HOG1 Mitogen-Activated Protein Kinase Pathway–Related Autophagy Induced by H2O2 in Lentinula edodes Mycelia

Mycelial ageing is associated with ROS and autophagy in Lentinula edodes. However, the underlying cellular and molecular mechanisms between ROS and autophagy remain obscure. This study induced autophagy in L. edodes mycelia through exogenous H2O2 treatment. Results showed that 100 μM H2O2 treatment for 24 h significantly inhibited mycelial growth. H2O2 caused the depolarisation of MMP and accumulation of TUNEL-positive nuclei, which was similar to the ageing phenotype of L. edodes mycelia. Transcriptome analysis showed that differentially expressed genes were enriched in the mitophagic, autophagic, and MAPK pathways. LeAtg8 and LeHog1 were selected as hub genes. RNA and protein levels of LeATG8 increased in the H2O2-treated mycelia. Using fluorescent labelling, we observed for the first time the classic ring structure of autophagosomes in a mushroom, while 3D imaging suggested that these autophagosomes surrounded the nuclei to degrade them at specific growth stages. Phospho-LeHOG1 protein can translocate from the cytoplasm to the nucleus to regulate mycelial cells, resisting ROS-induced oxidative stress. Furthermore, LeATG8 expression was suppressed when LeHOG1 phosphorylation was inhibited. These results suggest that the LeATG8-dependent autophagy in L. edodes mycelial is closely associated with the activity or even phosphorylation of LeHOG1.


Introduction
Autophagy is an evolutionarily conserved process in eukaryotes [1]. Due to its significance in maintaining intracellular physiological balance and stress resistance, autophagy has become a research hotspot. In 1997, the first autophagy-related gene Atg1 was discovered in yeast, and since then, 42 Atg genes have been reported in eukaryotes to be responsible for the formation and regulation of autophagosomes [2]. The ubiquitin-like protein encoded by the Atg8 gene (the homologous protein in mammalian cells is LC3) is a key component of autophagosomes [3]. ATG8 can bind to phosphatidylethanolamine (PE) under the action of ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3, thereby anchoring on the autophagosome membrane and outer membrane to help facilitate the extension and expansion of autophagosomes [1]. Therefore, ATG8 protein is widely used in labelling autophagosomes and evaluating the degree of autophagy [4]. At the late stage of autophagy, autophagosomes fuse with vacuoles to form autophagic lysosomes. The outer-membrane-bound ATG8 is released into the cytoplasm for repeated use, while the inner-membrane-bound ATG8 and its inclusions are degraded by vacuoles.
ATG8 protein plays a key role in the growth, development, cell differentiation, and secondary metabolism of fungi [1]. In Magnaporthe oryzae, ATG8-mediated autophagy is each treatment were assayed to evaluate treatment effects and mitochondrial function. The fluorochrome JC-1 (Beyotime Biotechnology, Shanghai, China) was used to estimate MMP. L. edodes mycelia were treated with 10 µM JC-1 in the dark for 30 min, washed twice with JC-1 buffer, and observed with green and red fluorescence channels under an IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan). DAPI (Sigma Aldrich, St. Louis, MO, USA) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) fluorescence staining (Beyotime Biotechnology, Shanghai, China) were performed to detect total nuclei and DNA fragments [18]. The results of nuclear labelling were observed under the blue fluorescence and green fluorescence channel using a fluorescence microscope, for DAPI and TUNEL, respectively. Each experiment was performed in triplicate.
( Figure 1A). To optimise H2O2 concentration, apical mycelia cultured as described above were treated with 1,2,3,4,5,8,30,50,100, and 200 µM H2O2. After culturing for 24 and 48 h, the growth rate, mitochondrial membrane potential (MMP), and nuclei phenotype of the mycelia under each treatment were assayed to evaluate treatment effects and mitochondrial function. The fluorochrome JC-1 (Beyotime Biotechnology, Shanghai, China) was used to estimate MMP. L. edodes mycelia were treated with 10 µM JC-1 in the dark for 30 min, washed twice with JC-1 buffer, and observed with green and red fluorescence channels under an IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan). DAPI (Sigma Aldrich, St. Louis, MO, USA) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) fluorescence staining (Beyotime Biotechnology, Shanghai, China) were performed to detect total nuclei and DNA fragments [18]. The results of nuclear labelling were observed under the blue fluorescence and green fluorescence channel using a fluorescence microscope, for DAPI and TUNEL, respectively. Each experiment was performed in triplicate.

