Modifying the Stripe Rust Resistance Gene Yr10 in Wheat (Triticum aestivum L.) by PCR Mutagenesis
Stripe rust is a disease in cereal crops caused by the fungal pathogen Puccinia striiformis which can have devastating effects on crop yields. Cereal plants have unique resistance (R) genes which code for resistance (R) proteins with coiled coil (CC), nucleotide binding site (NBS), and leucine rich repeat (LRR) domains. The CC and LRR domains are thought to be particularly important for protein function/interaction in the resistance response. The Yr10 gene is a stripe rust resistance gene in Triticum aestivum that codes for a unique CC-NBS-LRR sequence but no longer confers resistance to some races of rust. Previous studies indicate that by mutagenizing the DNA sequences that code for the CC and LRR domains it is possible to change the R protein’s sequence, and possibly the interaction during the resistance response3. Thus, the defeated R genes can confer resistance once again when mutagenized in these regions.
This study used PCR mutagenesis (by overlap extension) to mutagenize the Yr10 gene in the regions encoding the CC and LRR domains. Primers were designed to amplify Yr10 such that products contained mutated sequence as extensions, available for overlap. The amplifications were the expected size, indicted primers were successful. The PCR products containing the mutagenized sequences of Yr10 were then assembled with the pANIC 6D vector to be used in further experiments involving the transformation of wheat crops to test resistance.