Different Putative Methyltransferases Have Different Effects on the Expression Patterns of Cellulolytic Genes

Putative methyltranferase LaeA and LaeA-like proteins, conserved in many filamentous fungi, regulate fungal growth, development, virulence, the biosynthesis of secondary metabolites, and the production of cellulolytic enzymes. Penicillium oxaliucm is a typical fungus that produces cellulolytic enzymes. In this study, we reported the biological function of eight putative methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) containing a methyltransf_23 or methyltransf_25 domain, with a focus on their roles in the production of cellulolytic enzymes. In P. oxalicum, various methyltransferase genes displayed different transcriptional levels. The genes Pomtr23C and Pomtr25A exhibited high transcriptional levels, while Pomtr23D/E/F/G/H and Pomtr25B were transcribed constantly at low levels. The gene deletion mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) were constructed. Various mutants have different patterns in cellulolytic enzyme production. Compared to the WT, the largest increase in filter paper activity (FPA, indicating total cellulase activity) was observed in the Δmtr23G mutant, the only mutant with a cellulolytic halo surrounding the colony. Three mutants (Δmtr23C/D and Δmtr25A) also showed increased cellulolytic enzyme production. The Δmtr23E and Δmtr25B mutants displayed decreased FPA activity, while the Δmtr23F and Δmtr23H mutants displayed similar patterns of cellulolytic enzyme production compared with the WT. The assay of transcriptional levels of cellobiohydrolase gene Pocbh1 and β-1,4-endoglucanase Poeg1 supported that higher cellulolytic gene transcription resulted in higher production of cellulolytic enzymes, and vice versa. The transcriptional levels of two transcription factors, activator XlnR and repressor CreA, were measured. The high transcription level of the PoxlnR gene in the Δmtr23D mutant should be one reason for the increased transcription of its cellulolytic enzyme gene. Both XlnR and CreA transcriptional levels increased in the Δmtr23G mutant, but the former showed a more significant increase than the latter, indicating that the activation effect predominated. The PoMtr25A is localized in the nucleus. The catalytic subunit SNF2 of the SWI/SNF chromatin-remodeling complex was found as one of the interacting proteins of PoMtr25A via tandem affinity purification coupled with mass spectrometry. PoMtr25A may affect not only the transcription of repressor CreA but also by recruiting SWI/SNF complexes that affect chromatin structure, thereby regulating the transcription of target genes.


