Drought stress can have a major impact upon plant survival and crop productivity. Sorghum bicolor is an important cereal crop grown in the arid and semi-arid regions of >98 different countries. It is well adapted to the harsh drought-prone environments in which it is grown however; relatively few studies have investigated the molecular basis of these adaptations. Breeding programs have lead to the identification of ‘stay-green’ varieties, so-called due to their ability to maintain green photosynthetic leaf area for longer under drought conditions. However, despite extensive breeding efforts to select for this trait we have very little understanding of the fundamental biological processes that underlie it. Microarray analysis was used to identify gene expression changes in sorghum following heat stress, drought stress and combined heat and drought stress. These microarrays were additionally used to compare gene expression in stay-green (drought-tolerant) and senescent (drought-sensitive) sorghum lines. Ontological analysis of the genes expressed to higher levels in the stay-green lines identified key processes hypothesised to be associated with the trait. These include genes associated with proline and betaine biosynthesis, glutathione S-transferase (GST) activity and the regulation of stomatal aperture and density. Both proline levels and GST activity were found to be higher in the stay-green lines thus validating that the changes at the gene expression level result in changes at the protein level. Stay-green lines were also shown to have reduced transpiration and reduced numbers of stomata. Two signalling genes, DREB1A and SDIR1 were expressed to higher levels in the stay-green varieties. Transgenic lines overexpressing these genes were generated in order to test their function. Based on the gene expression data, putative mechanisms underlying two QTL for the stay-green trait (Stg1 and StgB) were generated. Further validation of these genes and processes could not only improve our understanding of drought tolerance mechanisms in sorghum, but also facilitate the improvement of future sorghum cultivars.

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Plants have a remarkable ability to resist the majority of pathogenic microbes they encounter. As such, they are described as nonhosts. Nonhost resistance is often conceptualised as a qualitative separation from host resistance. Classification into these two states is generally facile, as they fail to fully describe the range of states that exist in the transition from host to nonhost. This poses a problem when studying pathosystems that cannot be classified into either of these categories due to their intermediate status relative to the two extremes. Therefore, the terms intermediate host and intermediate nonhost have been proposed to describe pathosystems in the evolutionary transition between host and nonhost status. At present, a significant amount of research exists into the molecular genetics of host and nonhost pathosystems but very little is known about intermediate systems. The work in this Ph. D. thesis focuses on the interaction of barley with Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, as an intermediate host pathosystem. The first research chapter describes the development of two microscopic phenotypic assays used to quantify P. striformis f. sp. tritici in barley leaves challenged with the pathogen. These assays are then used to screen a large panel of barley accessions to define the intermediate host status of barley relative to a host pathosystem. Subsequently, these assays play a key role in determining that the genetic architecture of resistance in barley is underpinned by three major effect resistance loci: Rpst1, Rpst2, and Rpst3. Using a combination of classical map-based genetics and contemporary genomics information I identify a candidate NLR gene underlying Rpst2 resistance on chromosome 7HL. Furthermore, I show that distinct genes condition host and nonhost resistance in barley by mapping the host resistance gene, rps2 to chromosome 2HL.

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Transgenic maize MON810 is a maize variety that has been genetically modified to express Cry1Ab isolated from the soil bacterium Bacillus thuringiensis (Bt) to produce a natural insecticide (Bt toxin) which kills larvae of the European corn borer (ECB), a major pest of maize. The mode of action of Bt toxin in ECB is through binding to specific receptors on the epithelial cells of the highly alkaline midgut of the insect, resulting in pore-formation, osmotic imbalance, cell lysis and subsequent death of the insect. In contrast, this Bt toxin is considered to be harmless or nontoxic to mammals due to acidified gut pepsinolysis and the lack of Cry protein binding-sites on the mammalian gut epithelial cells. However, to date, no studies have investigated the cellular effects of these Cry proteins at the proteome level. The aim of this study was therefor to investigate the in vivo and in vitro effects of MON810 maize expressing the δ-insecticidal protein Cry1Ab from Bacillus thuringiensis, on differential gene expression at the proteome level in the epithelial cells of the small intestine of the rat as a model for mammals. Proteomic profiling techniques were included for the in vivo and in vitro studies to obtain a better understanding of the underlying molecular responses in rat to MON810. Transgenic Bt maize (MON810), the corresponding parental non-transgenic maize (MON CONV CORN), and 3 other maize varieties, MON Garst 8450, MON Gold HVST H8920 and MCert Rod commercial control (used as internal controls), were provided by Monsanto, USA. All diets were formulated by TestDiet and contained approximately 33% (w/w) corn grain; other diet components were adjusted to provide approximately equal levels of protein, calories and nutrients. Different reference varieties were used in this study to determine whether the changes that may occur with the consumption of MON810 maize lay within the expected range for several different unmodified reference varieties. Two rats feeding trials (7-day and 28-day) were conducted to assess the safety of MON810 maize using forty immature male Wistar rats (rats were between 6 to 7 weeks of age at the beginning of the study). Rats were assigned to the above 5 experimental groups based on body weight means. No adverse behavioural effects on rats were observed and there were no significant differences in absolute body weights, body weight gains, food consumption and feed conversion efficiency between rats fed MON810 in the diet when compared to rats fed diets containing grain from the other maize varieties. Thus the transgenic variety MON810 had no adverse effects on these parameters. Following these feeding studies, rats were sacrificed and the total proteins extracted from the small intestinal epithelial cells were separated by 2D gel electrophoresis. Differentially expressed proteins were identified using SameSpot Progenesis software followed by liquid chromatography–mass spectrometry (LC-MS/MS); the mass spectrometry data were analysed by Global Proteome Machine (GPM) search engine for protein identification. The same proteomic profiling techniques were used for the in vitro approaches for MON810 maize safety evaluation where primary intestinal epithelial cells and HCT116 cell line were used. In vivo effects of these different maize varieties on the proteome of the epithelial cells of the small intestine when all five groups were compared showed that there were 5, 4, 3, 0, 0, differentially expresed protein spots for Mcert, Mon Conv Corn, MON810, Mon Garst and MON Gold, respectively for the 7- day trial. Two stress-related proteins (LDLR chaperone MESD precursor and peroxiredoxin-6) were up-regulated (2.4 fold) in the MON810 group and 1 stress-related protein (thioredoxindependent peroxide reductase) was up-regulated (2.6 fold) in the Mcert group. For the 28-day trial, only 2 proteins spots (representing 6 proteins) were up-regulated in the small intestinal epithelial cells of rats fed MON810, one of which was a stress-related protein (stress-induced phosphoprotein 1; 3.1 fold). In vitro effects of MON810 and its near isogenic line on the proteome of the epithelial cell lines were negligible, with only 4 protein spots (5 stress-related protein) being up-regulated in the small intestinal primary epithelial cells (IE cells) when exposed to Bt maize extracts and 2 protein spots (1 stress-related protein) being up-regulated when exposed to non-Bt maize extracts. There were no differentially expressed spots between the HCT116 cell lines. The findings from the in vivo and in vitro studies both suggest that MON810 has negligible effects on rats at the cellular level. They also confirm the lack of mammalian toxicity when using rats as a model system.