RNA-Seq and Transcriptome Analysis
Five-day-old cultures of L. edodes mycelia were inoculated with 100 µM H2O2 in PDAcellophane at 22 °C and sampled at 0, 3, and 24 h intervals. Apical cells were collected, flash frozen using liquid nitrogen, and stored at −80 °C for transcriptome analysis.
The RNA of three mycelia samples (three independent replicates per sample) were extracted using TRIZOL ® Reagent (Invitrogen, Waltham, MA, USA). The quality and integrity of the RNA were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 bioanalyser (Agilent Technologies, Santa Clara, CA, USA), respectively. The library construction and RNA-seq were

RNA-Seq and Transcriptome Analysis
Five-day-old cultures of L. edodes mycelia were inoculated with 100 µM H 2 O 2 in PDAcellophane at 22 • C and sampled at 0, 3, and 24 h intervals. Apical cells were collected, flash frozen using liquid nitrogen, and stored at −80 • C for transcriptome analysis.
The RNA of three mycelia samples (three independent replicates per sample) were extracted using TRIZOL ® Reagent (Invitrogen, Waltham, MA, USA). The quality and integrity of the RNA were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 bioanalyser (Agilent Technologies, Santa Clara, CA, USA), respectively. The library construction and RNA-seq were conducted by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). cDNA libraries were constructed using NEBNext ® Ultra™ RNA Library Prep Kits for Illumina ® (NEB, Ipswich, MA, USA). After the library was qualified, different libraries were pooled according to the effective concentration and the machine data and then sequenced using an Illumina platform with 150 bp paired-end reads.

Antioxidant Activity and ROS Content of Induced and Inhibited Mycelia
A p38-MAPK inhibitor of SB203580 (10 µM) was used to pretreat the cellophane and identify the function of LeHOG1 in mycelial ageing. The total protein of L. edodes mycelia was extracted using a protein extraction kit (Bestbio, China) for microfungi. Total protein concentration was detected using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Superoxide dismutase (SOD) activity was measured using its corresponding kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Fresh L. edodes mycelia under different treatments were washed with PBS buffer three times and incubated with 10 µM 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, Beyotime Biotechnology, Shanghai, China) for 30 min at 25 • C in the dark. Images were obtained under a fluorescence microscope and analysed using Image J as previously described [22].
The loading amount of each sample was normalised according to the protein concentration of the sample. The loading protein was separated using 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane at 120 mA for 1 h. After blocking with 10% milk, the membrane was incubated with the anti-LeATG8 primary antibody and goat anti-rabbit IgG H&L secondary antibody (Abcam, Cambridge, MA, USA) in 1:1000 dilutions overnight at 4 • C. The immunoreactive bands were detected using the Chemidoc imaging system (Bio-Rad, Hercules, CA, USA).

Freezing Microtome Section and Immunofluorescence
L. edodes mycelia were washed twice with PBS and then fixed with glutaraldehyde (2.5% in PBS) for 20 min at 4 • C. After washing with PBS, the samples were immersed in protectants (5% trehalose and 10% glycerol) under vacuum and then incubated for 1 h. The embedded samples were cut into serial coronal sections (6 µm thick) using a freezing microtome (Leica CM1990, Nussloch, Germany). The sections were postfixed with paraformaldehyde (4% in PBS) for 30 min at 4 • C and then washed three times with PBS containing 0.1% Triton X-100 (PBST). Subsequently, the sections were incubated in immunol staining blocking buffer (Beyotime Biotechnology, Shanghai, China) for 1 h. After the sections were incubated at 4 • C overnight with anti-LeATG8 antibody, anti-P38 antibody, or anti-phospho-P38 antibody (1:1000 dilution), they were washed three times with PBST. The sections were incubated with goat anti-rabbit Alexa Fluor 488 dye (1:500 in PBST) for 2 h, counterstained with 8 µg/mL 4 ,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO, USA), and then mounted using Mowiol solution (10% Mowiol 4-88 and 20% glycerol in PBS). The sections were observed with a confocal microscope (LSM 900 with Airyscan2, Zeiss, Germany). Three repeated immunofluorescence stainings for each treated sample were performed. Each section observed 20 fields, and for each field, 8-16 images were taken for splicing. In order to observe all levels of mycelium, 40 images must be taken continuously by Z-stack. Z-stack images were taken in Airyscan mode in multiple fluorescence channel images with brightfield taken in confocal mode. We used the ZEN blue software surface mode of 3D VisArt for 3D imagination.