Introduction
Plants make up the overwhelming majority of biomass on Earth.Filamentous fungi, such as Trichoderma, Aspergillus, and Penicillium, have been developed for the industrial production of plant biomass-based biofuels and biochemicals due to their adequate production capacity of extracellular cellulolytic enzymes [1].Some Penicillium species are regarded as enzyme factories because they create a high representation of extracellular enzymes, including cellulases and hemicellulases involved in the degradation of plant cell walls, which are valuable in white biotechnology.Penicillium oxalicum (previously Penicillium decumbens), which produces a variety of lignocellulolytic enzymes, has a high potential for use in lignocellulose hydrolysis.Its cellulase hyperproducing mutant has been used to produce lignocellulolytic enzymes on an industrial scale [2,3].
The LaeA/Lae1 (loss of aflR expression) was initially identified as a secondary metabolic global regulator in Aspergillus nidulans.The lack of A. nidulans AnLaeA inhibited the expression of the TF AflR, which controls the secondary metabolic gene cluster in sterigmatocystin formation [9].Later, it was discovered that LaeA and its orthologs played roles in fungal growth, sexual or asexual development, the biosynthesis of secondary metabolite, virulence, and production of extracellular glycoside hydrolases in a variety of filamentous fungi, such as Aspergillus spp., Penicillium spp., Fusarium spp., and Trichoderma spp.[10].For example, the loss of laeA significantly reduced sporulation in Aspergillus fumigatus, Trichoderma reesei, and P. oxalicum [11][12][13].To date, all examined pathogenic fungi with laeA gene deletion have been found to display reduced virulence [10].
Additionally, LaeA regulated the expression of extracellular glycoside hydrolases (GHs), specifically the genes for cellulolytic enzymes.In addition to being a virulence factor, the insect pathogenic fungus Beauveria bassiana MtrA, an ortholog of AnLaeA, has been related to the production of various extracellular GHs, including chitinase and mannanase [14].T. reesei TrLae1 was necessary for the expression of at least 50 GH genes.All seven cellulases and auxiliary factors for the breakdown of cellulose were wholly lost from expression in the Trlae1 deletion strain [8].LaeA was also discovered to be crucial for extensive GH gene expression in cellulolytic fungi Myceliophthora thermophila and P. oxalicum, and its deletion reduced the ability to produce cellulolytic enzymes remarkably [15,16].LaeA also regulates TFs that can directly bind to the promoter region of cellulolytic genes and then directly activate or repress target gene expression.The expression of Xyr1, a transcriptional activator that activates cellulolytic gene expression, was Lae1-dependent in T. reesei [8].The transcriptional repressor CreA, which is involved in carbon catabolite repression (CCR) and inhibits cellulolytic gene expression, was significantly upregulated due to the deletion of PolaeA in P. oxalicum [16].
LaeA is considered a methyltransferase because it harbors a methyltrasf_23 domain and a conserved S-adenosylmethionine (SAM) binding site [9].Proteins similar to LaeA have been found in individual fungi containing LaeA.Thirteen LaeA-like methyltransferases (LLMs, AnLlmA to AnLlmJ) were found in the A. nidulans genome [17].P. oxalicum contained 11 LLMs with SAM sites similar to PoLaeA [18].According to studies on A. nidulans AnLimF and AnLlmG, Cochliobolus heterostrophus Llm1 (the ortholog of AnLlmF), and P. oxalicum LLM PoMtr23B, the regulatory functions of these LLMs were both similar to and different from those of LaeA [17][18][19][20].For instance, the positive secondary metabolite regulator AnLlmG, with an overexpression that boosted sterigmatocystin synthesis, shared a regulatory role similar to AnLaeA.In contrast, AnLlmF displayed characteristics opposite to those of AnLaeA, specifically, that AnLlmF repressed sterigmatocystin synthesis [17,19].Additionally, it has been found that LLMs have functions similar to LaeA, which can also affect the expression of GHs.P. oxalicum Mtr23B controls the production of extracellular GHs; deleting the Pomtr23B gene inhibited the expression of cellulolytic genes, including five cellulase genes and the xylanase gene [18].Silencing two LLMs (named PoLaeA2 and PoLaeA3) in Pleurotus ostreatus decreased cellulase production [21].Thus, the biological roles of LLMs are likewise important.However, research on LLMs is still sporadic and insufficient, unlike LaeA, where the detailed relationship between genotype and phenotype has been thoroughly examined in various fungi.Whether these LLMs have specialized functions or are redundant proteins is still being determined.
LaeA is a cryptic methyltransferase.It has been shown that in A. nidulans, the lack of LaeA leads to the silent sterigmatocystin gene cluster, which includes the occupation of the heterochromatin protein-1 (HepA) and is characterized by repressive histone H3 lysine (K) 9 trimethylation (H3K9me3) [22].Modulating H3K9 methylation levels was linked to M. thermophila LaeA [15].However, while the research indicates that laeA deletion mutants have changed chromatin modification, it does not prove that LaeA and histone modification are directly related.It was even found that T. reesei Trlae1 expression was unrelated to H3K9me3, H3K4me2, or H3K4me3 in regions where the carbohydrate-active enzyme coding gene was present [8].Although LaeA can self-methylate at the methionine residue at position 207 in vitro, its histone target is still unknown [23].Hence, LaeA is named as a "putative" histone methyltransferase.LLMs are also "putative" methyltransferase.Even though A. nidulans LLMs (AnLlmA to AnLlmJ) roles in regulating fungal development and the synthesis of secondary metabolites have been investigated [17]; it is unknown whether these proteins are true histone methyltransferases or some other kind of methyltransferase.
In this study, we investigated the fundamental biological roles of eight putative methyltransferases in P. oxalicum, emphasizing their effects on cellulolytic gene transcription and cellulolytic enzyme synthesis.Different methyltransferases have various effects on the expression of cellulolytic genes and differ in terms of the underlying mechanisms that regulate this expression.

Fungal Strains and Culture Conditions
The wild-type (WT) strain P. oxalicum 114-2 (CGMCC 5302), which was previously categorized as P. decumbens [2], was used as the parent strain for gene deletion.Fungal strains were routinely grown on modified Vogel's salt agar, supplemented with different carbon sources as indicated.Vogel's 50× salts (1 L) was used: 125 g Na

