Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes)

Abstract

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1, whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes.

Importance: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development.

Keywords: Basidiomycota; Coprinopsis cinerea; fruiting body; mushroom development; tissue differentiation; unannotated genes.

Fig 1

Cross-sections of developing fruiting bodies of wt and Δsnb1 Coprinopsis cinerea strains during the first 5 days post-light induction (pLI). Strains were grown on YMG medium with halved glucose content at 28°C. Scale bar = 1 mm.

Fig 2

Fruiting bodies of wt and Δsnb1 Coprinopsis cinerea strains after 2 weeks of growth in the dark. (A) The wt fruiting bodies elongated in darkness (dark stipes tilted by 90^o). (B) Δsnb1 fruiting bodies expanded laterally and remained spherical, and were unable to produce dark stipes. Scale bar = 2 mm.

Fig 3

The effect of LiCl on Coprinopsis cinerea fruiting body development. Pictures represent the final developmental stages after 2 weeks of incubation on a YMG medium with halved glucose content and increasing concentrations of LiCl. (A) Top view of wt and Δsnb1 colonies. The vegetative mycelia of the strains were grown under alternating dark and light cycles, leading to the formation of ring-like structures on the mycelia. (B) Whole and sectioned fruiting bodies of wt and Δsnb1 strain.

Fig 4

Structural and phylogenetic analysis of SNB1. Based on predictions made by (A) DeepTMHMM and (B) AlphaFold (https://alphafold.ebi.ac.uk/entry/A8N2R0), SNB1 possesses seven transmembrane domains. The N-terminus of SNB1 is oriented toward the extracellular environment, while the C-terminus faces the intracellular space. (C) Maximum likelihood phylogenetic tree showing copy numbers of snb1 in 109 species. Ascomycota was used as an outgroup. Coprinopsis cinerea AmutBmut1 is highlighted with red, which has four snb1 paralogs. For detailed data, see Fig. S4A.

Fig 5

Results of RNA-Seq analysis. (A) Heatmap of the 1,299 differentially expressed genes. (B) Functional categories of developmentally regulated genes according to Nagy et al. (2023). Asterisks indicate the significant enrichment of DEGs in a given category (one-tailed Fisher’s exact test, *P ≦ 0.05, **P ≦ 0.01). (C) Heatmap displaying the normalized expression levels of differentially expressed genes (DEGs) within the 14 functional categories.