Summary In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant−microbe communities, the mechanisms driving such multipartite interactions remain largely unknown. Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil‐based split‐root system. Fungal confrontation in soil resulted in induction of B. sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S. vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S. vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S. vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response. The activation of stress response genes and concomitant repression of putative effector gene expression in B. sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen's virulence potential before host plant infection.

Extracellular adenosine 5′‐triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane‐associated receptor proteins in animals and plants. eATP regulates plant growth, development, and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We show here that perception of extracellular nucleotides, such as eATP, is important in plant–fungus interactions and that during colonization by the beneficial root endophyte Serendipita indica eATP accumulates in the apoplast at early symbiotic stages. Using liquid chromatography–tandem mass spectrometry, and cytological and functional analysis, we show that S. indica secrets SiE5′NT, an enzymatically active ecto‐5′‐nucleotidase capable of hydrolyzing nucleotides in the apoplast. Arabidopsis thaliana lines producing extracellular SiE5′NT are significantly better colonized, have reduced eATP levels, and altered responses to biotic stresses, indicating that SiE5′NT functions as a compatibility factor. Our data suggest that extracellular bioactive nucleotides and their perception play an important role in fungus–root interactions and that fungal‐derived enzymes can modify apoplastic metabolites to promote fungal accommodation.