Directed Evolution of a Plant Immune Receptor for Broad Spectrum Effector Recognition

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. Competing Interest Statement E.Y.R. and P.C.R. are inventors on a provisional patent application (No. 63/692,024) related to this work. Footnotes The manuscript has been revised to include additional data - functional validation in planta.