Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi.

The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.

Comparative genomics reveals unique wood-decay strategies and fruiting body development in the Schizophyllaceae.

Agaricomycetes are fruiting body-forming fungi that produce some of the most efficient enzyme systems to degrade wood. Despite decades-long interest in their biology, the evolution and functional diversity of both wood-decay and fruiting body formation are incompletely known. We performed comparative genomic and transcriptomic analyses of wood-decay and fruiting body development in Auriculariopsis ampla and Schizophyllum commune (Schizophyllaceae), species with secondarily simplified morphologies, an enigmatic wood-decay strategy and weak pathogenicity to woody plants. The plant cell wall-degrading enzyme repertoires of Schizophyllaceae are transitional between those of white rot species and less efficient wood-degraders such as brown rot or mycorrhizal fungi. Rich repertoires of suberinase and tannase genes were found in both species, with tannases restricted to Agaricomycetes that preferentially colonize bark-covered wood, suggesting potential complementation of their weaker wood-decaying abilities and adaptations to wood colonization through the bark. Fruiting body transcriptomes revealed a high rate of divergence in developmental gene expression, but also several genes with conserved expression patterns, including novel transcription factors and small-secreted proteins, some of the latter which might represent fruiting body effectors. Taken together, our analyses highlighted novel aspects of wood-decay and fruiting body development in an important family of mushroom-forming fungi.

Dominant suppressor genes of p53-induced apoptosis in Drosophila melanogaster.

One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53-induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2, Rga, crol, and Spt5). In the other 3 (Orct2, Polr2M, and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Selective elimination/RNAi silencing of FMRF-related peptides and their receptors decreases the locomotor activity in Drosophila melanogaster.

Five neuropeptide genes are classified in the FMRF-related (FaRP) group: the Fmrf, dromyosuppressin (Dms), drosulfakinin (Dsk), neuropeptide F (npf) and short neuropeptide F (sNPF) genes coding for 8, 1, 2, 1 and 4 peptides, respectively. In order to compare their effects on the locomotor activity of Drosophila adults, we made RNAi knockdown of the peptides and their specific receptor genes. In addition, we constructed Gal4 drivers with three distinct parts of the Fmrf gene's 5' regulatory sequence (RS8-Gal4, RS11-Gal4, RS17-Gal4), and used them to ablate FMRF-positive neurons inducing apoptosis by expressing the reaper (rpr) gene. We examined the locomotor activity of flies by measuring the mean velocity of movement (MVM) following repeated air-puffs. Locomotor activity was decreased by RNAi knockdown induced in the CNS by the elav-Gal4 driver. According to the MVM curve profiles, RNAi knockdown most effectively decreased the velocity when the DmsR-1 and DmsR-2 genes were silenced together (DmsR-1-RNAi/elav-Gal4; DmsR-2-RNAi/+). Similar effect was observed in Dsk-RNAi/ elav-Gal4; DskR-2-RNAi/+, while moderate effects were found in three other combinations (Fmrf-RNAi/elav-Gal4; FR-RNAi/+, Dms-RNAi/ elav-Gal4;DmsR-2-RNAi/+, CCKLR-17D1-RNAi/elav-Gal4; CCKLR-17D3-RNAi/+), and weak effect in DmsR-2-RNAi/elav-Gal4; DmsR-1-RNAi/+. Male and female flies were not different in this respect. In the cell ablation experiment, the MVM profiles of the female flies were different from the controls when the UAS-rpr transgene was driven by RS8-Gal4 or RS17-Gal4. The RS11-Gal4 and Fmrf-Gal4 drivers were ineffective. In the males only the RS17-Gal4 showed a weak effect. RNAi silencing of the FaRP and FaRP-receptor genes effectively decreased the startle-induced locomotor activity of flies. Ablation of FMRF-positive neurons by the RS8-Gal4 and/or RS17-Gal4 drivers also decreased the flies' activity.

The Hatching Time of Broiler Chickens Modifies Not Only the Production Traits but Also the Early Bacteriota Development of the Ceca.

This trial was carried out to find out the effects of the parent flock and hatching time of broiler chickens on the production traits and bacteriota development of animals. Two sets of 730 hatching eggs were collected from two different parent flocks with ages of 25 and 50 weeks. In the hatchery, both groups were divided into two subgroups: those hatched during the first 10 and the subsequent 10 h of the hatching window. A feeding trial was carried out afterwards, using the four treatments in six replicate floor pens and feeding commercial starter, grower, and finisher diets that contained all the nutrients according to the breeder's recommendations. The day-old chickens of the older parent flock and those hatched later were heavier, and this advantage remained until the end of the production period. The different ages and origins of the parent flocks failed to modify the microbiological parameters of the chicken's ceca; however, the hatching time significantly influenced the different bacteriota diversity indices: the late-hatched chickens showed higher Bacteroidetes and lower Firmicutes and Actinobacteria abundances at day 11. These treatments resulted in differences in the main families, Ruminococcaceae, Lactobacillaceae, and Bacteroidaceae. These differences could not be found at day 39.

Gene age shapes the transcriptional landscape of sexual morphogenesis in mushroom-forming fungi (Agaricomycetes).

