2.1 Research materials

Normal and abnormal browning bags were used to isolate mycelial material Le-BP and Le-WP under laboratory conditions, respectively (Figure 1). The strain name of both kinds of bags is Qi he No. 7, coming from Qi he Edible Mushroom Co., Ltd. normal browning bag (Le-BB) and abnormal browning bags (Le-WB) were inoculated on the same date and cultured in the same laboratory in Qi he Edible Mushroom Co., Ltd, and the abnormal browning phenomenon of Le-WB began to be observed after 45 days. Le-BP and Le-WP were cultured on the maltose, yeast, and glucose (MYG) medium and preserved in the Institute of Applied Mycology, Huazhong Agricultural University.

2.2 Culture test

The mycelial disk was grown on MYG solid medium at 25°C in dark conditions. The culture characteristics were observed after incubation for 10, 20, 30, and 40 days, respectively. The mycelial growth rate was also measured. The experiment was performed using five biological replicates.

2.3 Observation of mycelia by scanning electron microscope and transmission electron microscope

The mycelial disk was inoculated on the center of sterile cellophane and covered on a sterile Petri dish containing solid MYG. When the mycelium grows to 1/2 of the Petri dish (9 cm in diameter), the cellophane containing the outermost edge of the colony was cut into small pieces of 2 mm² using sharp scissors, and then fixed with 2.5% (v/v) glutaraldehyde solution at 4°C for 48 h. Air was pumped for 30 min to completely submerge the pieces in the liquid. Subsequently, the mycelia were washed three times with 0.05 mol/L phosphate buffer (pH 6.8) and later dehydrated in a graded ethanol series (30%, 50%, 70%, 90%, and 100%, v/v) each for 20 min. Samples were then dried in liquid CO₂ at the critical point and sputter coated with palladium-gold, finally examined under scanning electron microscopy (SEM) (JSM-6390LV, JEOL). For the transmission electron microscopy (TEM) study, the mycelia were obtained similar to the SEM method. The samples were prefixed in a 2.5% glutaraldehyde solution adjusted to pH 7.4 with 0.1 M phosphate buffer. Air was pumped for 30 min to completely submerge the pieces in the liquid. The samples were placed in a refrigerator to fix overnight at 4°C. Subsequently, the mycelia were washed three times with 0.1 mol/L phosphate buffer solution for 30 min and then fixed in 1% (w/v) osmic acid (SPI, SPI Chem) solution for 2 h. Later, washed again three times with PBS for 30 min. Next, the mycelia pieces were dehydrated with gradient acetone (Sinopharm), then infiltrated with gradient Spurr resin (SPI, SPI Chem) acetone solution and polymerized and embedded with pure Spurr resin at 60°C for 48 h. The embedded mycelia were cut into 60–70 nm ultra-thin sections with an ultramicrotome (UC6, Leica). Then, the sections were fished on a copper net and 2% (w/v) uranyl acetate (SPI, SPI Chem) aqueous solution. The stain was used for 30 min under a voltage of 80 KV. A transmission electron microscope (H-7650, Hitachi) was used to observe the stained ultrathin sections. Later, the observation results were collected with a CCD camera (Model832 ORIUS, Gatan). At least three to nine independent mycelia plugs (5 mm) for each material were used. After that, at least three ultrathin sections for each mycelia plug (5 mm) were used for TEM observation.

2.4 Sequencing samples and transcriptome sequencing

Le-WP and Le-BP hyphae were respectively inoculated on the MYG solid medium covered with cellophane, and cultured at 25°C for 10 days. The sequenced mycelial samples of Le-BB and Le-WB were collected on the 60th day after inoculation. Then, the mycelia of the two strains were collected and stored at −80°C for further transcriptomic sequencing. Samples from three different Petri dishes or cultivated bags were defined as three biological repeats for each group. Isolation of total RNA was performed from each mycelium sample using the TRIzol method as per the manufacturer's instructions. The integrity of the RNA samples was determined through 1% denaturing agarose gel electrophoresis. The quality and concentration of the RNA samples were analyzed with a NanoDrop spectrophotometer (Denovix, DS-11). Library construction and sequencing were performed by Wuhan GooAl Gene Technology Co., Ltd. The complementary DNA (cDNA) library was established using NEBNext® UltraTM II RNA Library Prep Kit (New England Biolabs Inc.). Illumina X-Ten sequencing platform was used to sequence the L. edodes samples. No less than 6 GB of clean data were obtained for each sample.

