Pooled effector library screening in protoplasts rapidly identifies novel Avr genes

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years 1-3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts for rapid identification of interacting R/Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) by screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution by genotype surveillance and optimise R gene deployment and stacking strategies. This screening platform is broadly applicable to many crop pathogens, whilst also adaptable for screening genes involved in other protoplast-selectable traits.