Rust fungi are important pathogens of many crops, especially wheat and other cereals where they cause substantial economic losses worldwide. While progress in resistance (R) gene cloning and stacking has accelerated in recent years, crop breeding for durable disease resistance is challenging due to the rapid evolution of virulence in these pathogens. Known Avr genes in Melampsora lini (flax rust) and Puccinia graminis f. sp. tritici (Pgt) (wheat stem rust) encode secreted proteins that are co-ordinately expressed in haustoria structures during infection. However, broader Avr gene identification in rusts has been hindered due to the complex genomes of these dikaryotic fungi (containing two divergent haploid nuclei), their obligate biotrophic nature and the large number of potential effector genes they encode. To address these limitations, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. Using this platform we have screened libraries of candidate effectors from wheat stem rust and isolated several new Avr genes recognised by known stem rust resistance (Sr) genes. We have used a combination of structural prediction and functional analyses to investigate molecular recognition events between corresponding R and Avr proteins and re-engineer a defeated Sr gene to recognise an effector variant from a recently detected virulent rust strain. We have also uncovered a novel immune induction mechanism used by non-canonical Sr genes.