Engineered geminivirus replicons enable rapid in planta directed evolution

Directed evolution can rapidly generate genetic variants with new and enhanced properties, yet efficient platforms for performing such evolution directly in plant cells have been lacking. We developed G eminivirus R eplicon- A ssisted in P lanta Directed E volution (GRAPE), a system that links gene function to geminivirus rolling circle replication (RCR) to enable high-throughput selection for desired activities. GRAPE supports the screening of up to 10 5 variants on a single Nicotiana benthamiana leaf within four days. Using GRAPE, we evolved the immune receptor NRC3 to resist inhibition by the nematode effector SPRYSEC15 and broadened the recognition spectrum of the rice immune receptor Pikm-1 to recognize all six alleles of the fungal effector AVR-Pik. GRAPE provides a rapid, scalable, and generalizable platform for directed evolution of diverse genes in plant cellular context.