Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-020-11087-9 GENOMICS, TRANSCRIPTOMICS, PROTEOMICS Comparative transcriptional analyses of Pleurotus ostreatus mutants on beech wood and rice straw shed light on substrate-biased gene regulation Hongli Wu 1 & Takehito Nakazawa 1 & Haibo Xu 1 & Ruiheng Yang 2 & Dapeng Bao 2 & Moriyuki Kawauchi 1 & Masahiro Sakamoto 1 & Yoichi Honda 1 Received: 7 October 2020 / Revised: 15 December 2020 / Accepted: 28 December 2020 # The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021 Abstract Distinct wood degraders occupying their preferred habitats have biased enzyme repertoires that are well fitted to their colonized substrates. Pleurotus ostreatus, commonly found on wood, has evolved its own enzyme-producing traits. In our previous study, transcriptional shifts in several P. ostreatus delignification-defective mutants, including Δhir1 and Δgat1 strains, were analyzed, which revealed the downregulation of ligninolytic genes and the upregulation of cellulolytic and xylanolytic genes when compared to their parental strain 20b on beech wood sawdust medium (BWS). In this study, rice straw (RS) was used as an alternative substrate to examine the transcriptional responses of P. ostreatus to distinct substrates. The vp1 gene and a cupredoxin-encoding gene were significantly upregulated in the 20b strain on RS compared with that on BWS, reflecting their distinct regulation patterns. The overall expression level of genes encoding glucuronidases was also higher on RS than on BWS, showing a good correlation with the substrate composition. Transcriptional alterations in the mutants (Δhir1 or Δgat1 versus 20b strain) on RS were similar to those on BWS, and the extracellular lignocellulose-degrading enzyme activities and lignindegrading ability of the mutants on RS were consistent with the transcriptional alterations of the corresponding enzymeencoding genes. However, transcripts of specific genes encoding enzymes belonging to the same CAZyme family exhibited distinct alteration patterns in the mutant strains grown on RS compared to those grown on BWS. These findings provide new insights into the molecular mechanisms underlying the transcriptional regulation of lignocellulolytic genes in P. ostreatus. Key Points • P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates. • A demand to upregulate the cellulolytic genes seems to be present in ligninolysis-deficient mutants. • The regulation of some specific genes probably driven by the demand is dependent on the substrate. Keywords Lignin . Cellulose . Basidiomycete . Transcriptomes . White-rot Introduction Lignocellulosic biomass is an abundant source of carbon that has attracted increased interest due to its applicability as a * Takehito Nakazawa nakazawa.takehito.8u@kyoto-u.ac.jp 1 Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan 2 Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China renewable energy source and in chemical products. There are three main components of lignocellulosic biomass. Among these, lignin and hemicellulose provide a matrix in which cellulose gathers into a highly ordered network structure via various intermolecular and intramolecular forces, making lignocellulose resistant to enzymatic attacks and recalcitrant to decomposition (Zoghlami and Paës 2019). Whiterot fungi utilize not only cellulose and hemicellulose, but also uniquely depolymerize the most recalcitrant component of lignin, and are thus one of the key players in the global carbon cycle. White-rot fungi utilize lignocellulose by secreting a number of lignocellulose-degrading enzymes, including cellulolytic, xylanolytic, and pectinolytic enzymes, as well as Appl Microbiol Biotechnol oxidative enzymes, which function coordinately to decompose lignocellulose (Floudas et al. 2012; Rytioja et al. 2014). Most of these enzymes have been biochemically characterized (Lundell et al. 2010; Manavalan et al. 2015) and classified as carbohydrate-active enzymes (CAZymes) (Lombard et al. 2013). Six classes have been identified in CAZymes: glycoside hydrolases (GHs) (Henrissat 1991), glycosyltransferases (GTs) (Campbell et al. 1997), polysaccharide lyases (PLs) (Lombard et al. 2010), carbohydrate esterases (CEs) (Lombard et al. 2010), carbohydrate-binding modules (CBMs) (Boraston et al. 2004), and auxiliary activities (AAs) (Levasseur et al. 2013). Typically, white-rot fungi are thought to attack lignin by producing a series of oxidative enzymes, including manganese peroxidase (MnP, EC 1.11.1.13), versatile peroxidase (VP, EC 1.11.1.16), and lignin peroxidase (Lip, EC 1.11.1.14) (Janusz et al. 2017). In addition to these enzymes, enzymes such as glyoxal oxidases, pyranose dehydrogenases, and methanol oxidases may also play indispensable roles in lignin degradation (Kersten and Cullen 2014). The transcriptomes of white-rot fungi have been studied widely, and it has been suggested that their high variability is dependent on the substrates and growth period (Hori et al. 2014; Alfaro et al. 2016; Fernández-Fueyo et al. 2016; Rytioja et al. 2017). Colonizing various lignocellulosic biomasses, white-rot fungi obtain nutrients from the substrates, while these substrates affect the colonization strategies of these biomasses in return (Rytioja et al. 2017). Wood and herb biomass differ in both composition and structures. Generally, wood biomass contains higher amounts of lignin than herb biomass (Sjostrom 1993). There are also more guaiacyl and syringyl units and smaller amounts of p-hydroxyphenyl units in hardwood compared to softwood, in which guaiacyl units play a predominant role, with very small amounts of p-hydroxyphenyl units. All three types of lignin units are present in comparable amounts in herb biomass (Billa and Monties 1995; Vanholme et al. 2010; Kotake et al. 2015). In addition to lignin, hemicellulose, which is mainly composed of xylose units connected by β-1,4-glucosidic bonds, also differs in the fraction of hardwood and graminaceous plants. The hemicellulose of birchwood contains 89.3% xylose, 1% arabinose, 8.3% anhydrouronic acid, and 1.4% glucose (Kormelink and Voragen 1993). The hemicellulose in the bran of rice contains 46% xylose, 44.9% arabinose, and 6.1% galactose (Shibuya and Iwasaki 1985), whereas rice straw hemicellulose contains 43.2% glucose, 42.2% xylan, 11.2% arabinose, and 3.07% galactose (Xiao et al. 2001). In addition to xylan backbones, the side chains in hemicellulose also vary between hardwood and graminaceous plants. In plant heteroxylans, the arabinofuranosyl residues are esterified with p-coumaric acids and ferulic acids (Mueller-Harvey et al. 1986; Sun et al. 2001), while O-acetyl substitution commonly occurs in hardwood (Koutaniemi et al. 2013). Pleurotus ostreatus has been studied widely not only as a popular edible mushroom but also as one of the model whiterot fungi of the order Agaricales. Most members