Directed evolution of a plant immune receptor for broad spectrum recognition of pathogen effectors

Abstract Rapid development of immune receptors that protect crops from emerging pathogens is a critical challenge 1,2 . While novel immune receptors that recognize previously undetected pathogen effectors could provide protection against a wider range of pathogens, engineering such receptors has been constrained by the low throughput and speed of in planta testing. We established yeast surface display as a high throughput platform to recapitulate plant immune receptor-ligand interactions and evolve new binding capabilities. Using this directed evolution platform, we engineered the ligand binding domain of the rice immune receptor Pik-1 to recognize diverse effectors from the fast-evolving fungal pathogen Magnaporthe oryzae. Our approach yielded Pik-1 ligand binding domains with affinity for variants of the M. oryzae effector Avr-Pik that previously escaped detection by known rice alleles of Pik-1, with in planta assays confirming functional recognition of these effectors. Additional rounds of mutagenesis and selection led to a Pik-1 domain that binds all tested Avr-Pik variants as well as the evolutionarily divergent effector AvrPiz-t. These results demonstrate the potential of directed evolution to engineer immune receptors with new-to-nature recognition of a wide range of pathogen-derived ligands and accelerate development of broad spectrum resistance in crops.