DOI: 10.5061/dryad.v6wwpzgtn
title: Symbiotic nitrogen fixation in the reproductive structures of a basidiomycete fungus

description: 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.
description: This dataset is composed of multiple different data types,
 including: 1) vouchered specimens of the fungus, <em>Guyanagaster
 necrorhizus, 2) </em>nitrogen elemental analysis of <em>G.
 necrorhizus </em>gleba, 3) ethylene production of <em>G.
 necrorhizus </em>gleba (for the acetylene reduction assay), 4)
 bacterial community data of <em>G. necrorhizus</em>
 sporocarps, 5) plant cell wall degrading gene copy number in the genome of
 <em>G. necrorhizus</em> and other closely related species, and
 6) proteomics data from <em>G. necrorhizus</em> 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.
description: Data_S1.csv: master file sheet that includes all of the
 <em>G. necrorhizus</em> sporocarps that were used in this
 study, and the year collected. Data_S2.csv: This file
 contains the % N and stable N15 values of <em>G.
 necrorhizus</em> gleba tissue and the maturity group of the
 corresponding sporocarp. Data_S3.csv: Contains the
 ethylene measurements from gleba tissue, and the corresponding maturity
 group and replicate. The ethylene value was normalized to per hour and per
 gram weight. The ethylene value is a proxy for nitrogenase
 activity. Data_S4.csv: Contains the gene copy number
 for a group of plant cell wall degrading enzymes. Besides <em>G.
 necrorhizus</em>, there are five <em>Armillaria</em>
 species, and five additional closely related species. These data were
 derived from the CAZyme annotation pipeline in MycoCosm.
 Data_S5.csv: Contains the count data and taxonomy output from
 DADA2 for all samples (negative controls were removed).
 Spectral_Counts_Gn.csv: Contains the soluble spectral counts for
 all of the <em>G. necrorhizus</em> protein hits for three
 biological replicates at MG I and MG IV. NA indicates values where the
 peptide and/or spectral count was equal to 0.
 Data_S6.csv: Contains the fold change of the proteins hits
 between MG I and MG IV, and the <em>p</em>-value of the
 t-tests between the two treatments. While certain values were imputed (in
 Perseus) when missing in only several of the samples, NA indicates missing
 values that could not be imputed because the proteins were only present in
 one or two samples. ASVs_Gn_20200714.fa: A fasta file
 containing all of the ASVs after processing with DADA2.
 ASV3_Enterobacteriaceae.fa: alignment of the 16S fragment of ASV3
 and other Enterobacteriaceae species.