of Agaricales grow naturally on various lignocellulosic substrates (Fernández-Fueyo et al. 2016). Several studies have explored the effects of distinct substrates on the growth of P. ostreatus and fruiting body formation (Ashraf et al. 2013; Hoa et al. 2015). Transcriptome or secretome analyses of P. ostreatus PC9 on several substrates have recently been reported (Fernández-Fueyo et al. 2016; Alfaro et al. 2016, 2020). A comparative secretome analysis of poplar, wheat straw, and glucose medium suggested several specific genes that were highly expressed on lignocellulosic substrates (FernándezFueyo et al. 2016). Alfaro et al. (2016) also reported the transcriptome and secretome analysis of PC9 grown on sucrosemalt-yeast extract (SMY) medium under different culture conditions (shaken and static cultures). However, these studies are still not sufficient to fully elucidate the mechanism underlying lignocellulose degradation. In addition to transcriptome analysis, considerable efforts have been devoted to exploring the lignocellulose-degrading mechanisms using molecular genetics, especially since efficient gene targeting using homologous recombination has been established in P. ostreatus (Salame et al. 2012; Nakazawa et al. 2016). The genes and proteins involved in the transcriptional regulation of the ligninolytic system have been identified in some white-rot fungi (Álvarez et al. 2009; Toyokawa et al. 2016). In P. ostreatus, it was reported that the disruption and overexpression of cre1 enhanced and reduced the cellulolytic activity, respectively, in a liquid medium containing wheat straw (Yoav et al. 2018). Feldman et al. (2017) reported that the overexpression of the gene ssp1, which encodes a small secreted protein, elevated the expression of vp1 in glucose-peptone medium. In our previous studies, singlegene mutations in a putative chromatin remodeler gene chd1, a peroxisome biogenesis gene pex1, a histone chaperone gene hir1, and two putative Agaricomycetes-specific DNA-binding transcription factor (TF) genes, wtr1 and gat1, were shown to reduce the expression levels of specific mnp/vp genes as well as the wood lignin-degrading abilities of P. ostreatus on beech wood sawdust medium (BWS) (Nakazawa et al. 2017a, b, 2019; Wu et al. 2021). Additionally, certain cellulolytic genes were shown to be upregulated in most of the mutants (Wu et al. 2020). Gene expression in P. ostreatus could be induced variably across different substrate species (Alfaro et al. 2016). Thus, transcriptional alterations caused by the mutations could be different on BWS compared with the other substrates. Investigating mutants generated from the deletion of genes encoding TF or functional proteins on different substrates may cue us in which way or what extent these TF/functional proteins affects gene expressions. In this study, rice straw was used as the substrate. The transcriptional expression of P. ostreatus strain 20b grown Appl Microbiol Biotechnol on rice straw medium (RS) for 13 d was examined and compared with those grown on BWS. Two mutants obtained from our previous study, Δgat1 and Δhir1, were also cultured on rice straw medium to compare the transcriptional expression between 20b and the mutants when grown on rice straw medium, with the aim of improving our understanding of the transcriptional shifts of P. ostreatus in response to lignocellulosic substrates. Materials and methods P. ostreatus strains and media The strains used in this study are all monokaryon and are listed in Table 1. YMG agar medium [0.4% (w/v) yeast extract, 1% (w/v) malt extract, 1% (w/v) glucose, and 2% (w/v) agar] (Rao and Niederpruem 1969) was used for routine cultures. Rice straw (strain name is Koshihikari, one of the popular cultivars of Japonica rice in Japan) was purchased from Honda Nojo, Ishikawa, Japan. Rice straw medium [RS, 1.9 g size-fractionated rice straw (250−500 μm), 0.1 g wheat bran, and 6 mL H2O] was used as the substrate for the transcriptional analysis of the P. ostreatus strains. Beech wood sawdust media [BWS, 1.9 g beech wood sawdust (almost all of the particles were smaller than 1 mm), 0.1 g wheat bran, and 6 mL H2O] was used in our previous study of transcriptome analysis of Δgat1 and Δhir1 mutants (Wu et al. 2020, 2021). Each P. ostreatus strain was precultured on YMG agar medium and fresh mycelia were inoculated into 6-cm glass Petri dishes containing RS under solid conditions at 28°C for 13 d. The growth rate of the 20b strain on RS and BWS was almost the same, since it takes 13 d to fully cover the plate. The rice straw, beech wood sawdust, and wheat bran used in this study were subjected to organic solvent extraction by soaking the samples in toluene/ ethanol (2:1, v/v) for 1 h at 80°C, repeating 4 times. Wheat bran was added to the cultures to increase the production of ligninolytic enzymes (Tsukihara et al. 2006). The cultures were maintained under continuous darkness. Table 1 The P. ostreatus strains used in this study RNA-seq and bioinformatic analysis Each strain was grown on RS for 13 d, after which the total RNA was isolated from each sample in two biological replicates, using the RNeasy Plant Mini Kit (Qiagen, Venlo, Netherland). To remove genomic DNA, all RNA samples were subjected to DNase digestion using the Turbo DNAfree Kit (Ambion, Austin, TX). The kits used to prepare samples for sequencing and the accession numbers of the sequencing raw data are shown in Supplemental Table S1. Paired-end raw reads were quality trimmed by the CLC Genomics Workbench tool version 20.0 (Qiagen, Venlo, The Netherlands). Trimmed reads were mapped to the PC9 strain genome (http://genome.jgi.doe.gov/PleosPC9_1/ PleosPC9_1.home.html) using the RNA-seq analysis package in CLC with at least 80% sequence identity over at least 80% of the read lengths. The RPKM (reads per kilobase of transcript per million mapped reads) and CPM (count per million) values were exported from the CLC workbench for subsequent analysis. Differentially expressed genes (DEGs) were defined using edgeR (http://bioconductor.org) with the following parameters: fold change >4 and FDR (false discovery rate) <0.05. The “calcNormFactors” from R (Robinson et al. 2010) were used to perform data scaling with the trimmed mean of M values (TMM) method. CPM (count per million) was used for DEG assessment as required by the function of edgeR (Robinson et al. 2010). Heatmap analysis was calculated using Heml 1.0-Heatmap Illustrator (Deng et al. 2014) and the Euclidean squared distance metric was used. Gene annotations were based on the available PC9 genome on JGI (http://genome.jgi.doe.gov/PleosPC9_1/PleosPC9_1.home. html) and dbCAN (http://csb1.bmb.uga.edu/dbCAN). Hierarchical clustering analysis was performed using Euclidean distance. Principal component analysis (PCA) was performed using the RPKM values of each sample. Co-expression and putative shared motif analysis