Winter 2004-2005,
Isolation of RGAs and disease related gene fragments from wheat stripe rust resistant differential lines (http://www.crpmb.org/icrpmc11/abstracts.htm)
I took role as an undergrad researcher in the project below supervised by Mahinur Akkaya, Department of Chemistry, METU, Turkey
Stripe rust, caused by biotrophic Puccinia striiformis f. sp. tritici, has been the most devastating wheat disease for the last 20 years in Turkey and the region. So far, only the sequence of Yr10 has been released as wheat stripe rust resistance gene (AF149114).
Several approaches were pursued for the identification of genes involved in the resistance against stripe rust. Firstly, RGA fragments amplified from 17 differential Avocet-Yr lines (ICARDA and C. Wellings) were investigated. Radioactively labeled PCR products, amplified using various RGA primers, corresponding to the p-loop, kinase and LRR domains, and were separated on denaturing gels. Those fragments having unique profiles - present only in one differential line and absent in all the others including Avocet-S - were isolated, cloned and sequenced. One of the clones showed homology to rice Rim2 transcript, which accumulates in response to infection with Magnoporthe grisea. Also, the predicted protein sequence of another clone showed homology to the BIS1 protein of H. vulgare, which is up-regulated at rust infection sites.
Secondly, in order to identify those genes that are induced upon infection with Pst17 and Pst45 strains, presenting compatible interactions with Avocet-Yr10 and Avocet-Yr1 lines, respectively, differential display (DD) analysis was performed. Differentially expressed clones were isolated showing a high degree of homology to receptor like kinases and PR proteins.
Thirdly, the stripe rust differential lines used to isolate Yr10 gene homologs were investigated using primers to amplify the first 1500 bps of Yr10. PCR products of the expected size were sequenced from Avocet-Yr6 and Avocet-Yr11 lines. In addition, a longer PCR product was obtained from both lines, showing homologies to the 3’ end of the barley Rpg1 gene. Yr10 homologs from other Avocet differential lines will be sequenced.