2.5 Transcriptome analysis

FastQC v0.11.8 was used to examine the read data's quality. With Trimmomatic v0.39, reads were trimmed by excluding nucleotides with a quality score of less than 30 and keeping only reads longer than 50 nucleotides [9]. Clean reads were mapped to the L. edodes reference genome (https://mycocosm.jgi.doe.gov/LedB17_3/LedB17_3.home.html) using Hisat2 [10], duplicate reads were eliminated using Picard (http://broadinstitute.github.io/picard/), and the mapping rate was estimated using Qualimap [11]. By using HTseq [12], the mapped clean reads count was assessed, and TBtools translated the results to Transcripts Per Million (TPM) data [13]. EdgeR [14] was utilized to find differentially expressed genes (DEGs) with |log2 (fold change)| greater than 1 and a false discovery rate (FDR) below 0.01. Blast2GO annotated DEGs [15]. By using BLAST, all of the genes were mapped to the seqdb database [16], and gene2go was used to determine each gene's GO ID [17]. Gene ontology (GO) enrichment analysis was carried out with Fisher's test and FDR 0.01 correction. The soft of KEGG Orthology-Based Anotation System carried out Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis [18]. The ggplot2 was used to present GO and KEGG data (http://had.co.nz/ggplot2/).

2.6 Gene expression validation by quantitative real-time PCR (qRT-PCR)

RNA-Seq results were verified by selecting 12 genes on the basis of significant “q” values from the Kyoto encyclopedia of genes and genomes (KEGG) pathways and analyzed by qRT-PCR. The 12 genes included gene_2896, gene_8542, gene_20562, gene_41402, gene_31666, gene_32475, gene_17327, gene_5406, gene_12275, gene_29922, gene_22010, and gene_10464 (Supporting Information: Table S1). The primer pairs were designed using primer premium 5 (Premier Biosoft International) (Supporting Information: Table S1). LedActin was used as a reference gene for qRT-PCR validation. Reverse-transcription of RNA was performed using HiScript RT Supermix for qPCR (+gDNA wiper) (Vazyme Biotech) SYBR green fluorescent dye (Vazyme Biotech) was used to perform the qPCR reactions on a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories). The relative expression values of each gene were calculated using the $<math altimg="urn:x-wiley:0233111X:media:jobm202200405:jobm202200405-math-0001" wiley:location="equation/jobm202200405-math-0001.png" xmlns="http://www.w3.org/1998/Math/MathML"><mrow><mrow><msup><mn>2</mn><mrow><mo>\unicode{x02212}</mo><mi>\unicode{x00394}\unicode{x00394}</mi><msub><mi>C</mi><mi mathvariant="normal">t</mi></msub></mrow></msup></mrow></mrow></math>$ method. The experiment was performed using nine biological replicates [19].

2.7 Aniline blue assay of 1,3-beta-glucan

The enzymatic activity of 1,3-beta-glucan was calculated by performing an aniline blue assay, according to a previous study [20]. The fluorescence readings were recorded by using a fluorimeter (Tecan Infinite M200), with 460 nm emission and 405 nm excitation. The unit used for the expression of values is (mg/g) of the mycelial tissue, by using Le-BB, Le-WB, and Le-BP, Le-WP. The experiment was performed in triplicate.