Co-expression network analysis was performed using the Comparative Co-Expression Network Construction and Visualization (Cytoscape) tool with the Pearson correlation Strain Genotype/description Source 20b A2B1 ku80::CbxRa Salame et al. (2012) Δhir1 A2B1 ku80::Cbx hir1::hph / a hir1 disruptant derived from 20b Wu et al. (2021) Δgat12 A2B1 ku80::CbxR gat1::hph / a gat1 disruptant derived from 20b Nakazawa et al. (2017a) 1 R a CbxR indicates the carboxin resistance gene (Honda et al. 2000) 1 The strains hir1d#1 and hir1d#2 used in this study represent duplicate samples of the strain Δhir1 2 The strains gat1d#1 and gat1d#2 used in this study represent duplicate samples of the strain Δgat1 Appl Microbiol Biotechnol coefficient (Tzfadia et al. 2016). The RPKM values of DEGs, mainly CAZyme-encoding genes, were used as the input files. The gene gat1 (protein ID 83134 in the PC9 genome database) was used as a bait gene. We used a 1 kb DNA fragment in the promoter region of co-expressed genes associated with the gene gat1 to predict the putative shared motifs. The meme suite webserver was used to identify common binding motifs shared by co-expressed genes, allowing for a width from 6 to 10 with 1 or 2 mismatches (Bailey and Elkan 1994). The generated motifs were compared against the JASPAR Core (fungi) 2018 database using the TOMTOM webserver (Gupta et al. 2007). Quantitative reverse transcription-PCR (qRT-PCR) 20b as well as the mutant strains gat1d#1 and hir1d#1 were cultured on RS for 9, 13 and, 20 days; moreover, the parental strain 20b was also cultured on BWS for 9, 13, and 20 days. Afterward, total RNA was isolated from each sample in two biological replicates. Of note, rice straw with particle size larger than 500 μm (almost all of the particles were smaller than 2 mm), and the FastGene RNA Premium Kit (Nippon Genetics, Tokyo, Japan; this kit includes DNase) were used. qRT-PCR was performed as described by Nakazawa et al. (2019). Assay for extracellular enzyme activities Results P. ostreatus strains were cultured on RS for 13 d. The RS covered with mycelial cells was suspended in 0.1 M Nalactate buffer (pH 4.5) and then centrifuged at 2000×g to remove the rice straw and mycelial cells. The resulting supernatant fluids were used to determine the ligninolytic enzyme activities. Guaiacol (2-methoxyphenol) oxidation was used as the substrate to examine H2O2-independent oxidase and Mn2+-independent/dependent peroxidase activities, as described by Kamitsuji et al. (2004). One unit of guaiacol oxidation activity was defined as the amount of enzyme that increased the absorbance at 465 nm by 1.0 per min. The CMCase (carboxymethyl cellulase) and xylanase activities were assayed as described by König et al. (2002). Before the examination, the resulting supernatant fluids were subjected to ultrafiltration with a 10-kDa cutoff to remove molecules affecting the quantification of reducing sugars. A 3,5dinitrosalicylic acid reagent was used to quantify the reducing sugars. Xylans derived from beech wood (Nacalai Tesque, Kyoto, Japan) and CMC (carboxymethyl cellulose) were used as substrates to determine xylanase and CMCase activity, respectively. One unit of xylanase/CMCase was defined as the amount of enzyme required to liberate 1 μmol of reducing sugar as xylose/glucose per minute under the assay conditions. The protein concentration was estimated using the Bradford assay. Extracellular enzymes boiled at 95°C for 10 min were used as controls. The absorbance was measured and monitored using a Multiskan GO plate reader (Thermo Fisher Scientific, MA, USA). Quantification of Klason lignin content in rice straw medium Each P. ostreatus strain was grown on RS for 28 d, followed by solvent extraction using toluene and ethanol (2:1, v/v). The residual amount of acid-insoluble Klason lignin contained in each BWS solution after the cultivation of the P. ostreatus strains was quantified using the acidolysis method as described by Ritter et al. (1932). Transcriptional expression of CAZyme-encoding genes in the P. ostreatus strain 20b grown on RS and BWS White-rot fungi harness lignocellulolytic enzymes to forge sugars from the cell wall. These enzymes (or enzymeencoding genes) are characterized by high variety in protein families and redundancy in copy numbers. In this study, transcripts of such enzyme-encoding genes were examined to provide an overview of different lignocellulolytic strategies in P. ostreatus strain 20b on RS and BWS (Fig. 1a). Enzymes that had similar targeting polysaccharide-substrates were also identified (Fig. 1a). A lower transcriptional expression level (overall RPKM values in each CAZy family) of ligninolytic genes encoding enzymes from AA2 on RS was observed (Fig. 1b). However, genes encoding enzymes from AA3_2, AA3_3, and AA5 families, catalyzing the production of H2O2, which supports peroxidase oxidation processes, shared comparable expression levels when grown on RS and BWS. It was also suggested that genes which encode enzymes (CH6/7, GH10/11, AA9 families) targeting cellulose and xylan exhibited lower expression level on RS. We observed a more abundant accumulation of transcripts encoding laccases from AA1 family and β-glucuronidases from the GH79 family when grown on RS. DEG analysis of the P. ostreatus strain 20b grown on RS and BWS We also examined differentially expressed genes in 20b_RS versus 20b_BWS to identify various specific genes with distinct functions, which could provide more clues for elucidating gene regulation in the adaption of distinct substrates. A total of 139 and 67 down- and upregulated genes (Supplemental Table S2) (fold change [FC] > 4, FDR < 0.05) were observed in the fungus grown on RS compared with those grown on BWS, respectively. It is worth noting Appl Microbiol Biotechnol Fig. 1 RPKM values of CAZyme-encoding genes associated with plant cell wall degradation in P. ostreatus strain 20b grown on RS and BWS. a Heatmap analysis of genes belonging to each CAZyme families and substrates predicted to be associated with each CAZymes are also listed; heatmap was built using the result generated from the formula [Log10(RPKM values)] of genes belonging to each CAZy families. b Overall expression levels shown by each substrate. RS, rice straw medium; BWS, beech wood sawdust; PLs, pectate lyase, pectinase; ABF, arabinofuranosidase that the gene vp1 (corresponding to protein ID 116738), which was expressed lightly in BWS, was strikingly upregulated in RS (Supplemental Table S2). The expression level of one cupredoxin-encoding gene (protein ID 87572) was also found to be extremely high (RPKM value, 5148.3) on RS (Supplemental Table S2). We also obtained some intriguing findings among the downregulated genes. It is worth noting that 15 putative peptidase-encoding genes were downregulated on RS (Supplemental Table S2). Apart from peptidaseencoding genes, many genes encoding sugar transporters