The emergence of the multi-species NIP1 effector in Rhynchosporium was accompanied by high rates of gene duplications and losses.

Plant pathogens secrete effector proteins to manipulate the host and facilitate infection. Cognate hosts trigger strong defence responses upon detection of these effectors. Consequently, pathogens and hosts undergo rapid coevolutionary arms races driven by adaptive evolution of effectors and receptors. Because of their high rate of turnover, most effectors are thought to be species-specific and the evolutionary trajectories are poorly understood. Here, we investigate the necrosis-inducing protein 1 (NIP1) effector in the multihost pathogen genus Rhynchosporium. We retraced the evolutionary history of the NIP1 locus using whole-genome assemblies of 146 strains covering four closely related species. NIP1 orthologues were present in all species but the locus consistently segregated presence-absence polymorphisms suggesting long-term balancing selection. We also identified previously unknown paralogues of NIP1 that were shared among multiple species and showed substantial copy-number variation within R. commune. The NIP1A paralogue was under significant positive selection suggesting that NIP1A is the dominant effector variant coevolving with host immune receptors. Consistent with this prediction, we found that copy number variation at NIP1A had a stronger effect on virulence than NIP1B. Our analyses unravelled the origins and diversification mechanisms of a pathogen effector family shedding light on how pathogens gain adaptive genetic variation.