Orthrus: a Pumilio-family gene involved in fruiting body and dark stipe development in Coprinopsis cinerea

Abstract

Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures whose formation is regulated by a developmental program that dynamically responds to environmental changes, such as light intensity. However, the genetic architecture and regulation of this developmental program are poorly known. Here, we characterize a novel Pumilio family gene, ort2, which influences fruiting body development, particularly the formation of dark stipes, a light-dependent alternative developmental trajectory. Phylogenetic analysis of this RNA-binding protein family in fungi revealed a distinct subfamily structure, with high conservation of each subfamily within Agaricomycetes. Reverse genetics experiments in the model species Coprinopsis cinerea revealed that ort2 disruptants produced fruiting bodies, but were deficient in dark stipe formation, whereas the overexpression mutants produced significantly more dark stipes. The gene was named after Orthrus, the two-headed dog of classical mythology, based on rare but reproducible branching fruiting body phenotypes observed upon overexpression. Our findings reveal fruiting-related functions for ort2, a novel conserved RNA-binding protein, and may serve as a novel entry point for understanding the molecular basis of dark stipe development.

Keywords: Pumilio; RNA-binding proteins; dark stipe; fruiting body formation; language model.

Figure 1

Unrooted maximum likelihood gene tree of fungal Pumilio proteins and expression dynamics of Coprinopsis cinerea Pumilio genes. The phylogenetic analysis comprises protein sequences from 17 fungal species. Pumilio proteins form five conserved subfamilies. Subfamilies are named after Sch. pombe proteins. Clade colors match those of the expression diagrams of the corresponding C. cinerea genes. In each clade, only terminal nodes belonging to C. cinerea, Cr. neoformans, S. cerevisiae, and Sch. pombe are labeled by JGI identifiers or common names. For more details, see Supplementary Figure S1 . For the expression plots, we used Krizsán et al.’s RNA-Seq dataset (2019). The developmental stages are as follows: VM, vegetative mycelium; HK, hyphal knot; P1, stage 1 primordium; P2, stage 2 primordium; YFB, young fruiting body; MFB, mature fruiting body. In earlier stages (VM to P2), bulk expression values are shown (indicated by filled circles), in more developed stages (YFB and MFB), tissue-specific expression is plotted. In the YFB stage, squares, triangles, and diamonds indicate expression in stipe, cap, and gills, respectively. In the MFB stage, squares similarly indicate expression in the stipe tissue, while empty triangles mark combined cap and gill expression. Expression values are expressed as CPM on all plots.

Figure 2

Phenotypic characterization of Δort2 disruptants. (a) Violin plot: total number of fruiting bodies in stages from P4 to MFB on light-grown jar cultures of the wild-type and Δort2 strains. (b) Cross-sections of wild-type (left panel) and Δort2 (right panel) primordia.

Figure 3

Phenotypic characterization of the ort2OE mutants. (a) Violin plots illustrating the total number of fruiting bodies (P4 to MFB) on light-grown Petri dish cultures of the wild-type and ort2OE strains. (b) Violin plots of the number of dark stipes exceeding 1 cm in length, formed by the wild-type and ort2OE strains. (c) Comparison of WT and ort2OE dark-grown cultures. (d) An overexpression-related abnormality; the reason we chose ort2 (two-headed Orthrus) as a name for C. cinerea 354109. The branched fruiting bodies were more pronounced in dark stipes (pictured). For other overexpression-related abnormalities, see Supplementary Figure S3 .

Figure 4

In silico prediction of Ort2 target genes. (a) S. cerevisiae Puf3p recognition motif (based on (Gerber et al., 2004)) (left) and Ort2 recognition motif predicted by the language model (right). Nucleotide frequencies of the aligned sites were transformed to information content. (b) qPCR measurement of mtDNA levels, relative to a nuclear gene, in the Δort2 and ort2OE strains, compared to a wild-type control. The relative mtDNA abundance of the wild type corresponds to 1 on the plot. Due to the necessity of averaging during the calculation of FC values, standard deviations of FC values cannot be assessed. See raw data in Supplementary Table S5 . (c) Euler diagram of five C. cinerea protein sets: those with mitochondrial GO annotations (mt_GO), proteins with mitochondrial localization predicted by DeepLoc 2.0 (mt_DeepLoc), proteins with mitochondrial localization predicted by TergetP 2.0 (mt_TargetP), Ort2 targets predicted by Karollus et al.’s language model (targets_LM), and Ort2 targets predicted using FIMO (targets_FIMO). See gene sets in Supplementary Table S6 . Euler plots are model-based, and the model might not include low-cardinality intersections. For the precise overlap analysis, see Supplementary Figure S4 .