Symbiotic nitrogen fixation in the reproductive structures of a basidiomycete fungus

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 the sexual structures of species outside of lichens.2-6  The basidiomycete, Guyanagaster necrorhizus, is a sequestrate fungus endemic to the Guiana Shield.7 Much like the root rot-causing species in its sister genera, Armillaria and Desarmillaria, G. necrorhizus sporocarps fruit from the roots of decaying trees (Figures 1A, 1B and 1C),8 and genome sequencing is consistent with observations that G. necrorhizus is a white-rotting decomposer. This species also represents the first documented instance of an arthropod-dispersed sequestrate fungus. Numerous species of distantly related wood-feeding termites, which scavenge for N-rich food, feed on mature G. necrorhizus gleba, the interior spore-bearing tissue. 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 N2 content within mature sporocarps may offer a dietary inducement for termites in exchange for spore dispersal to woody substrates. These results show that the flexible metabolic capacity of fungi may facilitate N2-fixing associations, as well as higher-level organismal associations.

Methods
This dataset is composed of multiple different data types, including: 1) vouchered specimens of the fungus, Guyanagaster necrorhizus, 2) nitrogen elemental analysis of G. necrorhizus gleba, 3) ethylene production of G. necrorhizus gleba (for the acetylene reduction assay), 4) bacterial community data of G. necrorhizus sporocarps, 5) plant cell wall degrading gene copy number in the genome of G. necrorhizus and other closely related species, and 6) proteomics data from G. necrorhizus gleba. Data from 1, 2, 3, 5 and 6 are raw data. Data from the microbiome dataset includes the count table and taxonomy table output from DADA2.