Statistical Analysis and Data Availability
The SOD activity and DCFH-DA-labelled ROS contents obtained in this study are presented as means ± standard deviation (SD). The numbers of biological replicates in each experiment are noted in the figure legends. Statistical analysis was performed using ANOVA. The significant differences between means were determined by Duncan's multiplerange test. Unless otherwise stated, the differences were considered statistically significant at p < 0.01.
All data generated or analysed during this study are included in this article and its supplementary information files. The raw sequencing data of RNA-seq are available at the National Genomics Data Center, China National Center for Bioinformation, under BioProject ID PRJCA012101 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA012101, accessed on 17 March 2023).

H 2 O 2 Treatment Altered Mycelial Morphology and Microstructure
In the long-term cultured ageing mycelium of L. edodes, with the increase of autophagy protein LeATG8 expression, PCD phenomena, such as weakened mycelium growth, mycelium agglutination, decreased MMP, and increased TUNEL-positive nuclei, were observed (Gao et al., 2023). The above phenotypic changes were used as screening markers to determine the optimal time and concentration of H 2 O 2 treatment when establishing an induced autophagy model of L. edodes.
First, we used a high concentration of H 2 O 2 (200 µM) to treat the mycelium and cultured it for 20 days ( Figure 1A MMP was determined using JC-1 staining to measure the mitochondrial depolarisation induced by H 2 O 2 ( Figure S1). H 2 O 2 increased the oxidative stress and induced ageing by initiating mitochondrial dysfunctions. JC-1 exhibits red fluorescence in polarised healthy mitochondria and green fluorescence in depolarised mitochondria. In the control group, JC-1 showed intense red fluorescence and weak green fluorescence. The fluorescence of JC-1 changed from red to green after treatment of the mycelia with a high (100 µM) or low (8 µM) concentration of H 2 O 2 . This result suggests mitochondrial damage and depolarisation caused by H 2 O 2 . Therefore, we selected 100 µM H 2 O 2 for subsequent experiments. We observed the mycelial cell structure after treatment with 100 µM H 2 O 2 . After 3 h of treatment, MMP fluorescence showed green fluorescence indicating that the mitochondrial membrane potential had decreased ( Figure S2A,B). However, the results of TUNEL and DAPI combined labelling showed that the hyphae after 3 h of treatment were mainly DAPI-labelled blue nuclei ( Figure S2C). After 24 h of treatment, a large number of TUNEL-positive nuclei appeared in the mycelial cells, indicating that the DNA had been fragmented ( Figure S2D).

