valentyna.klymiuk@usask.ca, k.wiebe@usask.ca, Harmeet.Chawla@umanitoba.ca, jennifer.ens@usask.ca, rajagopal.subramaniam@AGR.GC.CA, curtis.pozniak@usask.ca

Rapid cloning and functional characterization of genes controlling important agronomic traits in polyploid wheat are facilitated by recent technological innovations. We have applied a combination of bulk-segregant analysis sequencing (BSA-Seq), fine mapping, PacBio genome sequencing, long-read amplicon sequencing and EMS-mutagenesis to reveal genes underlying Yr84-mediated stripe rust resistance. We have confirmed that the expression of resistance requires two nucleotide-binding and leucine-rich repeat genes (NLRs): CC-NB-ARC-LRR (CNL) and NB-ARC-LRR (NL).

In NLR pairs, one protein, designated as the sensor NLR (sNLR), detects the presence of the pathogen, while the other, known as the helper NLR (hNLR), transmits the signal to downstream cascades. In the case of Yr84, expression studies revealed that only the NL was upregulated in the presence of the pathogen, whereas CNL exhibited constitutive expression.

These results are consistent with expression of paired NLRs in rice and Arabidopsis, where the hNLR is upregulated while the sNLR is constitutively expressed. Therefore, we hypothesize that in the Yr84 NLR pair, CNL functions as the sNLR, while NL serves as the hNLR. Although these NLRs are physically linked on chromosome arm 1BS, they exhibit structural and evolutionary divergence. Several susceptible alleles have been identified in natural populations of wild emmer wheat for both genes, whereas resistance alleles were rarely detected.

An analysis of predicted CNL and NL protein structures revealed that both proteins contain conserved amino acid motifs in the NB-ARC and LRR domains, with conservation being more pronounced for the CNL compared to the NL protein. An analysis of the interactions within the Yr84 NLR pair using the yeast two-hybrid system did not confirm the general trend of direct homo- or hetero-interaction between the sNLR and hNLR observed in other pathosystems.

However, we are in the process of verifying this finding through co-immunoprecipitation experiments (co-IP) conducted using Tobacco infiltration experiments. We have developed a Yr84 functional molecular marker, located within the CNL gene, which has been utilized for introgressions into multiple durum and bread wheat genetic backgrounds, including those important for breeding programs and genetic/pathogenicity studies.