and CAZymes showed lower expression level when parental strain 20b were grown on RS. Global transcriptional expression of mutant strains grown on RS In our previous study, we observed transcriptional alterations with respect to lignocellulolytic genes in two single-gene disruptants, Δgat1 and Δhir1, when grown on BWS (Wu et al. 2020, 2021). However, it remains unclear whether these alterations are consistent with other substrates. Thus, the comparative transcriptional analysis of parental strain 20b and mutant strains Δgat1 and Δhir1 grown on RS was performed. Hierarchy cluster and PCA analysis were performed using transcriptomics of mutants Δhir1 and Δgat1 and parental strain 20b (Fig. 2a, b). Two major clusters were observed: parental strain 20b and mutant strains, which revealed some co-upregulated or downregulated genes caused by mutations. PCA analysis showed a strong clustering of two biological replicates in all strains. A clear separation of parental strain 20b and mutants Δgat1 and Δhir1 was identified on the distribution along PC1. Along the distribution of PC2, Δgat1 and Δhir1 exhibited some differences. Furthermore, some strains from the same substrate were found to cluster together, which suggested that gene expression was more dependent on the type of strains other than the substrates they were colonized on. Differentially expressed genes were examined in each mutant strain. There were 427 and 591 upregulated and downregulated genes identified in Δgat1, respectively, while 362 and 326 up- and downregulated genes were observed in hir1 disruptants. Transcriptional alterations of CAZyme-encoding genes in Δgat1 grown on RS The expression patterns of CAZyme-encoding genes in Δgat1 versus 20b were determined by carrying out five Appl Microbiol Biotechnol Fig. 2 The global transcriptional profiles of P. ostreatus strains grown on RS for 13 d. a Heatmap analysis showing the clustering of 6 samples by calculating the Pearson’s correlation values. b Principle component analysis (PCA) was built based on the normalized transcriptional expression level of 12 samples. Samples grown on BWS were used for comparison with those on RS. c Bar plot showing the number of DEGs in two pairwise comparisons, Δgat1 vs 20b and Δhir1 vs 20b groups (AA, AA9, GH, PL, and GL-encoding genes) (Fig. 3) to evaluate the transcript alterations of genes encoding enzymes from these CAZyme families. The RPKM values of all CAZyme-encoding genes are provided in Supplemental Table S3. AA9 was excluded from the AA families because it represents an oxidative mechanism that likely plays a role in cellulose attack other than lignin degradation. There were 158 CAZyme-encoding genes identified as DEGs in Δgat1. Among them, AA-encoding genes accounting for 15.7%, AA9-encoding genes accounting for 39.2%, and GHencoding genes accounting for 43.1% were upregulated, while the proportion of these identified groups for downregulated genes was 30.2%, 0%, and 62.8%, respectively (Fig. 3b). Genes encoding enzymes from AA9 families exhibited high expression levels in the mutant Δgat1 versus 20b on RS. The AA-encoding genes, which are mainly involved in the lignin oxidation process, showed a high proportion among downregulated genes. Among them, the vp2 (protein ID 60432) gene, which was predominantly accumulated in the 20b strain, was significantly downregulated in the Δgat1 mutant strain. Other vp/mnp genes, which were not highly expressed in parental strain 20b, were also inactivated in the mutant Δgat1. Six genes (protein IDs 62347, 67424, 77373, 89214, 96655, and 134564) encoding putative glyoxal oxidases from AA5 families were also strikingly downregulated. The RPKM values and fold changes of the genes encoding putative enzymes from the AA2 and AA5 families are shown in Table 2. These transcriptional alterations were consistent with those observed in BWS (Wu et al. 2020). In addition to downregulated genes, upregulated genes also revealed some intriguing findings. Some cellulolytic (protein IDs 43698, 49686, and 130231, Table 3) and xylanolytic genes (protein IDs 81650, 89740, 110996, and 125911, Table 4), which encode putative endo-β-1,4 glucanases and xylanases, respectively, were found to be strongly upregulated in the mutants Δgat1 when grown on RS, supporting the changes in the expression pattern found on BWS. However, the transcriptional alterations of some genes were not consistent on these two different substrates. For example, one gene (protein ID 83320, GH7, Table 3) was highly upregulated (121-fold) in Δgat1 when grown on BWS but not remarkably activated (2-fold) when grown on RS. In contrast, three genes (protein IDs 47406, 100231, and 129772, GH7, Table 3) that were not activated in Δgat1 when grown on BWS, were highly upregulated on RS. We also observed similar transcriptional alterations in the expression of genes encoding lytic polysaccharide monooxygenases Appl Microbiol Biotechnol Table 2 Fold changes of ligninolytic genes including vps/mnps and cros in hir1 and gat1 disruptants versus 20b strain, respectively CAZy Gene or protein IDa FC (RS)b FC (BWS)c Table 3 Fold changes of cellulolytic genes encoding endo-β-1,4 glucanases belonging to GH6 and GH7 in hir1 and gat1 disruptants versus 20b strain, respectively CAZy AA2 AA5 Δgat1 Δhir1 Δgat1 Δhir1 vp1 0.01 1.40 1.80 418.32 vp2 vp3 mnp1 mnp2 mnp3 mnp4 mnp5 mnp6 101121 134564 98389 121363 62166 62347 67424 77373 84350 88952 0.00 0.03 0.39 0.03 0.00 0.53 0.05 0.06 0.28 0.20 1.15 0.82 0.67 0.05 0.07 0.04 1.04 1.11 0.00 0.10 0.26 0.27 0.15 0.77 0.04 0.85 0.90 0.97 0.84 0.90 0.70 0.05 0.18 0.04 1.11 1.30 0.00 0.01 0.71 0.01 0.01 1.06 0.13 0.08 0.94 0.03 1.48 1.93 1.47 0.04 0.13 0.09 1.57 1.22 0.01 0.29 1.28 1.70 2.87 1.82 0.18 4.60 0.61 0.59 0.60 1.83 0.66 0.04 0.94 0.15 1.19 1.13 89214 91068 94009 96655 99670 0.01 1.25 15.93 0.01 0.78 0.03 0.81 1.29 0.01 0.38 0.04 2.28 2.58 0.01 0.17 0.17 1.27 0.72 0.05 0.31 a A genomic fragment containing the genes that corresponds to each Protein ID were from the genome database of strain PC9 (JGI Pleurotus ostreatus PC9 v1.0, https://genome.jgi.doe.gov/PleosPC9_ 1/PleosPC9_1.home.html) b FC, fold change. Ratios were calculated by comparing the RPKM value of mutants and that of the parental control strain 20b grown on rice straw c FC, fold change. Ratios were calculated by comparing the RPKM value of mutants and that of the parental control strain 20b grown beechwood sawdust (Wu et al. 2020, 2021) (LPMOs) from the AA9 family (Protein IDs 96461, and 97339, Table 4) on RS compared with that on BWS. Transcriptional alterations of CAZyme-encoding genes in Δhir1 grown on RS A total of 125 CAZyme-encoding genes were identified as DEGs in mutant strain Δhir1. In the mutant Δhir1, 7.0%, 29.6%, and 57.8% AA, AA9, and GH-encoding genes were upregulated, while those for downregulated genes were 39.0%, 0%, and 56%, respectively (Fig. 3b). AA9-encoding genes