Symbiotic nitrogen fixation in the reproductive structures of a basidiomycete fungus

Abstract

Nitrogen (N) fixation is a driving force for the formation of symbiotic associations between N₂-fixing bacteria and eukaryotes.¹ Limited examples of these associations are known in fungi, and none with sexual structures of non-lichenized species.^2-6 The basidiomycete Guyanagaster necrorhizus is a sequestrate fungus endemic to the Guiana Shield.⁷ Like the root rot-causing species in its sister genera Armillaria and Desarmillaria, G. necrorhizus sporocarps fruit from roots of decaying trees (Figures 1A-1C),⁸ and genome sequencing is consistent with observations that G. necrorhizus is a white-rotting decomposer. This species also represents the first documentation of an arthropod-dispersed sequestrate fungus. Numerous species of distantly related wood-feeding termites, which scavenge for N-rich food, feed on the mature spore-bearing tissue, or gleba, of G. necrorhizus. During feeding, mature spores adhere to termites for subsequent dispersal.⁹ Using chemical assays, isotope analysis, and high-throughput sequencing, we show that the sporocarps harbor actively N₂-fixing Enterobacteriaceae species and that the N content within fungal tissue increases with maturation. Untargeted proteomic profiling suggests that ATP generation in the gleba is accomplished via fermentation. The use of fermentation-an anaerobic process-indicates that the sporocarp environment is anoxic, likely an adaptation to protect the oxygen-sensitive nitrogenase enzyme. Sporocarps also have a thick outer covering, possibly to limit oxygen diffusion. The enriched N content within mature sporocarps may offer a dietary inducement for termites in exchange for spore dispersal. These results show that the flexible metabolic capacity of fungi may facilitate N₂-fixing associations, as well as higher-level organismal associations.

Keywords: Basidiomycota; Enterobacteriaceae; Guiana Shield; Physalacriaceae; genome sequencing; insect fungiculture; mutualism; trophic interactions; tropical ecology; white rot.