Transcriptome Changes after H 2 O 2 Treatment
The global DEGs in L. edodes mycelia after 0, 3, and 24 h of 100 µM H 2 O 2 treatment were determined via high-throughput sequencing. A total of 384 Mb clean reads with Q20 values greater than 98% were obtained, and the average total reads mapped were 89.26% (Tables S1 and S2). The lowest Pearson correlation coefficient between biological replicates was 0.9856, indicating the reliability and validity of the RNA-seq data ( Figure S3). Three DEG categories of LeHH3h vs. LeHH0h, LeHH24h vs. LeHH0h, and LeHH24h vs. LeHH3 were compared to understand the mechanism by which H 2 O 2 affected the mycelia. In total, 2398 DEGs were identified in the three categories, accounting for 23.0% of all transcripts. Compared with LeHH0h, LeHH3h had 351 upregulated genes and 194 downregulated genes (Table S3). LeHH24h had 1032 DEGs (619 upregulated and 413 downregulated) compared with LeHH0h and 821 DEGs (456 upregulated and 365 downregulated) compared with LeHH3h (Tables S4 and S5).
The Venn diagram showed that 83 genes were common among the three categories ( Figure S4 and Table S6). The numbers of DEGs uniquely expressed in LeHH3h vs. LeHH0h, LeHH24h vs. LeHH0h, and LeHH24h vs. LeHH3h were 154, 271, and 182, respectively. Cluster analysis was performed to divide the DEGs into three main clusters and six subclusters according to the duration of H 2 O 2 treatment ( Figure S5). The genes in cluster 1 were downregulated after H 2 O 2 treatment, whereas the main genes in cluster 3 were upregulated after H 2 O 2 treatment. In addition, the genes in cluster 2 were first downregulated after 3 h and then upregulated after 24 h of treatment. Moreover, the sample of LeHH3h clustered with that of LeHH0h, highlighting the similarity between the two samples.
The DEGs in LeHH24h vs. LeHH0h were enriched to analyse their functional features. The top 20 enriched GO terms included cell wall, nucleosome, ribosome, chromatin, mitochondrial inner membrane, and mitochondrial envelope, which belonged to 16 cellular component categories ( Figure S6 and Table S7). Besides cellular components, the GO terms were also enriched in oxidation-reduction, structural constituent of cell wall, and structural molecule activity, which belonged to biological process and molecular function. Furthermore, the DEGs were mapped to the KEGG pathway to identify the active metabolism pathways involved in H 2 O 2 response. The top 20 enriched KEGG terms included ribosome, glycolysis/gluconeogenesis, tyrosine metabolism, glyoxylate and dicarboxylate, oxidative phosphorylation, glycerolipid metabolism, fatty acid degradation, and mitophagy ( Figure S7 and Table S8).

LeHOG1 Phosphorylation Participated in LeATG8-Dependent Autophagy Induced by H2O2
H2O2 induces oxidative stress by exerting cytotoxic effects and increasing intracellular superoxide (O2·ˉ) levels [31]. In the present study, treatment with H2O2 for 24 h significantly increased the antioxidant activity of superoxide dismutase (SOD) activity ( Figure  3A). Moreover, DCFH-DA dyeing results showed that the treatment increased the ROS The KEGG pathway enrichment network of autophagic, mitophagic, and MAPK signal pathways was visualised to understand the relationship among these pathways and identify interactive hub genes ( Figure 2B). The MAPK signal pathway had the greatest number of genes (n = 7), followed by the mitophagy pathway (n = 6) and the autophagy pathway (n = 2). Hub genes LeAtg8 and LeHog1 were involved in two of the three pathways. LeAtg8 was mapped on the autophagic and mitophagy pathways, whereas LeHog1 was mapped on the mitophagy and MAPK signal pathways. Considering the possible important roles of these genes in H 2 O 2 -induced autophagy, we focused on them in subsequent analyses.

LeHOG1
Phosphorylation Participated in LeATG8-Dependent Autophagy Induced by H 2 O 2 H 2 O 2 induces oxidative stress by exerting cytotoxic effects and increasing intracellular superoxide (O 2 · ) levels [31]. In the present study, treatment with H 2 O 2 for 24 h significantly increased the antioxidant activity of superoxide dismutase (SOD) activity ( Figure 3A). Moreover, DCFH-DA dyeing results showed that the treatment increased the ROS content of the mycelia ( Figure S9). The high-osmolarity glycol (HOG) pathway may be associated with oxidative stress. Hog1 is activated by dual phosphorylation on a tripeptide motif (Thr-X-Tyr) via promoting high levels of ROS [32]. Therefore, Western blotting was used to verify the expression levels of LeATG8 and LeHOG1 and the expression level of phospho-LeHOG1 in H 2 O 2 -induced ageing mycelia. The expression of LeATG8 and LeHOG1 showed no change in the mycelia after 3 h of H 2 O 2 treatment compared with the control ( Figure 3B) but was upregulated after 24 h of treatment, indicating that autophagy was induced by H 2 O 2 . Meanwhile, the phosphorylation level of LeHOG1 increased as early as 3 h and continued to increase until 24 h ( Figure 3B). This immediate and long-lasting phosphorylation of LeHOG1 suggested that LeHOG1 played a crucial part in response to oxidative stress and the activation of LeATG8-dependent autophagy. Furthermore, we used the HOG1 inhibitor SB203580 to inactivate LeHOG1 selectively. SB203580 (10 µM)pretreated cellophane and untreated cellophane were used for mycelial culture, which was then treated with 100 µM H 2 O 2 and incubated for 24 h. SB203580 slightly relieved the growth inhibition caused by exogenous H 2 O 2 treatment ( Figure 3C). At the protein level, SB203580 reduced LeHOG1 phosphorylation and LeATG8 expression ( Figure 3D). However, LeHOG1 expression was increased. According to statistical analysis, on adding the SB203580 inhibitor, the intracellular ROS was significantly reduced compared with the H 2 O 2 treatment group ( Figures 3E and S9). Inactivation of LeHOG1 might inhibit the function of LeATG8, demonstrating that the LeATG8-dependent autophagy in L. edodes was strongly correlated with LeHOG1 MAPK activity or even phosphorylation.

