plante ‘n hoër luis vrugbaarheid tot gevolg het. In die studie is gevind dat onder alleenlik verwondingkondisies, plante wat Dn1 bevat, ‘n groter hipersensitiewe respons vertoon, as vatbare kontroles. Daar is verder gevind dat askorbien peroksidase ‘n belangrike rol tydens verwondings-geïnduseerde weerstand in Dn1-plante speel. Daar is verder bevind dat die onderdrukking van superoksied dismutase Cu/Zn (SOD) en ‘n tilakoïed-geassosïeerde askorbien peroksidase (tAPX). Onder luis-infestasie kondisies, geen effek op luisvrugbaarheid gehad het nie en dus nie direk by die weerstandsrespons betrokke is nie. Die onderdrukking van ‘n phi-klas glutatioon-S-transferase F6 (TaGSTF6) het egter ‘n groot invloed op luis-vrugbaarheid gehad en kan dus ‘n rol in Dn1-weerstand speel. Die moontlike weerstands gene, geïdentifiseer as nukleotied bindings proteïen (NBP) en weestandsgeen anoloog 2 (T10rga2-1D), is getoets onder luis-infestasie kondisies. Die analise van NBP het getoon dat dit ‘n integrale deel van die yster homeostase meganisme in die sitosol, wat vir Fe-kluster samestelling verantwoordelik is, vorm. Onderdrukking van beide die NBP en T10rga2-1D het tot die uitdrukking van ‘n vatbare fenotipe aanleiding gegee. T10rga2-1A is ‘n NBS-LRR proteïen wat bekend is om noodsaaklik te wees tydens roes weerstandigheid in teenwoordigheid van die weerstandsgeen Lr10. T10rga2-1D-onderdrukte behandelings het tot vatbaarheid aangeiding gegee en daartoe gelei dat plante na luis-infestasies doodgaan. Hierdie resultate dui dus ‘n rol vir T10rga2-1D in Dn1-weerstandigheid aan, en suggereer verder dat hierdie geen ‘n goeie stroom-op kandidaat in Dn1-weerstandigheid is.