Transcriptomic and Physiological Profiling Reveals Metabolic Determinants and Key Regulatory Hubs of Fruiting Body Degeneration in Lentinula edodes

Abstract

Frequent strain degeneration during subcultivation, characterized by impaired sporulation and fruiting body formation, represents a major constraint in fungal agricultural production. Our study systematically investigated two naturally degenerated Lentinula edodes strains classified as abortive (Abt: L808-13, L808-14) and malformed (Abn: L808-18) fruiting-body phenotypes, through comprehensive phenotypic characterization, enzymatic profiling, thermotolerance assessment, and transcriptomic analysis. While vegetative growth remained unaffected, degenerated strains exhibited premature hyphal knotting, significantly reduced thermotolerance, and Abn-specific suppression of carboxymethyl cellulase (CMCase) activity. Comparative transcriptomics revealed 1239 and 582 differentially expressed genes (DEGs) in Abt and Abn groups, respectively, accompanied by a global dysregulation in carbohydrate catabolism, phospholipid metabolism, and redox homeostasis. Furthermore, protein-protein interaction (PPI) networks and RT-qPCR data highlighted 12 core hub genes enriched in glycoside hydrolysis, cytochrome P450 signaling, and membrane lipid dynamics. These findings provide mechanistic insights into the molecular basis of fruiting body degeneration and establish a foundation for developing diagnostic indicators to screen for early-stage degeneration in industrial mushroom production.

Keywords: Lentinula edodes; fruiting body; metabolic dysregulation; strain degeneration; transcriptomics.

Figure 1

Phenotypic characteristics of Lentinula edodes L808 strains. (A) Normal strain L808-9 and degenerated strains L808-13, L808-14, L808-18 with abnormal fruiting body development. (B) Malformed, undifferentiated fruiting body of degenerated strain L808-18. (C) Hyphal morphology observation of L. edodes L808 strains. (D) Fruiting body yield per bag (left) and daily mycelial growth rate (right). Data are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences (p < 0.05). Scale bars: (A) = 5 cm; (B) = 20 mm; (D) = 10 cm.

Figure 2

Analysis of enzymatic activities and thermotolerance in Lentinula edodes L808. (A) Determination of relative laccase activity. (B) Determination of relative carboxymethyl cellulase (CMCase) activity. (C) Statistical analysis of enzyme activities was quantified based on the diameter of the oxidation zone (laccase) or hydrolysis halo (CMCase). (D) Assessment of mycelial thermotolerance. Data are presented as mean ± SD (n = 3). Different lowercase letters indicate statistically significant differences (p < 0.05); ** p < 0.01. Bars = 10 cm in (A,B).

Figure 3

Transcriptomic analysis of normal and degenerated Lentinula edodes strains. (A) PCA analysis. (B) Volcano plots showing DEGs in Nle _vs_Abt (normal strains L808-6/L808-9 vs. abortive strains L808-13/L808-14) comparison groups. (C) Volcano plots showing DEGs in Nle_vs_Abn (normal strains L808-6/L808-9 vs. malformed strain L808-18) comparison groups.

Figure 4

GO enriched pathway diagram of DEGs in Lentinula edodes L808.

Figure 5

KEGG enriched pathway diagram of DEGs in Lentinula edodes L808. (A) Top 20 enriched KEGG bubble chart and annotation table for the DEGs in the abortive comparison group (Nle_vs_Abt). (B) Top 20 enriched KEGG bubble chart and annotation table for DEGs in the abnormal comparison group (Nle_vs_Abn). The size of the circles indicates the number of enriched genes. The orange line indicates the significance threshold (p-value = 0.05).

Figure 6

Weighted gene co-expression network analysis (WGCNA) of Lentinula edodes L808. (A) Hierarchical cluster tree. Each leaf represents a gene, and each major branch represents a co-expression module, with distinct colors indicating different modules. Genes not assigned to any module are shown in gray. (B) Module-trait correlation heatmap. The color scale on right shows module-trait correlation from −1 (blue) to 1 (red). The top and bottom numbers in each module represent the correlation coefficient and the corresponding p-value, respectively. (C) Scatter plot of module membership (MM) versus gene significance (GS) for the degeneration trait. Correlation coefficients for the turquoise and tan modules are 0.89 and 0.87, respectively.

Figure 7

Identification and functional analysis of core genes associated with fruiting body degeneration. (A) Venn diagrams of DEGs from Nle vs. Abt and Nle vs. Abn; the overlap defines co_diff. (B) Venn diagram showing the intersection between common DEGs (co_diff) and genes from the turquoise and tan WGCNA modules. (C) KEGG pathway enrichment analysis of 111 shared genes identified from intersections in (B). (D) Heatmap of DEGs within the top four enriched KEGG pathways from (C). (E) Protein–protein interaction network for genes from the top four pathways, highlighting functional clusters: carbohydrate metabolism (GHs, α-AMYs), cytochrome P450 (CYP450), phospholipid metabolism (PSD), and aldehyde/acetyl-CoA processing (ALDH, ACAT). PSD, phosphatidylserine decarboxylase; ACAT, acetyl-CoA acetyltransferase; CYP450, cytochrome P450 family; GH, glycoside hydrolase; ALDH, aldehyde dehydrogenase; α-AMY, α-amylase.

Figure 8

Verification of the relative expression levels of DEGs by RT-qPCR. Expression patterns of 6 DEGs were performed. Data are presented as mean ± SD (n = 3). Different lowercase letters indicate statistically significant differences (p < 0.05).