Subcellular Localisation of LeATG8, LeHOG1, and Phospho-LeHOG1 during H 2 O 2 Treatment
We evaluated the cellular localisations of LeATG8, LeHOG1, and phospho-LeHOG1 using immunofluorescence. We labelled these proteins using the same antibodies used in the Western blot analysis and evaluated the nuclear localisation using DAPI. Without H 2 O 2 treatment, LeATG8, LeHOG1, and phospho-LeHOG1 localised in the cytoplasm as control (Figures 4A,B and S10A). After a 3 h treatment, they were still located in the cytoplasm, while the phospho-LeHOG1 in the cytoplasm aggregated ( Figures 4C,D and S10B). After 24 h of H 2 O 2 treatment, the positive fluorescence of LeAtg8, LeHOG1, and phospho-LeHOG1 was stronger than that after 3 h H 2 O 2 and control treatments, which agreed with the Western blot results (Figures 4E,F and S10C). In nearly 90% of visual fields, LeHOG1 and phospho-HOG1 were clearly located in the nucleus. This result suggested that, upon exposure to H 2 O 2 , phospho-LeHOG1 was translocated into the nucleus, where it directly targeted several transcription factors, as observed in yeasts under hyperosmotic stress [33]. Additionally, in nearly 60% of visual fields, we clearly observed a ring-shaped autophagosome structure in L. edodes hyphae by labelling the LeATG8 protein ( Figure 5A), and a 3D reconstruction of the Z-stack images of immunofluorescence staining was obtained ( Figure 5B). The results of 3D imaging clearly showed that LeAtg8-labelled autophagosomes (green) were in the process of enveloping the nucleus (blue).
which was then treated with 100 µM H2O2 and incubated for 24 h. SB203580 slightly re-lieved the growth inhibition caused by exogenous H2O2 treatment ( Figure 3C). At the protein level, SB203580 reduced LeHOG1 phosphorylation and LeATG8 expression ( Figure  3D). However, LeHOG1 expression was increased. According to statistical analysis, on adding the SB203580 inhibitor, the intracellular ROS was significantly reduced compared with the H2O2 treatment group (Figures 3E and S9). Inactivation of LeHOG1 might inhibit the function of LeATG8, demonstrating that the LeATG8-dependent autophagy in L. edodes was strongly correlated with LeHOG1 MAPK activity or even phosphorylation.

Subcellular Localisation of LeATG8, LeHOG1, and Phospho-LeHOG1 during H2O2 Treatment
We evaluated the cellular localisations of LeATG8, LeHOG1, and phospho-LeHOG1 using immunofluorescence. We labelled these proteins using the same antibodies used in the Western blot analysis and evaluated the nuclear localisation using DAPI. Without H2O2 treatment, LeATG8, LeHOG1, and phospho-LeHOG1 localised in the cytoplasm as control ( Figures 4A,B and S10A). After a 3 h treatment, they were still located in the cytoplasm, while the phospho-LeHOG1 in the cytoplasm aggregated ( Figures 4C,D and S10B). After 24 h of H2O2 treatment, the positive fluorescence of LeAtg8, LeHOG1, and phospho-LeHOG1 was stronger than that after 3 h H2O2 and control treatments, which agreed with the Western blot results ( Figures 4E,F and S10C). In nearly 90% of visual fields, LeHOG1 and phospho-HOG1 were clearly located in the nucleus. This result suggested that, upon exposure to H2O2, phospho-LeHOG1 was translocated into the nucleus, where it directly targeted several transcription factors, as observed in yeasts under hyperosmotic stress [33]. Additionally, in nearly 60% of visual fields, we clearly observed a ring-shaped autophagosome structure in L. edodes hyphae by labelling the LeATG8 protein ( Figure 5A), and a 3D reconstruction of the Z-stack images of immunofluorescence staining was obtained ( Figure 5B). The results of 3D imaging clearly showed that LeAtg8-labelled autophagosomes (green) were in the process of enveloping the nucleus (blue).