were mostly found in the upregulated group, and AA- GH6 GH7 Protein IDa FC (RS)b FC (BWS)c Δgat1 Δhir1 Δgat1 Δhir1 45206 130231 43698 100231 100398 107842 9.41 17.51 202.96 24.73 1.19 21.78 36.39 31.93 243.72 16.76 0.39 5.79 7.65 15.86 858.68 1.06 13.02 26.24 6.38 14.75 93.95 1.85 0.66 1.86 114771 129772 129783 47406 49445 83320 83849 83987 90281 90565 94368 47295 49686 1.94 24.84 14.21 57.14 0.41 1.99 0.86 1.30 0.93 0.52 0.71 0.44 12.45 14.85 2.18 1.95 33.99 11.04 472.33 144.53 0.85 1.61 0.39 0.56 0.51 60.87 1.52 1.71 3.94 0.35 0.05 121.23 7.98 1.22 1.81 1.85 1.59 0.17 10.82 2.62 1.15 0.81 0.55 2.22 270.89 65.82 0.83 2.48 1.11 0.83 0.17 7.75 a A genomic fragment containing the genes that corresponds to each Protein ID were from the genome database of strain PC9 (JGI Pleurotus ostreatus PC9 v1.0, https://genome.jgi.doe.gov/PleosPC9_1/ PleosPC9_1.home.html) b FC, fold change. Ratios were calculated by comparing the RPKM value of mutants and that of the parental control strain 20b grown on rice straw c FC, fold change. Ratios were calculated by comparing the RPKM value of mutants and that of the parental control strain 20b grown beechwood sawdust Wu et al. 2020, 2021) encoding genes were mostly identified in the downregulated group. We observed similar transcriptional alterations with the mutant Δgat1, in which most cellulolytic (AA9, GH6, and GH7) and xylanolytic genes (GH10 and GH11), were upregulated, while most ligninolytic genes (AA2 and AA5, Table 2) were downregulated. However, we also observed some differences in the transcriptional alterations of several genes (AA2) in Δhir1 grown on RS compared to those grown on BWS. The mnp3 and mnp6 genes were both upregulated by 2.87and 4.60-fold in BWS (Δhir1 vs 20b) and could be considered redundantly in the ligninolytic system (Wu et al. 2021), while these genes were both downregulated on RS (Δhir1 vs 20b). As mentioned previously, the vp1 gene (protein ID 116738, Table 2), which was slightly expressed in parental strain 20b on BWS, was upregulated (81.7-fold) on RS. As for the mutant Δhir1, the vp1 gene was only slightly upregulated (1.4fold) when it was cultured on RS, while it was highly Appl Microbiol Biotechnol Table 4 Fold changes of genes encoding LPMOs belonging to AA9 and xylanases belonging to GH10 and GH11 in hir1 and gat1 disruptants versus 20b strain, respectively CAZy AA9 GH10 and GH11 Protein IDa FC (RS)b 100006 and 122311, AA9) (Tables 3, 4) also exhibited distinct transcriptional alterations in the Δhir1 mutant in response to RS and BWS. FC (BWS)c Δgat1 Δhir1 Δgat1 Δhir1 100006 100072 117057 122311 125666 130437 31.79 4.64 1.89 22.19 26.57 2.99 130.93 38.28 0.73 67.20 7.68 2.26 15.58 3.33 1.43 0.61 0.34 191.12 7.59 1.84 1.00 6.78 1.68 2.26 134258 134259 20839 21077 21397 44265 45362 46220 46385 56431 59310 82144 82613 83777 83978 84841 87701 90424 2.09 1.27 0.37 0.50 136.65 88.76 4.14 21.01 2.64 12.26 175.74 2.92 54.22 41.32 12.36 0.82 132.87 109.69 1.15 0.83 0.48 0.24 139.37 273.07 3.64 102.94 2.27 87.82 274.68 25.25 27.73 61.79 11.57 20.23 132.96 309.49 30.41 6.29 34.09 16.53 4.42 61.40 0.30 12.47 75.05 68.41 0.73 522.70 176.82 53.35 3.81 53.70 62.57 92.73 1.34 0.67 0.40 0.14 13.99 34.51 1.55 10.26 1.14 4.60 11.04 3.24 3.20 26.28 3.35 7.90 39.67 98.88 90521 94095 94230 96461 97339 125911 81650 96691 110996 89740 33.23 31.28 25.73 103.56 39.82 52.04 5.81 0.14 77.61 4.41 12.63 15.71 37.32 181.59 69.72 104.96 36.26 0.08 126.27 62.27 15.58 3.33 1.43 0.61 0.34 23.97 2.45 0.10 394.47 4.38 6.90 2.19 13.98 43.34 52.34 19.25 2.12 0.10 45.56 3.62 a A genomic fragment containing the genes that corresponds to each Protein ID were from the genome database of strain PC9 (JGI Pleurotus ostreatus PC9 v1.0, https://genome.jgi.doe.gov/PleosPC9_1/ PleosPC9_1.home.html) b FC, fold change. Ratios were calculated by comparing the RPKM value of mutants and that of the parental control strain 20b grown on rice straw c FC, fold change. Ratios were calculated by comparing the RPKM value of mutants and that of the parental control strain 20b grown beechwood sawdust Wu et al. 2020, 2021) upregulated (300 to 400-fold) on BWS (Wu et al. 2021). Other cellulolytic genes (protein IDs 47406 and 100231, GH7; Transcriptional analysis of small secreted proteinencoding genes and hydrophobin genes CAZymes are key players in the lignocellulose degrading process. Some intriguing findings on CAZyme-encoding genes have been identified in our previous and current studies. However, in addition to CAZyme-encoding genes, there are a number of genes that may play important roles in lignocellulose degradation processes that have not been extensively studied (Nagy et al. 2020). In addition to the enzymatic degradation of substrates by CAZymes, the interaction between substrates and fungi is facilitated through, for example, small secreted proteins (SSPs) and hydrophobins (Bayry et al. 2012; Pellegrin et al. 2015). SSPs and hydrophobins all feature short-chain amino acids (aa): less than 300 aa with a conserved secretion signal peptide for SSPs (Kim et al. 2016) and approximately 100 aa in length with eight conserved cysteine residues for hydrophobins (Wösten 2001). In this study, we compared the transcription of genes that encode SSP and hydrophobins (Fig. 4) to find more clues about the wooddecay process in mutants and parental strains grown on RS and BWS. There are totally six ssp genes predicted in the genome of P. ostreatus PC9 strain currently (Feldman et al. 2017). We observed a very low expression level for all ssp genes in the parental strain 20b grown on both RS and BWS (RPKM value <30). However, the ssp1 gene (protein ID 65712), which was slightly expressed in parental strain 20b, was found to be significantly upregulated in the mutant Δgat1 on both RS (330.3-fold) and BWS (3518.4-fold) (Fig. 4, Supplemental Table S4). This upregulation was not observed in the Δhir1 strain. Twenty-five hydrophobin-encoding genes were predicted in the genome of P. ostreatus PC9 strain, among which three genes (protein IDs 74127, 80078, and 114483) were predominantly accumulated in the parental strain 20b on both RS and BWS (Fig. 4, Supplemental Table S4). Among these three genes, two genes (protein IDs 74127 and 80078) have been identified and designated as vmh2 and vmh3, respectively (Peñas et al. 2002). The vmh3 gene was reported to be the most highly expressed hydrophobin-encoding genes in P. ostreatus PC9 grown on SMY liquid medium under both shaken and static conditions (Alfaro et al. 2016), different from the parental strain 20b grown on BWS. All three genes were significantly downregulated in the Δgat1 mutant on both substrates, especially vmh2, which was downregulated by 17.2- and 31.0-fold in RS and BWS, respectively. In contrast, we did not observe a significant downregulation of the three genes in the Δhir1 mutant on both substrates. In addition, Appl Microbiol Biotechnol Fig. 3 Heatmap