2.8 Statistical analysis

The statistical analysis was performed by using Statistix 8.1 (Analytical Software Inc.). The standard error and mean values were calculated using Excel (Microsoft Corp.). Multiple comparisons with least significant difference test at significant difference p < 0.05 were used to determine significant differences among the different treatments, and one-way analysis of variance (ANOVA) was performed to find the expression values of genes by qRT-PCR. Origin Pro 8.5.1 (Origin Lab Corporation) was used for making the graphs.

3.1 Comparison of culture characteristics between the colony phenotypes of Le-BP and Le-WP

The colony phenotypes of Le-BP (normal) and Le-WP (abnormal) had different changes visible to the naked eye during the growth process (Figure 2). The Le-BP hyphae germinated the next day after inoculation, mycelia appeared grey-floss-shaped. The Le-WP mycelium appeared close-packed and radiating outward, and the colonies were whiter than the ones of Le-BP. After 20 days of culture, some “mycelium aggregates” were formed on the surface of Le-BP colonies. As the number of cultivation days increased, the “mycelium aggregates” in Le-BP colonies grew together and formed thicker mycoderma, and pigment deposition increased. However, the Le-WP colony had no obvious changes, except for the colonies became denser with the increase of culture time (Figure 2). The growth rates of Le-BP showed an upward growth trend, while the Le-WP strain showed a downward growth trend. The growth rate of abnormal mycelium Le-WP was slower than that of normal mycelium Le-BP (Figure 3).

3.2 Comparison of ultrastructure characteristics between the colony phenotypes of Le-BP and Le-WP

The differences in microscopic features between the normal Le-BP and the abnormal Le-WP mycelia are revealed more clearly observed by scanning electron microscopy (SEM) (Figure 4). The surface of the Le-BP colony is flat and has no “spherical mass formations” (Figure 4a), and the hyphae are regular (Figure 4b). However, multiple “spherical mass formations” can be observed on the surface of the Le-WP colony (Figure 4c), and hyphae adhesions are commonly observed in Le-WP (Figure 4d), and some hyphae of Le-WP are twisted together (Figure 4d), and become swollen (Figure 4d,e). Some of the adhesive hyphae become wrinkled and obvious uneven thickness, and some hyphae appeared completely shrunk (Figure 4e,f). Observation under TEM revealed, that Le-WP hyphae cells had some signs of aging or degeneration characteristics, such as separation of cytoplasm, increment of lipid droplets, cell shrinkage and vacuolization, cytoplasmic vacuolar and thicker and loose cell wall structures in some cells (Figure 5).

3.3 RNA sequencing data and screening of differentially expressed genes

In Le-BB, Le-WB, Le-BP, and Le-WP, total raw reads, clean reads, total clean bases, clean read ratio, total mapping, and unique mapping average values are shown in Table 1. Data were divided into three replicates. To reflect the correlation of gene expression between samples, Pearson correlation coefficients for all gene expressions between every two samples were calculated. In the case of correlation between samples, we compared Le-BB and Le-WB (Supporting Information: Figure S1). In Le-BB versus Le-WB total number of DEGs found was 262, among them 179 were upregulated, and 83 were downregulated. In Le-BP versus Le-WP, 908 DEGs were identified, 520 were upregulated and 388 were downregulated (Figure 6a). There were 82 overlapping genes between these two comparisons (Figure 6b). In addition, the volcano map is showing the degree of significance and up and downregulation of the DEGs between Le-BB versus Le-WB and Le-BP versus Le-WP. It can be seen clearly that the significant degree of DEGs in the Le-BP versus Le-WP is much higher than those in the Le-BB versus Le-WB (Figure 6c,d).

3.4 Go function and enrichment analysis revealed catalytic activity and metabolic process were enriched in Le-WP

In Le-BP versus Le-WP, there was a total of 11 different biological processes, in which various DEGs were expressed. Among these DEGs, 250 DEGs were expressed in the metabolic process and 120 DEGs were expressed in the single-organism process, in the cellular component, the most DEGs were involved in membrane (50 DEGs) and membrane part function (40 DEGs). In molecular function, DEGs expression was found in nine functions, having the maximum number (280 DEGs) in catalytic activity followed by the binding activity with 150 DEGs.