Discussion
ROS are direct triggers of oxidative stress [34] generated by the mitochondria, which are the main inducers of autophagy under oxidative stress [35]. ROS-induced damage of cellular constituents leads to mitochondrial dysfunction, DNA damage, chromatid breaks and mutations, and reduced metabolic efficiency [36]. The accumulation of damaged mitochondria in the cell can cause cellular oxidative stress and eventually lead to cell death [37]. When L. edodes mycelia were grown in the presence of H2O2, ROS blocked mycelial growth due to cellular damage in the early stage. After 20 days of treatment, mycelia agglomerated to form knoblike protuberances, which are similar to mycelial knots in spawn bags (Figure 1). In L. edodes cultivation, mycelia transform from vegetative growth to reproductive growth after mycelial maturation. Mycelial knots are the precursor of a primordium or fruiting body initiation, and mycelial knot formation in spawn bags usually takes 60 days. In the present study, H2O2 treatment caused the premature formation of knoblike protuberances, suggesting that H2O2 accelerated the ageing of L. edodes mycelia. Moreover, ROS accumulation decreases MMP and triggers apoptosis [36,38,39]. In L. edodes, the ratio of JC-1 aggregates to JC-1 monomers (red/green) gradually reduced, indicating mitochondrial depolarisation of mycelia treated with H2O2 ( Figure S1). Moreover, the nucleus in the mycelium also became TUNEL positive after 100 µM H2O2 treatment ( Figure S2). Antioxidant enzymes, such as SOD, could metabolise ROS to increase resistance to oxidative stress [40].
This phenomenon was confirmed by the significantly enhanced SOD activity and ROS content in the H2O2-treated L. edodes mycelia (Figure 3). In our previous study, the expression of autophagy key protein LeATG8 was significantly increased in long-term cultured mycelium. The later stage of culture showed that the degree of autophagy was deepened, with similar changes of cell structure and SOD activity to those after 24 h H2O2 treatment. Our findings indicated that 24 h H2O2 treatment induced rapid artificial ageing of L. edodes mycelia. The expression of LeATG8 protein in the mycelium significantly increased after 24 h treatment, indicating that this model can be used as an induction model