analysis of CAZyme-encoding genes in the parental strain and mutants grown on rice straw for 13 d. a Heatmap analysis of two groups: Δgat1 vs 20b and Δhir1 vs 20b. b The proportion of five groups: AA, AA9, GH, PL, and GL-encoding genes in up- and Fig. 4 Heatmap analysis of hydrophobin-encoding genes and small secreted protein-encoding genes predicted in the JGI P. ostreatus PC9 genome (http:// genome.jgi.doe.g.,ov/PleosPC9_ 1/PleosPC9_1.home.html) grown on BWS and RS downregulated sets, respectively. The percentage was calculated using the number of genes in each group divided by the number of all up- or downregulated genes Appl Microbiol Biotechnol vmh2, which was strikingly downregulated in the Δgat1 mutant, was found to be slightly upregulated in the Δhir1 mutant. Time-course expression analysis of some of the DEGs using qRT-PCR In this study, transcriptomic analysis of the strains grown for 13 days was performed. However, different growth periods may result in gene expression bias in some wood-decaying fungi (Hori et al. 2014; Alfaro et al. 2016; Fernández-Fueyo et al. 2016). Therefore, to examine the time-course expression patterns of some of the DEGs identified in this study (17 genes; Supplemental Table S6), qRT-PCR was performed. As shown in Supplemental Fig. S1, different relative expression levels at different culture periods were observed in the context of most of the analyzed genes, indicating that different growth periods can indeed affect gene expression; however, the transcriptomic alterations regarding the DEGs were maintained. The relative expression levels of up- and downregulated genes identified by RNA-seq analysis were also higher and lower in the mutant strain(s), respectively, with two exceptions: 59310 was not significantly upregulated in hir1d#1 grown on RS, and 87582 was not highly expressed in 20b grown on BWS. Overall, these qRT-PCR results mostly validated the RNA-seq data, further supporting the reliability of the transcriptome analysis performed in this study. Promoter analysis of genes co-expressed on distinct substrates Transcription factor alterations could affect the transcriptional expression of specific genes (Hobert 2008). As the gene gat1 encodes a putative Agaricomycete-specific DNA-binding transcription factor, we identified co-expressed genes using the gene gat1 (protein ID 83134) as the bait gene in the DEG lists of Δgat1 versus 20b grown on BWS and RS, respectively, to determine whether most transcriptional alterations are associated with the gat1 gene. There were 257 and 72 genes co-expressed on BWS and RS, respectively (Fig. 5, Supplemental Table S5). These numbers are quite small when compared to the DEGs identified on each substrate, which suggested that most genes are regulated indirectly by Gat1. Among these co-expressed genes, 43 and 13 genes were CAZyme-encoding genes. Using these co-expressed CAZymes genes, we searched for putative shared-regulatory elements using de novo motif discovery in the promoter regions (Fig. 5b, d). However, considering that the number of co-expressed CAZyme-encoding genes is smaller on RS than that on BWS, which could make the shared motifs less ubiquitous, we added 13 more DEGs from the co-expressed datasets of RS samples. As a result, a total of 43 and 26 genes from the DEG lists identified from Δgat1 versus 20b on RS and BWS, respectively. One motif in each co-expressed set covered almost all co-expressed genes and was identified on BWS and RS. The consensus binding motifs within the 1-kb promoter regions of co-expressed CAZyme-encoding genes showed conservation of two critical bases: C and T (Fig. 5). Measurement of extracellular enzyme activities of mutants grown on RS To determine whether lignocellulolytic enzymes were affected by the transcript alterations of lignocellulolytic genes on RS, we compared the extracellular lignin-modifying enzymes, cellulase, and xylanase activities of the mutants Δgat1 and Δhir1 with the parental strain 20b. We cultivated the mutants Δgat1 and Δhir1, as well as the parental strain 20b, for 13 d on RS, and subsequently examined the activities of extracellular guaiacol oxidizing activities (H2O2-independent, Mn2+independent/dependent peroxidase activities), and the specific activities of carboxymethylcellulase (CMCase) and xylanase. As shown in Fig. 6, extracellular laccase activities were almost lost in the Δgat1 mutant, but increased in the Δhir1 mutant. Mn2+-independent and Mn2+-dependent peroxidase activities were all inactivated in both mutants, which was consistent with the transcriptional alterations observed in the mutants. Both specific activities of CMCase and xylanase were higher in Δgat1 and Δhir1 than in 20b. These results suggest that the up- and downregulation of lignocellulolytic genes are correlated with the increase and decrease of lignocellulolytic enzymes in both RS and BWS. Examination of lignin-degrading abilities of mutants grown on RS In a previous study, Δgat1 and Δhir1 showed total defects and decreased lignin-degrading ability on BWS compared to the parental strain 20b (Nakazawa et al. 2019; Wu et al. 2021). In this study, we examined lignin-degrading capacity again by evaluating the Klason lignin loss after culturing the strains on RS. As shown in Fig. 6c, similar results were obtained as those on BWS, where Δgat1 lost the ability to degrade lignin and Δhir1 retained approximately 20% of the lignin-degrading ability. These results suggest that changing the substrate from BWS to RS did not change their lignin-degrading capacity, despite the fact that changes in the transcriptional expression of several specific genes were observed. Discussion In this study, we aimed to examine whether distinct substrates affect the transcriptional alterations present in the ligninolysisdeficient mutants. Comparative transcriptional analysis of the parental strain 20b grown on BWS and RS was firstly examined. Genes which encode enzymes from AA2 family Appl Microbiol Biotechnol Fig. 5 Co-expressed transcription factor (Gat1)-related genes of P. ostreatus strains grown on beech wood (a) and rice straw (c). The sequenced logos showing motifs shared by all co-expressed CAZyme-encoding genes associated with the gene gat1 on beech wood (b) and rice straw (c) Fig. 6 The extracellular enzyme activities and Klason lignin loss in the indicated strains grown on RS for 13 d (n = 3) and 28 d (n = 3), respectively. a Extracellular lignin-modifying enzyme activities of indicated strains, one unit of activity for guaiacol oxidation was defined as the amount of enzyme that increased the absorbance at 465 nm by 1.0 per min. b Extracellular CMCase and xylanase activities, one unit of xylanase/CMCase (U) is defined as the amount of enzyme required to liberate 1 μmol of reducing sugar as xylose/glucose per minute under the assay conditions. c Klason lignin loss was determined by detecting the decrease