Protists are key players in the utilization of protein nitrogen in the arbuscular mycorrhizal hyphosphere.

While largely depending on other microorganisms for nitrogen (N) mineralization, arbuscular mycorrhizal fungi (AMF) can transfer N from organic sources to their host plants. Here, we compared N acquisition by the AMF hyphae from chitin and protein sources and assessed the effects of microbial interactions in the hyphosphere. We employed in vitro compartmented microcosms, each containing three distinct hyphosphere compartments amended with different N sources (protein, chitin, or ammonium chloride), one of which was enriched with N isotope. All hyphosphere compartments were supplied with Paenibacillus bacteria, with or without the protist Polysphondylium pallidum. We measured the effect of these model microbiomes on the efficiency of N transfer to roots via the AMF hyphae. We found that the hyphae efficiently took up N from ammonium chloride, competing strongly with bacteria and protists. Mobilization of N from chitin and protein was facilitated by bacteria and protists, respectively. Notably, AMF priming significantly affected the abundance of bacteria and protists in hyphosphere compartments and promoted mineralization of protein N by protists. Subsequently, this N was transferred into roots. Our results provide the first unequivocal evidence that roots can acquire N from proteins present in the AMF hyphosphere and that protists may play a crucial role in protein N mineralization.