Discussion
ROS are direct triggers of oxidative stress [34] generated by the mitochondria, which are the main inducers of autophagy under oxidative stress [35]. ROS-induced damage of cellular constituents leads to mitochondrial dysfunction, DNA damage, chromatid breaks and mutations, and reduced metabolic efficiency [36]. The accumulation of damaged mitochondria in the cell can cause cellular oxidative stress and eventually lead to cell death [37]. When L. edodes mycelia were grown in the presence of H 2 O 2 , ROS blocked mycelial growth due to cellular damage in the early stage. After 20 days of treatment, mycelia agglomerated to form knoblike protuberances, which are similar to mycelial knots in spawn bags (Figure 1). In L. edodes cultivation, mycelia transform from vegetative growth to reproductive growth after mycelial maturation. Mycelial knots are the precursor of a primordium or fruiting body initiation, and mycelial knot formation in spawn bags usually takes 60 days. In the present study, H 2 O 2 treatment caused the premature formation of knoblike protuberances, suggesting that H 2 O 2 accelerated the ageing of L. edodes mycelia. Moreover, ROS accumulation decreases MMP and triggers apoptosis [36,38,39]. In L. edodes, the ratio of JC-1 aggregates to JC-1 monomers (red/green) gradually reduced, indicating mitochondrial depolarisation of mycelia treated with H 2 O 2 ( Figure S1). Moreover, the nucleus in the mycelium also became TUNEL positive after 100 µM H 2 O 2 treatment ( Figure S2). Antioxidant enzymes, such as SOD, could metabolise ROS to increase resistance to oxidative stress [40].
This phenomenon was confirmed by the significantly enhanced SOD activity and ROS content in the H 2 O 2 -treated L. edodes mycelia (Figure 3). In our previous study, the expression of autophagy key protein LeATG8 was significantly increased in long-term cultured mycelium. The later stage of culture showed that the degree of autophagy was deepened, with similar changes of cell structure and SOD activity to those after 24 h H 2 O 2 treatment. Our findings indicated that 24 h H 2 O 2 treatment induced rapid artificial ageing of L. edodes mycelia. The expression of LeATG8 protein in the mycelium significantly increased after 24 h treatment, indicating that this model can be used as an induction model for studying autophagy in L. edodes.
Damaged cellular constituents and organelles must be selectively removed by autophagy to protect cells from excessive oxidative stress and cell death [37]. A possible relationship between autophagy and mycelial ageing in L. edodes has been previously suggested [18]. However, data on the determination of autophagosomes in L. edodes remain limited. As Atg8 has been used as a protein marker of the double-layer membrane autophagosome in yeast and mammalian cells, in the present study, using fluorescent labelling, we observed the classic ring structure of autophagosomes in L. edodes hyphae for the first time ( Figure 5). Moreover, 3D imaging revealed that LeATG8 was enriched on the nuclear periphery ( Figure 5), suggesting that autophagosomes surrounded these nuclei to degrade them at specific stages. This result provided strong evidence for the importance of LeATG8 in nuclear degradation. Meanwhile, LeATG3, which encodes a homologue of an E2-conjugating enzyme that generates ATG8-PE, was also identified via the enrichment network of the ageing mycelia ( Figure 2). These results implied the formation of autophagosomes and the occurrence of autophagy in the ROS-induced mycelia.
When the selective cargo is the mitochondria, autophagy becomes mitophagy [41,42]. In the present study, exogenous H 2 O 2 treatment increased ROS production in L. edodes mycelia, which resulted in MMP loss, oxidative stress, and ageing (Figures 1, 3, S1 and S8). MMP loss is obvious because ROS-induced mitophagy requires mitochondrial depolarisation [37]. Furthermore, mitophagy-related genes (LeAtg8, LeHog1, LeRpd3, LeCk2, LeFis1, and LeUbp3) were overexpressed in ageing mycelia, and an enrichment network of autophagic, mitophagic, and MAPK signal pathways was constructed ( Figure 2). ATG8 is essential in all autophagic pathways, including mitophagy. Therefore, LeATG8 in the present study acted as a hub gene involved in autophagy and mitophagy. In addition, mitophagy is specifically mediated by ATG32, which is anchored in the mitochondrial outer membrane, acting as a receptor for ATG8 to recruit the autophagy machinery to the mitochondrial surface [41]. ATG32 interaction with scaffolding component ATG11 marks mitochondrial degradation [25,43]. When ATG11 engages the mitochondria with ATG8-PE, the Fis1-Mdv1-Dnm1 molecular complex is necessary for mitochondrial fission and fragmentation [25]. The high expression of LeFIS1 in the present study confirmed that the mycelia with ROS-induced ageing underwent mitophagy ( Figure 2).
Aside from the autophagy machinery, upstream signalling regulation is also indispensable for mitophagy. Rapamycin complex 1 (TORC1) in yeast is a negative regulator of autophagy and mitophagy [41]. Under mitophagy-inducing conditions, TORC1 is suppressed, and its downstream Ume6-Sin3-Rpd3 complex releases ATG32 transcription repression, resulting in ATG32 expression [43]. LeRPD3, a homologue of Rpd3 of histone deacetylase complex in L. edodes, was 1.63-fold overexpressed in the present study (Figure 2), indicating the regulation of mitophagy through protein kinase TORC1. Similarly, it was also previously suggested that LeRPD3 and LeTORC1 are involved in oxidative stress and autophagy of the L. edodes brown film formation process through proteomic quantification analysis [15]. Different from TOR signalling, the MAPK signalling pathway regulates mitophagy but not non-selective autophagy [41]. The ATG32/ATG11 interaction is strictly regulated via ATG32 phosphorylation through kinase CK2 [25,43]. During mitophagy, LeCK2 expression showed a 1.36-fold upregulation in the ROS-induced ageing mycelia of L. edodes, suggesting the active phosphorylation of LeATG32 by LeCK2 (Figure 2). Activation of CK2 depends on the MAPKs of the HOG pathway [41].
In yeast and other filamentous fungi (e.g., Aspergillus), HOG1 acts as a kinase in the MAPK cascade, and high intracellular concentrations of H 2 O 2 can activate HOG1 [44,45]. Transcriptome, RT-qPCR, and Western blot analyses showed that H 2 O 2 treatment increased the relative expression level of LeHOG1 and regulated the downstream mitophagic pathway (Figures 2, 3 and S7). Based on antioxidant response, H 2 O 2 induces major changes in protein phosphorylation [46]. Cytoplasmic HOG1 is phosphorylated by dual specific kinase PBS2 and subsequently translocates to the nucleus and activates the downstream transcription factor, thereby altering gene expression under stress [24]. In the present study, H 2 O 2 treatment increased the phosphorylation of LeHOG1 as early as 3 h and lasted into 24 h, whereas the protein level of non-phosphorylated LeHOG1 increased until 24 h treatment ( Figure 3). Phospho-LeHOG1 agglutinated after 3 h of treatment and then translocated into the nucleus after 24 h, with the time course matching its phosphorylation status (Figure 4). LeHOG1 also translocated to the nucleus following H 2 O 2 treatment. Similarly, in fungi (e.g., Saccharomyces cerevisiae, Alternaria, and Magnaporthe), the threonine (Thr) and tyrosine (Tyr) residues on the Hog1 protein are double phosphorylated under the action of ROS. LeHOG1 and phospho-LeHOG1 transfer to the nucleus to regulate gene transcription and increase cell resistance to oxidative stress and autophagy [33,44,[46][47][48]. Additionally, the HOG1 inhibitor SB203580 blocked LeATG8 and phospho-LeHOG1, which consequently upregulated LeHOG1 expression (Figure 3). This result suggests a link between the LeATG8-dependent autophagy in L. edodes mycelial and LeHOG1 phosphorylation. While inhibiting HOG1 phosphorylation, SB203580 also reduced intracellular ROS content. Similar results that the inhibition of P38 (HOG1 homologous protein in mammals) phosphorylation can inhibit ROS content in human or animal cells have been reported [49][50][51]. However, in addition to the HOG-MAPK pathway, there are many ways to respond to oxidative stress in fungi. In this study, we also found that the transcription of catalase (CTT1) was upregulated ( Figure 2B). Therefore, we suggested that inhibition of autophagy and increase in the protein expression of non-phosphorylated LeHOG1 triggered other antioxidant responses. Thus, the ROS content in the hyphal cells was reduced, which increased the survival of the cells under oxidative stress.

