Detection of differentially expressed genes upon compatible and incompatible inoculation of wheat with yellow rust using suppression subtractive hybridization (SSH)

Çelik, İlay
Yellow rust disease is one of the most important problems in wheat production. It causes substantial yield losses throughout the world. There are resistant and susceptible wheat varieties to various yellow rust pathotypes. In this thesis genes that are induced in wheat, in virulence and avirulence conditions upon yellow rust inoculations were investigated. Consequently, it was aimed to identify genes that may be playing critical roles in the disease resistance mechanism. The strategy was to construct subtracted cDNA libraries from resistant and susceptible plants and analyse the sequences obtained from these libraries. The subtraction approach in this study differs from the common subtraction designs implicated in plant-pathogen interactions; instead of comparing a compatible or an incompatible interaction with a control, one of the subtractions in this study is done taking a compatible interaction as the tester and an incompatible one as the driver, and the second subtraction, vice versa. Therefore, it was intended to compare the transcriptomes from compatible and incompatible plant-pathogen interactions directly. Suppression Subtractive Hybridization method was used to construct subtracted cDNA libraries. Two subtractions were performed; SSH1 (D-R), taking a compatible interaction as the tester sample and an incompatible one as the driver sample, and SSH2 (R-D), taking an incompatible interaction as the tester sample and a compatible one as the driver. In the end, two subtracted cDNA libraries, SSH1 (D-R) library (1536 clones) and SSH2 (R-D) library (1152 clones) were obtained and the libraries were sequenced. Sequence results were subjected to BlastN and BlastX analysis. We looked for a group of genes that were frequently emphasized in plant disease related studies when we searched within the Blast N homology results of the two libraries. We found out that 19 such genes are present in our libraries. We discussed supposed induction of these genes in the interactions investigated in our study. The fact that these genes were found to be present in our libraries enhances the reliability of our results suggesting that the gene sequences we found indeed belong to genes differentially expressed in the respective comparisons investigated in our study. As such, it also implies that other sequences that were found similar to genes of known functions may represent candidate genes as subjects of further studies investigating wheat-yellow rust interactions.