Engineering the plant intracellular immune receptor Sr50 to restore recognition of the AvrSr50 escape mutant

Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici . The receptor recognizes the pathogen effector AvrSr50 through its C-terminal leucine-rich repeat domain, initiating a localized cell death immune response. However, this immunity is compromised by mutations in the effector, as in the escape mutant AvrSr50 ^QCMJC , which evades Sr50 detection. In this study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering of Sr50 to gain recognition of AvrSr50 ^QCMJC . Following an initial structural hypothesis driven by molecular docking, we identified the Sr50 ^K711D single mutant, which induces an intermediate immune response against AvrSr50 ^QCMJC without losing recognition against AvrSr50. Increasing gene expression with a stronger promoter enabled the mutant to elicit a robust response, indicating weak effector recognition can be complemented by enhanced receptor expression. Further structural refinements led to the creation of five double mutants and two triple mutants with dual recognition of AvrSr50 and AvrSr50 ^QCMJC with greater immune response intensities than Sr50 ^K711D against the escape mutant. All effective mutations against AvrSr50 ^QCMJC required the K711D substitution, indicating that multiple solutions exist for gain of recognition, but the path to reach these mutations may be confined. Furthermore, this single substitution alters the prediction of AlphaFold 2, allowing it to model the complex structure of Sr50 ^K711D and AvrSr50 that match our final structural hypothesis. Collectively, our study outlines a framework for rational engineering of NLR systems to overcome pathogen escape mutations and provides datasets for future computational models for NLR resurrection.