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.