Identification of specificity-defining amino acids of the wheat immune receptor Pm2 and powdery mildew effector AvrPm2.

Plant nucleotide-binding, leucine-rich repeat receptors (NLRs) act as intracellular sensors for pathogen- derived effector proteins and trigger an immune response, frequently resulting in hypersensitive cell death response (HR) of the infected host cell. The wheat (Triticum aestivum) NLR Pm2 confers resistance against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) if the isolate contains the specific RNase-like effector AvrPm2. We identified and isolated seven new Pm2 alleles (Pm2e - i) in the wheat D-genome ancestor Aegilops tauschii and two new, natural AvrPm2 haplotypes from Bgt. Upon transient co-expression in Nicotiana benthamiana, we observed a variant-specific HR of the Pm2 variants Pm2a and Pm2i towards AvrPm2 or its homolog from the AvrPm2 effector family, BgtE-5843, respectively. Through the introduction of naturally occurring non-synonymous SNPs and structure-guided mutations, we identified single amino acids, in both the wheat NLR Pm2 and the fungal effector proteins AvrPm2 and BgtE5843 responsible for the variant-specific HR of the Pm2 variants. Exchanging these amino acids led to modified HR of the Pm2-AvrPm2 interaction and allowed the identification of the effector head epitope, a 20 amino acid long unit of AvrPm2 involved in HR. Swapping of the AvrPm2 head epitope to the non HR-triggering AvrPm2 family member BgtE-5846 led to gain of HR by Pm2a. Our study presents a molecular approach to identify crucial effector surface structures involved in HR and demonstrates that natural and induced diversity in an immune receptor and its corresponding effectors can provide the basis for understanding and modifying NLR - effector specificity.

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