Multicellularity has been one of the most important innovations in the history of life. The role of gene regulatory changes in driving transitions to multicellularity is being increasingly recognized; however, factors influencing gene expression patterns are poorly known in many clades. Here, we compared the developmental transcriptomes of complex multicellular fruiting bodies of eight Agaricomycetes and Cryptococcus neoformans, a closely related human pathogen with a simple morphology. In-depth analysis in Pleurotus ostreatus revealed that allele-specific expression, natural antisense transcripts, and developmental gene expression, but not RNA editing or a 'developmental hourglass,' act in concert to shape its transcriptome during fruiting body development. We found that transcriptional patterns of genes strongly depend on their evolutionary ages. Young genes showed more developmental and allele-specific expression variation, possibly because of weaker evolutionary constraint, suggestive of nonadaptive expression variance in fruiting bodies. These results prompted us to define a set of conserved genes specifically regulated only during complex morphogenesis by excluding young genes and accounting for deeply conserved ones shared with species showing simple sexual development. Analysis of the resulting gene set revealed evolutionary and functional associations with complex multicellularity, which allowed us to speculate they are involved in complex multicellular morphogenesis of mushroom fruiting bodies.

Hallmarks of Basidiomycete Soft- and White-Rot in Wood-Decay -Omics Data of Two Armillaria Species.

Wood-decaying Basidiomycetes are among the most efficient degraders of plant cell walls, making them key players in forest ecosystems, global carbon cycle, and in bio-based industries. Recent insights from -omics data revealed a high functional diversity of wood-decay strategies, especially among the traditional white-rot and brown-rot dichotomy. We examined the mechanistic bases of wood-decay in the conifer-specialists Armillaria ostoyae and Armillaria cepistipes using transcriptomic and proteomic approaches. Armillaria spp. (Fungi, Basidiomycota) include devastating pathogens of temperate forests and saprotrophs that decay wood. They have been discussed as white-rot species, though their response to wood deviates from typical white-rotters. While we observed an upregulation of a diverse suite of plant cell wall degrading enzymes, unlike white-rotters, they possess and express an atypical wood-decay repertoire in which pectinases and expansins are enriched, whereas lignin-decaying enzymes (LDEs) are generally downregulated. This combination of wood decay genes resembles the soft-rot of Ascomycota and appears widespread among Basidiomycota that produce a superficial white rot-like decay. These observations are consistent with ancestral soft-rot decay machinery conserved across asco- and Basidiomycota, a gain of efficient lignin-degrading ability in white-rot fungi and repeated, complete, or partial losses of LDE encoding gene repertoires in brown- and secondarily soft-rot fungi.

Megaphylogeny resolves global patterns of mushroom evolution.

Mushroom-forming fungi (Agaricomycetes) have the greatest morphological diversity and complexity of any group of fungi. They have radiated into most niches and fulfil diverse roles in the ecosystem, including wood decomposers, pathogens or mycorrhizal mutualists. Despite the importance of mushroom-forming fungi, large-scale patterns of their evolutionary history are poorly known, in part due to the lack of a comprehensive and dated molecular phylogeny. Here, using multigene and genome-based data, we assemble a 5,284-species phylogenetic tree and infer ages and broad patterns of speciation/extinction and morphological innovation in mushroom-forming fungi. Agaricomycetes started a rapid class-wide radiation in the Jurassic, coinciding with the spread of (sub)tropical coniferous forests and a warming climate. A possible mass extinction, several clade-specific adaptive radiations and morphological diversification of fruiting bodies followed during the Cretaceous and the Paleogene, convergently giving rise to the classic toadstool morphology, with a cap, stalk and gills (pileate-stipitate morphology). This morphology is associated with increased rates of lineage diversification, suggesting it represents a key innovation in the evolution of mushroom-forming fungi. The increase in mushroom diversity started during the Mesozoic-Cenozoic radiation event, an era of humid climate when terrestrial communities dominated by gymnosperms and reptiles were also expanding.

Author Correction: Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria.

In the version of this Article originally published, it was incorrectly stated that "16,687 protein-coding genes were inferred for the most recent common ancestor (MRCA) of Armillaria"; the value was incorrect and it should have read "15,787". This has now been corrected.

Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria.

Armillaria species are both devastating forest pathogens and some of the largest terrestrial organisms on Earth. They forage for hosts and achieve immense colony sizes via rhizomorphs, root-like multicellular structures of clonal dispersal. Here, we sequenced and analysed the genomes of four Armillaria species and performed RNA sequencing and quantitative proteomic analysis on the invasive and reproductive developmental stages of A. ostoyae. Comparison with 22 related fungi revealed a significant genome expansion in Armillaria, affecting several pathogenicity-related genes, lignocellulose-degrading enzymes and lineage-specific genes expressed during rhizomorph development. Rhizomorphs express an evolutionarily young transcriptome that shares features with the transcriptomes of both fruiting bodies and vegetative mycelia. Several genes show concomitant upregulation in rhizomorphs and fruiting bodies and share cis-regulatory signatures in their promoters, providing genetic and regulatory insights into complex multicellularity in fungi. Our results suggest that the evolution of the unique dispersal and pathogenicity mechanisms of Armillaria might have drawn upon ancestral genetic toolkits for wood-decay, morphogenesis and complex multicellularity.