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Determination of genes involved in the yellow rust disease of wheat

Bozkurt, Osman
It is important to understand the underlying plant defense mechanisms in order to establish best strategies to reduce losses due to diseases in cereals. The current available information is mostly on model organisms and their plant-pathogen interactions. However, this study is focused on the identification of genes involved in the resistance mechanism of one of the most devastating diseases of wheat, yellow rust. The strategy undertaken was to use differential display method (DD) together with microarray technology, on yellow rust differential lines of wheat (Avocet-Yr1 and Avocet-Yr10) infected with the virulent and avirulent Puccinia striiformis f. sp. tritici races (Pst: PST17, PST45, 169E136 and 232E137) together with appropriate control infections. DD primer combinations of ninety allowed the detection of fourteen differentially expressed genes which were also confirmed by real-time QRT-PCR analysis. All of but one were found to be novel sequences in wheat genome. Among those, two very important genes were identified as full ORF including 5’ and 3’ end untranslated regions (UTR); namely cyclophilin like protein (putative antifungal activity) and ubiquitin conjugating enzyme (E2). The sequence homology analysis of the cloned gene fragments reveled that the genes detected have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance, pathogen related responses, including a few with no known function. In addition to DD analysis, using wheat Affymetrix “GeneChip”, we identified 93 differentially expressed ESTs of wheat in response to avirulent pathogen attack. We also investigated the differential expression profiles of wheat leaves during the virulent infections and determined 75 differentially regulated ESTs. 1Selected ESTs were further analyzed using QRT-PCR analysis and 15 were confirmed to be differentially regulated. For the further characterization of the identified genes, such as determination of their putative roles in disease response, functional studies have to be performed. For this purpose, BSMV (Barley Stripe Mosaic Virus) mediated virus induced gene silencing (VIGS) method is optimized in this thesis for wheat. We have successfully managed to silence the endogenous PDS gene (Phytoene desaturase) of wheat which can be used as a positive control for the monitoring of silencing of the genes we have identified. Our results show that BSMV mediated VIGS can be used efficiently and effectively to silence wheat genes that we identified through differential display and microarray analysis and can be used to study the functions of those genes.