Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

Abstract

h Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.

Symbiotic adaptations in the fungal cultivar of leaf-cutting ants

“…The present transcriptome analysis supports the hypothesis that carbohydrate-active enzymes important for plant material degradation are upregulated in gongylidia and adds the contig03063 GH12 xyloglucanase xeg1 (UniProt accession code U6NF03) to the list of significantly upregulated extracellular gongylidia enzymes (Supplementary Table 3). Expression of this xyloglucanase peaks in the bottom of gardens 55 where many more cellulolytic enzymes are overexpressed 26,53,55 , but the overexpression in gongylidia suggests that xyloglucanase may also be transferred to the top of fungus gardens via faecal droplets.…”

“…Recent studies have shed considerable light on the biochemical and molecular details of degradation processes in attine fungus gardens 14,18,26,53,54 , emphasizing the importance of fungal carbohydrate-degrading enzymes for rapid and efficient leaf degradation. Gongylidia are important in this process because of the produced and stored enzymes for later ant ingestion and transfer to newly ant-processed plant substrate via faecal fluid 19 .…”

“…Most transcripts mapped to the genomic sequence database of a L. gongylophorus strain from another A. echinatior colony (90%, 20,807/23,110, NCBI accession code PRJNA62733) 20 and an L. gongylophorus strain from an Atta cephalotes colony (90%, 20,690/23,110, NCBI accession code PRJNA48091) 26 , both from the same location in Panama, confirming that our laboratory procedures primarily extracted material form L. gongylophorus, and that symbiont strains reared by sympatric Acromyrmex colonies are very similar, consistent with earlier findings 27 . Of the 17,621 transcripts with a positive hit in the UniProtKB database, 99% (17,384) mapped to the Ascomycota and Basidiomycota (Table 1), but we retained all 23,110 transcripts to avoid missing potentially novel transcripts not covered in the incomplete (7-15 Â coverage) draft genome assemblies that were available.…”

Small genome of the fungus Escovopsis weberi , a specialized disease agent of ant agriculture

“…Interestingly, proteomic, transcriptomic, and draft genome sequencing have identified some of these missing enzymes to be present and highly expressed in the ant-cultivated fungus Leucoagaricus gongylophorus (Fig. 3 and SI Appendix, Table S10) (2,3). Taken together, these losses are consistent with previous findings that the specialized mycoparasite Escovopsis breaks down fungal but not plant material (7) and suggest that E. weberi has lost the ability to feed on lignocellulosic plant material, an ability retained by other microbial members of fungus-growing ant gardens (46).…”

“…The capacity of the cultivated fungus to break down plant material gives ant agriculturalists access to the vast nutrient stores locked within neotropical plants (Fig. 1A) (2)(3)(4)(5). The symbiosis between fungus-growing ants and their cultivated fungi has persisted for at least 50 million years (6).…”