Bradley, G.Njom, Henry Akum2016-07-262016-07-262016http://hdl.handle.net/20.500.11837/187Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40% by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9% confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P. triticina race 3SA145 infection including a key enzyme of the Calvin cycle, ribulose bisphosphate carboxylase/oxygenase (RubisCO. These changes observed may reflect the reallocation of metabolites from normal growth processes to defensive functions after induction of plant responses by aphid feeding as well as pathogen infection. Some of the differentially accumulated proteins were found to be involved in ATP synthesis. The increased energy demand by the infected or infested plants was to cope with the impeding stress. Also, a few of the differentially regulated proteins were found to be related to stress. Most of these proteins were expressed early in the resistant cultivar but only occurred much later in the moderately resistant cultivar. These results suggest that RWA SA1 infestation and leaf rust infection of wheat may result in an increase photosynthesis, photorespiration, ATP synthesis and production of stress related proteins to cope with the stress. The effect of leaf rust infection on wheat plant colonization with RWA was evaluated. Two wheat cultivars, SST 347 (resistant to both leaf rust race 3SA145 and Russian wheat aphid biotype SA1) and SST 356 (susceptible to both 3SA145 and RWA SA1) were grown to the 2-3 leaf stage. Treatments consisted of untreated control (wheat seedling infested with aphids only) and test (wheat seedlings inoculated with urediniospores of Puccinia triticina race 3SA145 suspended in Soltrol-170® and later infested with aphids at day 3, 5, 7 and 9 post infection). Aphid population on each plant was determined by counting the total number of aphids on each leaf with the aid of a hand lens for 21 days post infestation. Disease response in inoculated seedlings was scored using the 0 to 4 infection type scale. Results obtained showed infection types; 1+ and 3++ for SST 347 and SST 356 respectively. Few aphids chose to colonise previously infected plants at the beginning as aphid numbers on the preinfected SST 347 only reached >10 after day 16 whereas on preinfected SST 356 reached >10 after day 13-15. We therefore concluded that preinfection with leaf rust delayed aphid colonization of both the resistant SST 347 and susceptible SST 356 cultivars to the same extent. Therefore prior infection of wheat plants under laboratory conditions appears to induce resistance to Russian wheat aphids (antixenosis) in both resistant and susceptible cultivars. However, this resistance was later overcome with doubling of aphid populations occurring at a higher rate on the susceptible cultivar as compared to the resistant cultivar. This was the first study to show that infection of plants with rust primed resistance to insect herbivores.engThesis