Symbiotic nitrogen fixation in the reproductive structures of a basidiomycete fungus

<p><span>Nitrogen (N) fixation is a driving force for the formation of symbiotic associations between N</span>_{<span>2</span>}<span>-fixing bacteria and eukaryotes.</span>^{<span>1</span>}<span> Limited examples of these associations are known in fungi, and none with the sexual structures of species outside of lichens.</span>^{<span>2-6</span>}<span>  The basidiomycete, </span><i><span>Guyanagaster necrorhizus</span></i><span>, is a sequestrate fungus endemic to the Guiana Shield.</span>^{<span>7</span>}<span> Much like the root rot-causing species in its sister genera, </span><i><span>Armillaria</span></i><span> and </span><i><span>Desarmillaria</span></i><span>, </span><i><span>G. necrorhizus</span></i><span> sporocarps fruit from the roots of decaying trees (Figures 1A, 1B and 1C),</span>^{<span>8</span>}<span> and genome sequencing is consistent with observations that </span><i><span>G. necrorhizus</span></i><span> 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, </span><a><span>feed on </span></a><span>mature </span><i><span>G. necrorhizus</span></i><span> gleba, </span><span><span>the interior spore-bearing tissue</span></span><span>. 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 N</span>_{<span>2</span>}<span>-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</span>_{<span>2</span>}<span> 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 N</span>_{<span>2</span>}<span>-fixing associations, as well as higher-level organismal associations.</span></p>