in the amount of Klason lignin in the substrate relative to the nofungus control plate. Error bars represent the standard deviations of three bioreplicates; *p < 0.05 and **p < 0.01 were determined by t test Appl Microbiol Biotechnol exhibited lower expression level on RS (Fig. 1). Considering previous reports that a lower number and expression level of ligninolytic genes in wheat bran and cotton seed hulls in the white-rot fungus Dichomitus squalens were detected (Rytioja et al. 2017), ligninolytic genes might also be more likely induced by specific components present in wood biomass in P. ostreatus. However, most genes encoding putative H2O2producing enzymes possibly ascribed to lignin-degrading system exhibited comparable amount on both substrates (Janusz et al. 2017). Given that lignin-modifying enzymes exhibited lower expression levels on RS, a possible explanation may be that these H2O2-producing enzymes may have other functions exemplified by supporting LPMOs in cellulose oxidative degradation (Kracher et al. 2016). Rytioja et al. (2017) examined the transcript responses of D. squalens to distinct substrates, and found a similar cellulose/xylan response by the fungus with a lower expression level of (hemi)-cellulolytic genes on wheat bran substrates compared to spruce substrates when grown for 16 d. This is similar with our findings in P. ostreatus which showed lower transcript accumulations of genes targeting cellulose and xylan on RS when compared with that on BWS. It has been documented that graminaceous plants and softwood consist mainly of arabino-4-Omethylglucuronoxylans, while hemicellulose of hardwoods contains O-acetyl-4-O-methylglucuronoxylan, with a lower amount of methylgulcuronic acid (Sunna and Antranikian 1997). This may explain why P. ostreatus expressed more β - g l uc ur on i da s e- en co di n g g en es on R S t ha n o n methylgulcuronic acid-rich BWS. Considering that the total accumulation of transcripts targeting each substrate on BWS was higher than that on RS, we assume that P. ostreatus may be more suited to beech wood substrates, as this fungus is commonly found on wood in nature. However, many factors could be involved to affect the growth of P. ostreatus. To confirm the above-mentioned hypothesis, a more complex and comprehensive analysis is needed. We did not observe a large amount of DEGs when P. ostreatus was grown on RS compared with that on BWS (Supplemental Table S2). But some specific genes showing significantly different expression levels on distinct substrates may cue us that P. ostreatus may switch their transcriptional preferences in response to various substrates. Two genes which encode VP1 and cupredoxin were significantly upregulated on RS. The expression of vp1 was reported to be predominant in P. ostreatus PC9 after being cultured on Mn2+deficient GP liquid medium (Knop et al. 2014). However, when the 20b and PC9 strains were cultured on BWS and Mn2+ amendment cotton stalk solid substrates, respectively, the vp2 transcript accumulated most abundantly among the vp genes (Salame et al. 2014; Nakazawa et al. 2017a). The secretion of VP1 was higher when P. ostreatus PC9 was cultured on poplar wood and wheat straw for 21 d compared to glucose medium (Fernández-Fueyo et al. 2016). These reports suggested that vp1 or vp2 are two predominant expressed genes in P. ostreatus and which one is selected to play a predominant role is dependent on the substrate or the culture stage. Cupredoxin-encoding genes were shown to be highly expressed when the PC9 strain was cultured on SMY liquid medium in trophophase (Alfaro et al. 2016), but not by strain 20b on BWS in this study. The altered expression of vp1 and the cupredoxin-encoding gene in response to different substrates suggests some gene regulations induced by the different components of these substrates. Among downregulated genes on RS, many peptidase-encoding genes were found (Supplemental Table S2). Fungi secrete peptidases to breakdown proteins and polypeptides into small molecules to support osmotrophic nutrition of the growing hyphae (Petrini et al. 1993). It has been reported that the secretion of peptidases could be affected by trophic status and phylogeny (Semenova et al. 2017). However, our study suggests that different substrates may also affect the expression of genes encoding peptidases. A secretomic analysis showed that the production of several peptidases differs when the P. ostreatus PC9 strain was grown on lignocellulosic substrates and glucose medium, especially for the protein ID 71759, which was abundantly secreted on glucose medium, but completely absent from lignocellulosic substrates (Fernández-Fueyo et al. 2016). Notably, in the list of identified DEGs above, there are still many genes that encode enzymes with unknown functions. These genes could also provide key information in this study, as described by Nagy et al. (2020). Thus, more attention should be paid on the study of these genes in the future. The transcriptional alterations of ligninolysis-deficient mutants (Δgat1/Δhir1 vs 20b) grown on RS were also examined and compared with that on BWS. The downregulation of ligninolytic genes in Δgat1 grown on RS was consistent with that on BWS, suggesting that the transcriptional alterations of vp/mnp genes in gat1 disruptants on BWS (Wu et al. 2020) were also found on RS. However, as for the mutant Δhir1, the vp1 gene was only slightly upregulated when it was cultured on RS, while it was highly upregulated on BWS (Wu et al. 2021); in addition, some mnp genes upregulated on BWS were shown to be downregulated on RS. These results suggest that a redundant ligninolytic system in the mutant strain Δhir1 may not be induced properly when grown on RS. It has been reported that putative CRE-A regulatory elements were only found in the promoter region of the vp1 gene among nine vp/ mnp genes (Fernández-Fueyo et al. 2014). The regulation system of vps is different, especially between the genes vp1 and vp2, which could result in distinct transcriptional expression patterns. This should be confirmed with promoter analysis in the future. Regarding the upregulation of the vp1 gene in the Δhir1 mutant on RS, we assumed that the expression level of the vp1 gene had a threshold in the Δhir1 mutant strain, such that when the expression reached a certain level on RS, the elevation became limited. Cellulolytic and xylanolytic genes Appl Microbiol Biotechnol were also upregulated in both mutants (Δgat1 and Δhir1) grown on RS as they were observed on BWS, suggesting that upregulation of cellulolytic or xylanolytic genes seems to be a demand in ligninolysis-deficient mutant strains. However, we observed some specific cellulolytic genes which are quite differently regulated regarding to distinct substrate. Environmental stimuli and adaption to different substrates may be the reason why mutants alter the expression bias