Conclusions
In the present study, we successfully induced autophagy in L. edodes mycelia through exogenous H 2 O 2 application. We comprehensively studied the expression and subcellular localisation of ROS and autophagy-related proteins during H 2 O 2 treatment of mycelia. Using fluorescent labelling, we observed, to the best of our knowledge, for the first time, the classic ring structure of autophagosomes in L. edodes hyphae. Moreover, 3D imaging showed that LeATG8 was enriched on the nuclear periphery, suggesting that autophagosomes surrounded these nuclei to degrade them at specific stages. Our results indicated that both LeATG8 and LeHOG1 are involved in the mycelial autophagy of L. edodes. LeATG8 expression could be blocked by inhibiting LeHOG1 phosphorylation, which suggested a relationship between autophagy and oxidative stress in L. edodes. Further, in-depth exploration of the interaction between LeHOG1 and LeATG8, as well as the correlation between MAPK and autophagy pathways, will provide deeper insights into ROS-induced autophagy involvement in the ageing or pigmentation of L. edodes.
Funding: This research was supported by National Natural Science Foundation of China (32202568), and Beijing Academy of Agriculture and Forestry Sciences (KJCX20230808 and QNJJ202317), China. This work was partially funded by Beijing Natural Science Fundation (623008) and China agriculture research system (CARS-20).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analysed during this study are included in this published article and its supplementary information files. The raw sequencing data of RNA-seq are available at the National Genomics Data Center, China National Center for Bioinformation, under BioProject ID PRJCA012101 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA012101, accessed on 17 March 2023). The LeHH0h sample was labelled as LeC1.