Mitonuclear interactions are crucial in governing mitochondrial function, development and responses to stress in eukaryotic organisms. In this study, we explored how varying mitochondrial haplotypes affect the phenotype and oxidative stress response using hybrids of the basidiomycete Pleurotus ostreatus ( P. ostreatus ) as a model system. By performing reciprocal crosses between monokaryotic strains with distinct nuclear and mitochondrial genomes, we identified notable differences in growth rates, accumulation of reactive oxygen species (ROS) and gene expression patterns. Hybrids with incompatible mitonuclear combinations displayed slower growth and elevated expression of genes — some showing transgressive inheritance — associated with the Electron Transport Chain (ETC) and antioxidant defences. Mitochondria-dependent heterosis was observed in hybrids sharing the same nuclear background, but differing in mitochondrial genome, suggesting that mitonuclear incompatibilities can result in oxidative imbalance and compromised fungal performance. This experimental approach opens wide possibilities for exploring mitonuclear interactions and highlights the significance of mitonuclear co-adaptation in an edible mushroom, offering valuable insights for enhancing hybrid breeding programmes by accounting for the role of mitonuclear interactions in shaping quantitative traits related to mushroom yield.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

Mitochondrial genomes play a central role in fungal physiology and adaptation, yet their evolutionary dynamics during domestication remain poorly understood. Here, we performed a comparative mitogenomic and gene-expression analysis of three Pleurotus ostreatus dikaryotic strains differing in origin and degree of adaptation to laboratory conditions: the long-term commercial strain dkN001, the laboratory-maintained wild isolate dkF515, and the recently collected wild strain dkN009. High-throughput Illumina sequencing enabled complete assembly of circular mitochondrial genomes, revealing substantial size variation among strains, where the dkN001 strain exhibited the second smallest mitogenome reported for the genus Pleurotus. Comparative analyses showed >99% sequence identity between wild isolates and ~95% identity relative to the commercial strain. Variations in genome size among strains were associated with intron dynamics in the cox1 and rnl genes, as well as intron loss predominantly in the commercial strain dkN001, consistent with mitochondrial genome streamlining during domestication. Expression profiling of mitochondrial protein-coding genes (PCGs) under multiple culture conditions revealed conserved transcriptional responses in dkN001 and dkF515 that contrasted sharply with those of dkN009. The differences observed, which affected components of the electron transport chain, suggested shifts in energy metabolism associated with long-term laboratory maintenance. Therefore, our results demonstrate that domestication in P. ostreatus involves both structural remodelling of the mitogenome and changes in regulation of mitochondrial PCGs, highlighting the importance of mitonuclear interactions in fungal adaptation to controlled environments.

Wood-decaying fungi are key to forest ecosystems, breaking down lignocellulose—the cellulose, hemicellulose, and lignin matrix of plant cell walls. This process recycles nutrients, supports plant growth, and has industrial applications in biofuels and bioproducts. Agaricomycetes use two main decay strategies for lignocellulose degradation. White rot fungi (WRF) degrade lignin and carbohydrates, achieving complete lignocellulose breakdown, while brown rot fungi (BRF) mainly attack carbohydrates and leave lignin intact. This study compared three WRF (Pleurotus ostreatus, Phanerochaete chrysosporium, Heterobasidion irregulare) and two BRF (Fomitopsis schrenkii, Rhodonia placenta) through transcriptomics, enzyme family analysis, and metabolic profiling under glucose and wood growth conditions. WRF showed strong induction of lignin-degrading enzymes such as peroxidases and laccases, while BRF lacked these genes and relied on oxidative chemistry with limited cellulases. GH16 glucanases were the only enzyme family consistently upregulated across all fungi, highlighting their conserved but versatile role in carbohydrate metabolism. Enzyme repertoires were highly species-specific, with transcriptomes dominated by a few strongly expressed genes. In P. ostreatus, AA9 lytic polysaccharide monooxygenases (LPMOs) formed a large, diverse family regulated by promoter motifs and influenced by nearby transposable elements, suggesting dynamic evolution and fine-tuned control. Metabolic analysis showed that WRF used robust oxidative pathways (glycolysis, PPP, TCA cycle, glyoxylate shunt), supporting efficient carbon utilization. BRF favored partial sugar oxidation and fermentation, reflecting their carbohydrate-focused decay mode. These metabolic differences align with ecological strategies: WRF dismantle lignin-rich substrates, while BRF optimize rapid carbohydrate extraction. In summary, this work clarifies the molecular, enzymatic, and metabolic bases of fungal wood decay. It reveals contrasting strategies between WRF and BRF, the conserved central role of GH16 enzymes, the diversity and regulation of AA9 LPMOs, and distinct carbon metabolism adaptations. These insights advance understanding of fungal ecology and provide valuable knowledge for biotechnology and biomass conversion.