The threat to UK food security due to cereal diseases is serious. Diseases can affect crops and have a serious impact on the economic output of a farm and on food. Among cereal diseases, Fusarium Head Blight (FHB) and Fusarium Crown Rot (FCR) disease are two of the most widespread and damaging diseases of cereal crops. This thesis reports the effect of Piriformospora indica on Fusarium diseases of wheat, both head blight and crown rot, with the purpose of developing a solution to control crop diseases by using natural microorganisms. Piriformospora indica is a root endophyte belonging to the Sebacinaceae (Sebacinales, Basidiomycota). It was originally found in the Thar desert of Rajasthan, in India. P. indica forms mutualistic symbioses with a broad range of host plants, increasing their biomass production and resistance to fungal pathogens. Glasshouse experiments and controlled environmental chambers with conditions adjusted to UK autumn conditions were used to determine the effect of P. indica on FCR disease of wheat, both Fusarium culmorum and F. graminearum. P. indica reduced damage to wheat seedlings by restricting growth of pathogen in the root. The effect of P. indica on FHB disease of winter (cv. Battalion, NABIM group 2) and spring (cv. Paragon, Mulika, Zircon (NABIM group 1), Granary, KWS Willow (NABIM group 2) and KWS Kilburn (NABIM group 4)) hard wheat and subsequent contamination by the mycotoxin deoxynivalenol (DON) were examined in the pots under UK weather conditions. P. indica application reduced FHB disease severity and incidence and mycotoxin DON concentration of inoculated winter and spring wheat samples. P. indica also increased above-ground biomass, thousand grain weight and total grain weight. The effects were similar at different fertiliser levels. The effect of P. indica was compatible with the arbuscular mycorrhizal fungus Funneliformis mosseae and foliar fungicide Aviator Xpro (Bayer CropScience, UK; with active ingredients of prothioconazole and bixafen) application. P. indica reduced severity and incidence of naturally arising infection by Septoria leaf blotch (caused by Zymoseptoria tritici), yellow rust (caused by Puccinia striiformis f. sp. tritici) and powdery mildew (caused by Blumeria graminis f.sp. tritici). The nutrient analysis of soil and plant tissue samples showed that P. indica did not have any effects on phosphorus, nitrogen and potassium status and uptake were not significantly affected by P. indica inoculation. P. indica mRNA for the elongation factor (TEF gene) was used as an indicator of P. indica viability in soil. P. indica was still alive after four and eight months in pots of soil from the Reading area, which had been left open to winter-summer weather conditions without host plants, but not after 15 months. PCR-denaturing gradient gel electrophoresis of DNA extracted from root zone or from bulk soil, in which P. indica-infected wheat had been grown, showed P. indica increased the root and soil fungal and bacterial species diversity. Test on arable weeds, black-grass, wild-oat and cleavers, showed that on average over species P. indica increased root biomass by 35 %; but above-ground biomass was not significantly affected by P. indica. The average above-ground competitiveness of the weeds with wheat was slightly decreased. My results suggest that P. indica could be used to control wheat diseases in field settings in the UK. However, extensive data would be needed to determine ecological and agronomical safety and persistence, before release on a field scale was commercialised.

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