Lentinula edodes is a tetrapolar basidiomycete with two haploid nuclei in each cell during most of their life cycle. Understanding the two haploid nuclei genome structures and their interactions on growth and fruiting body development has significant practical implications, especially for commercial cultivars. In this study, we isolated and assembled the two haploid genomes from a commercial strain of L. edodes using Illumina, HiFi, and Hi-C technologies. The total genome lengths were 50.93 Mb and 49.80 Mb for the two monokaryons SP3 and SP30, respectively, with each assembled into 10 chromosomes with 99.63% and 98.91% anchoring rates, respectively, for contigs more than 100 Kb. Genome comparisons suggest that two haploid nuclei likely derived from distinct genetic ancestries, with ~30% of their genomes being unique or non-syntenic. Consistent with a tetrapolar mating system, the two mating-type loci A (matA) and B (matB) of L. edodes were found located on two different chromosomes. However, we identified a new but incomplete homeodomain (HD) sublocus at ~2.8 Mb from matA in both monokaryons. Our study provides a solid foundation for investigating the relationships among cultivars and between cultivars and wild strains and for studying how two genetically divergent nuclei coordinate to regulate fruiting body formation in L. edodes.

In basidiomycete fungi, the number of nuclei and their ploidy level per nucleus can vary tremendously among species; however, within species, nuclear number and ploidy levels are traditionally considered fixed in their vegetative hyphae. In the edible mushroom Lentinula edodes, the hyphae are classified as either monokaryotic or dikaryotic, with each monokaryotic hyphal cell containing one haploid nucleus, and each dikaryotic hyphal cell containing two haploid nuclei. The dikaryotic hyphae are the results of mating between two genetically distinct monokaryons with different mating types. In this study, we examined the nuclear number and size (a potential correlate to ploidy) of L. edodes mycelia throughout its vegetative growth. We found that the number of nuclei within individual hyphal cells varied widely from non-nucleated to uninucleated, dinucleated, and multinucleated. Additionally, different nuclei within the same cell appeared very different in size, with a maximum nucleus cross-sectional area of 4.94 μm² and the minimum nucleus cross-sectional area at only 0.37 μm². Moreover, as culture time increased, more cells appeared to be devoid of any nuclei, with transmission electron microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays of late-stage cultures showing autophagosomes fusing and dissolving the nuclei and resulting in a large number of TUNEL-positive DNA fragments in non-nucleated cells. These results indicated that non-nucleated cells were likely caused by autophagy and apoptosis-like activities within aging L. edodes hyphae.

Temperature type is one of the key traits determining the cultivation regime of Lentinula edodes. However, the molecular and metabolic basis underling temperature type remain unclear. Here, we investigated the phenotypic, transcriptomic, and metabolic features of L. edodes with different temperature types under both control (25 °C) and high (37 °C) temperature conditions. We found that under the control condition, the high- and low-temperature types of L. edodes harbored distinct transcriptional and metabolic profiles. The high-temperature (H-)-type strain had a higher expression level of genes involved in the toxin processes and carbohydrate binding, while the low-temperature (L-)-type strain had a high expression level of oxidoreductase activity. Heat stress significantly inhibited the growth of both H- and L-type strains, while the latter had a higher growth inhibition rate. Upon exposure to heat, the H-type strain significantly up-regulated genes associated with the components of the cellular membrane, whereas the L-type strain markedly up-regulated genes involved in the extracellular region and carbohydrate binding. Metabolome data showed that thermostress altered purine and pyrimidine metabolism in the H-type strain, whereas it altered cysteine, methionine, and glycerophospholipid metabolism in the L-type strain. Transcriptome and metabolome integrative analysis was able to identify three independent thermotolerance-related gene–metabolite regulatory networks. Our results deepen the current understanding of the molecular and metabolic basis underlying temperature type and suggest, for the first time, that thermotolerance mechanisms can be temperature-type-dependent for L. edodes.

The spawn of Lentinula edodes and other basidiomycete fungi tend to age with long-term culture. This causes heavy yield losses if aging spawn is used for propagation. In this study, we cultivated dikaryotic L. edodes mycelia in plates for 60 days to produce intrinsic aging phenotypes. We found that intracellular reactive oxygen species levels increased in contrast to mitochondrial depolarization and also observed greater DNA fragmentation with longer culture time. Transcriptome analysis of mycelia at different growth stages revealed pronounced expression differences between short- and long-term cultures. In particular, “phenylalanine, tyrosine, and tryptophan biosynthesis”, “mitophagy and autophagy”, “MAPK signaling pathway”, and “ABC transporter” were among the enriched terms in the mycelial aging process. Weighted correlation network analysis identified LeAtg8, LeHog1, LePbs2, and LemTOR as key genes during aging. Western blotting confirmed that LeATG8 and phosphorylated LeHOG1 protein levels were significantly upregulated in aging mycelia. Our combined analytical approach provides insights into the mechanisms that regulate mycelial aging, indicating that autophagy/mitophagy plays a major role in counteracting the effects of age on mycelial growth development.