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Structure-guided protein engineering extends immune receptor recognition of effectors from the rice blast fungus

Juan Carlos De la Concepcion: John Innes Centre

<div>Pathogens deliver an array of molecules, termed effectors, to manipulate host cellular processes for their own benefit. Perception of effectors by intracellular immune receptors from the NLR (Nucleotide-binding, Leucine-rich Repeat) family triggers immune responses, leading to cell death and preventing the spread of the pathogen. The engineering of new NLR receptors with improved recognition capabilities has been a long-term goal in plant biotechnology. However, it remains elusive due to the narrow recognition spectrum of NLRs, and the lack of mechanistic knowledge about immune activation. The rice NLR Pikp recognizes the rice blast pathogen effector AVR-PikD by direct binding to an integrated Heavy Metal Associated (HMA) domain, resulting in disease resistance. However, polymorphic effector variants have lower binding affinity, and evade immune recognition. Here, we exploit this knowledge to investigate the mechanistic basis of a natural allele of rice Pik, Pikm, with broader recognition specificities for effector variants. Biochemical characterization of Pikm/AVR-Pik interactions revealed a causal link between higher binding affinities <em>in vitro </em>and immune response <em>in planta</em>. The crystal structures of five different receptor/effector combinations uncovered the key amino acids that underpin differential binding specificities. We applied this information to generate mutations in the rice NLR Pikp that increased binding affinity for the effector variants. These mutants acquired gain of immune response to previously unrecognized effectors. Therefore, we have successfully engineered new immune receptors with the potential to improve disease resistance in crops.</div>