In Le-BB versus Le-WB, there were nine different biological processes, in which various DEGs were expressed. Among these DEGs, 70 DEGs were involved in the metabolic process and 30 DEGs were recorded in the single-organism process. There was a total of six different cellular component functions in Le-BB versus Le-BP, containing 15 DEGs involved in membrane activities. In the case of molecular function (Le-BB vs. Le-WB), there was a total of eight functions in which DEGs were expressed. Among these functions, catalytic activity contains 78 DEGs and the binding function contains 38 DEGs. Most of the genes involved in the metabolic process and single organism process through the molecular function of catalytic and binding activities were more expressed in Le-BP versus Le-WP and were significant (Figure 7), suggesting that these parameters are of great significance.

GO enrichment terms were selected on the basis of Q value less than 0.005. In both Le-BP versus Le-WP and Le-BB versus Le-WB, the highly enriched and significant terms included catalytic activity and metabolic process, with 229 and 217 DEGs, and with 77 and 73 DEGs, respectively (Supporting Information: Figure S2).

3.5 KEGG enrichment analysis revealed pentose and glucuronate interconversions and starch and sucrose metabolism were enriched in Le-WP

KEGG enrichment analysis revealed that starch and sucrose metabolism were enriched in Le-BP versus Le-WP and Le-BB versus Le-WB treatment, while the pentose and glucoronate interconversions pathway was only enriched in Le-BP versus Le-WP (Supporting Information: Figure S3). The genes having log2 values of more than twofold change were selected.

Pectinesterase and galacturonan 1,4-alpha-galacturonidase were involved in the synthesis of uridine diphosphate (UDP)-glucose in the pentose and glucuronate interconversions pathway. In the pentose and glucuronate interconversions pathway, two genes were identified encoding for pectinesterase (gene_2896, EC 3.1.1.11) and galacturonan 1,4-alpha-galacturonidase (gene_20562, EC 3.2.1.67), and only the latter gene was upregulated in Le-WP.

UDP-Glucose acted as a substrate to initiate the starch and sucrose metabolism pathway from three ends. Three ends included 1,3-beta-glucan, beta-d-glucoside, and cellulose, respectively. In starch and sucrose metabolism pathway, 10 genes were identified encoding for 1,3-beta-glucan synthase component (gene_31666), cellulase (gene_17327 and gene_32475, EC 3.2.1.4), beta-glucosidase (gene_5406, gene_12275, gene-21409, gene-46088 and gene_29922, EC 3.2.1.21), and cellulose 1,4-betacellobiosidase (gene_22010 and gene_10464, EC 3.2.1.91), respectively. 1,3-beta-glucan was synthesized by 1,3-beta-glucan synthase component (gene-31666), was upregulated in Le-WP, and was responsible for d-glucose synthesis. Cellulose was synthesized by cellulase (gene_17327 and gene_32475) and was upregulated in Le-WP. Cellulose was leading to two ends. At one end, it acted as substrate of cellodextin and was responsible for d-glucose synthesis, cellulase (gene_32475), beta-glucosidase (gene_5406, gene_12275 and gene_29922), and cellulose 1,4-betacellobiosidase (gene_22010 and gene_10464) were upregulated in Le-WP. At the second end, cellulose was directly responsible for d-glucose synthesis, at this end, beta-glucosidase (gene-21409) and cellulose 1,4-betacellobiosidase (gene_22010 and gene_10464) were upregulated in Le-WP (Figure 8). qRT-PCR was conducted to verify the above results, and the results showed that it was in compliance with the transcriptome data analysis (Figure 9).

3.6 Aniline blue assay of 1,3-beta-glucan

The enzyme analysis unveiled that the enzymatic activity of the 1,3-beta-glucan component was 28.30 mg/g in Le-WP comparison to Le-BB, Le-WB, and Le-BP, having an enzymatic activity of 16.5, 11.3, and 16 mg/g, respectively (Figure 10). These results were similar to those observed in transcriptome data analysis.