of specific genes (Jaenisch and Bird 2003). Antoniêto et al. (2014) reported that many DEGs identified in the Δcre1 mutant versus the Trichoderma reesei wild type strain were quite different on glucose and cellulose (Avicel) medium. Thus, distinct substrates could induce different regulation patterns. Regarding to which specific gene is going to be significantly upregulated in the mutant strains, it seems to be decided depending on the transcriptional regulation systems in response to different substrates independent from gat1 or hir1. Furthermore, the results of qRT-PCR (Supplemental Table S1) suggested that different growth periods are associated with gene expression bias in P. ostreatus in the context of RS and BWS media; therefore, time-course expression patterns should be investigated in detail in the future. In addition to CAZyme-encoding genes, we also observed some genes which encode SSPs and hyrophobins differentially expressed in mutants grown on both substrates. The gene ssp1 was found highly upregulated in Δgat1 mutant grown on both substrates. A previous study found that the expression level of ssp1 was also slightly expressed in P. ostreatus PC9 in either static or shaken cultures on SMY liquid medium (Alfaro et al. 2016). Potential functions of SSPs have been suggested, including an “effector” function in pathogenic fungi (review in Dodds et al. 2009), degradative capabilities in T. reesei (Saloheimo et al. 2002), and a symbiosis role in Laccaria bicolor (Plett et al. 2011). Feldman et al. (2017) proposed that SSPs may function as partial regulators of the ligninolytic system in P. ostreatus PC9 after demonstrating that modifications to ssp1 expression affected the transcriptional expression of genes encoding aryl-alcohol dehydrogenases, aryl alcohol oxidases, and VPs. In our study, all strains were cultured on RS and BWS solid medium, and the levels of gene expression were quite different from those of the strains grown on GP liquid medium. However, the transcriptional alterations of the ssp gene in our study strengthened our belief that these genes play a direct or indirect role in the lignocellulolytic system. Hydrophobins allow fungi aerial hyphae to escape from aqueous environments and come into contacting with the air (Wösten 2001). In wood decay fungi, the role of hydrophobins in wood colonization has yet to be fully elucidated (Peddireddi et al. 2006) and evidence to support the necessity of having a hydrophobic surface for lignocellulose decomposition is still needed. In this study, hydrophobinencoding genes in the Δgat1 mutant was found to be remarkably downregulated on both substrates while that in the Δhir1 mutant was slightly upregulated. In Aspergillus nidulans, hydrophobins were shown to promote biofilm formation on sugarcane bagasse and may enhance lignocellulose utilization by promoting the close-packed structure of enzyme-substratefungi (Brown et al. 2016). Transcriptome analysis of Aspergillus niger also revealed that the upregulation of hydrophobins when grown on wheat straw substrate may play a role in recruiting hydrolases to the surface of wheat straw (Delmas et al. 2012). Zong et al. (2016) reported that pretreating cellulases with hydrophobins improved the bioconversion of cellulose in corn stover. Thus, the downregulation of hydrophobin-encoding genes in the mutant Δgat1 may contribute to its defects in lignocellulose degradation. Our results support the hypothesis that hydrophobin-encoding genes in P. ostreatus are involved in the lignin-degrading process. The co-expressed genes exhibited differences on distinct substrates, suggesting that different substrates could affect the alterations associated with TF. In the brown rot fungi Wolfiporia cocos, TFs have been shown to play important roles in regulating biased expression on different substrates (Wu et al. 2019). Motifs shared by co-expressed genes suggest common regulatory mechanisms with respect to the TF Δgat1. The two consensus motifs provided in this study provide targets for further experimental testing to confirm these hypotheses. The extracellular enzyme activities identified in this study on RS were consistent with the alteration of transcriptional alterations and also showed consistency with that we observed on BWS (Wu et al. 2020, 2021). This strengthened our belief that there seems a demand to elevate the cellulolytic and xylanolytic systems. This should be confirmed in the future. To conclude, our study found that P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates. The overall expression level of most genes from the same CAZyme families was higher on BWS than on RS. However, the different levels of expression of some specific genes, such as the upregulation of β-glucuronidasesencoding genes, indicates that P. ostreatus adjusted its transcriptome in response to different substrates. Changing the substrate from BWS to RS did not significantly affect the trend of transcriptional shifts identified in Δgat1 and Δhir1 when grown on BWS. However, we observed that the activation of genes encoding cellulolytic or xylanolytic enzymes from the same CAZyme families differs on distinct substrates, suggesting that the upregulation of cellulolytic or xylanolytic genes seems to be a demand in ligninolysis-deficient mutant strains. Recently, Alfaro et al. (2020) showed that the wooddependent induction of the secretion of lignocellulolytic enzymes is suppressed in the presence of glucose in P. ostreatus in the context of liquid media; therefore, comparative transcriptome using the ligninolysis-deficient mutants grown on the medium containing lignocellulose and/or Appl Microbiol Biotechnol monosaccharides, such as glucose, may provide some mechanistic clues. In regard to the specific gene that will be significantly upregulated in the mutant strains, this seems to depend on the transcriptional regulation systems in response to different substrates, independent of gat1 or hir1. As such, this study provides insights into the regulatory mechanisms of lignocellulolytic genes. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00253-020-11087-9. Acknowledgments We would like to thank Prof. Yitzhak Hadar (Hebrew University of Jerusalem, Israel) for providing P. ostreatus strain 20b. Authors’ contributions TN conceived and designed the study. HLW carried out the experiment and drafted the manuscript. HLW, HBX, RHY, DPB and MK performed the analyses, TN, MK, MS, and YH provided editorial suggestions and revisions. All authors read and approved the final manuscript. Funding This work was supported in part by the Institute for Fermentation, Osaka [to T.N.], JSPS KAKENHIs [16K18729 and 19H03017 to T.N.], and the China Scholarship Council [to H.W.]. Data availability All data supporting the claims of this manuscript are presented and made available in this manuscript. 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