Interactions of Wheat Powdery Mildew Effectors Involved in Recognition by the Wheat NLR PM3

To successfully colonize the living tissue of its host, the fungal wheat powdery mildew pathogen produces diverse effector proteins that are suggested to reprogram host defense responses and physiology. When recognized by host immune receptors, these proteins become avirulence (AVR) effectors. Several sequence-diverse AVRPM3 effectors and the suppressor of AVRPM3-PM3 recognition (SVRPM3 a1/f1 ) are involved in triggering allele-specific, Pm3-mediated resistance, but the molecular mechanisms controlling their function in the host cell remain unknown. Here, we describe that AVRPM3 b2/c2 , AVRPM3 a2/f2 , and SVRPM3 a1/f1 form homo- and heteromeric complexes with each other, suggesting that they are present as dimers or higher-order multimers in the host cell. Alphafold2 modeling substantiated previous predictions that AVRPM3 b2/c2 , AVRPM3 a2/f2 , and SVRPM3 a1/f1 all adopt a core RNase-like fold. We found that a single amino acid mutation in a predicted surface-exposed region of AVRPM3 a2/f2 resulted in recognition by the PM3b immune receptor, which does not recognize wild-type AVRPM3 a2/f2 . This indicates that differential AVRPM3 recognition by variants of the highly related PM3 immune receptors is due to subtle differences in similar protein surfaces of sequence-diverse AVRs. Our study reveals complex molecular interactions between powdery mildew effectors. These findings suggest that structural similarity, rather than sequence conservation, underlies both the promiscuous multimerization of these effectors and their recognition by specific PM3 immune receptors.