Phylogenetic Tree Analysis and Domain Architecture Analysis
Putative LaeA-like methyltransferases were identified in Uniprot database via BLASTp (https://www.uniprot.org/blast,accessed on 10 August 2019), using the protein sequence of P. oxalicum PoLaeA (PDE_00584, UniProt accession: S7Z542) as a query with E-Threshold 0.0001, restrict by taxonomy P. oxalicum (strain 114-2/CGMCC 5302).Pfam search (http:// pfam.xfam.org/,accessed on 20 August 2019) and SMART (a Simple Modular Architecture Research Tool) (http://smart.embl-heidelberg.de/,accessed on 20 August 2019) were used to analyze the domain architectures of LaeA-like methyltransferases [24,25].For phylogenetic tree analysis, multiple sequence alignment was performed using the software MEGA 7.0.The tree was constructed using the Neighbor-Joining method in MEGA 7.0 [26].Positions containing alignment gaps and missing data were deleted.Statistical confidence of the inferred phylogenetic relationships was assessed by performing 1000 bootstrap replicates.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Construction of Different Mutants
All primers used for genetic manipulations are listed in Supplementary Table S1.The strategies for the construction of mutants are shown in Supplementary Figure S1.An overlap PCR method was used to create the fragment for targeted gene deletion.P. oxalicum WT genomic DNA was used as the template for the PCR amplification of the 5 -and 3 -flanking regions of the targeted genes.For Pomtr23C gene deletion, primer pairs mtr23C-UF/mtr23C-UR and mtr23C-DF/mtr23C-DR were used to amplify the 5 -and 3 -flanking regions of the Pomtr23C gene.Primer pair ptrA-F/ptrA-R was used to amplify the marker gene pyrithiamine hydrobromide (ptrA) from plasmid pME2892 [27].Then, the three PCR fragments (5 -and 3 -flanking regions of the Pomtr23C gene and ptrA) were fused with fusion PCR.The PCR product was then amplified using nested primers mtr23C-NF/mtr23C-NR and was transformed into the WT to obtain the ∆mtr23C mutant.P. oxalicum protoplast preparation and transformation were performed as previously described [28].
To construct the strain (Mtr25A-GPF) of PoMtr25A fused with a green fluorescent protein (GPF), we used primer pairs GFP-UF/GFP-UR, GFP-MF/GFP-MR, and GFP-DF/GFP-DR to amplify the promoter region and the gene of Pomtr25A fused with the GFP sequence, selection marker gene hygromycin B (hph) from the plasmid Psilent1 [29], and terminator sequence, respectively.The PCR fragments were fused and amplified using nested primers GFP-NF/GFP-NR.The PCR product was directly transformed into the WT to displace the native Pomtr25A gene to produce the Mtr25A-GFP strain.To construct the strain (Mtr25A-TAP) for tandem affinity purification, we used primer pairs TAP-UF/TAP-UR, TAP-MF/TAP-MR, and TAP-DF/TAP-DR to amplify the promoter region and the gene of Pomtr25A fused with the HA-FLAG sequence, selection marker gene hph, and terminator sequence, respectively.The PCR fragments were fused and amplified using nested primers TAP-NF/TAP-NR.The PCR product was directly transformed into the WT to displace the native Pomtr25A gene to produce the Mtr25A-TAP strain.The proper integration of GFP tag and HA-FLAG tag was verified via DNA sequencing.

Fungal Colony, Conidiation, and Biomass Analysis
Vogel's salt agar, supplemented with 2% glucose (w/v) (VSG), 2% glycerol (VSGly), or 1.0% ball-milled cellulose (VGC) as the sole carbon source, was used to monitor the fungal colony.Additionally, 2 µL conidial suspension (10 6 conidia mL −1 ) was spotted onto each plate and then grown at 30 • C for 3 to 5 days.For the conidiation test, 5 × 10 6 conidia were spread out on 9 cm VSG plates and cultivated at 30 • C for 5 days.Then, colony agar plugs with a 5 mm diameter were taken from the plates.Conidia were then harvested by flooding the plugs with sterile distilled H 2 O containing 0.02% Tween 80. Conidial concentrations were determined using a hemocytometer.The biomass of all mutants was measured as previously described [30].Briefly, the WT and mutants were pre-grown in VSG liquid at 30 • C, 200 rpm for 24 h.Then, 0.3 g vegetative mycelia was collected via vacuum filtration and transferred to the 100 mL of VSG liquid freshly prepared for another 4, 8, 12, 24, 48, and 72 h cultivation.Mycelia were then filtrated to remove the culture broth and dried at 70 • C to constant weight.All analyses were performed in biological triplicates.

Determination of Enzyme Activities
The strains were pre-grown in 100 mL VSG liquid at 30 • C, 200 rpm for 24 h.Then, 0.3 g vegetative mycelia was collected via vacuum filtration and added to 100 mL of liquid Vogel's salts supplemented with 1% wheat bran and 1% microcrystalline cellulose as carbon resources for fermentation at 30 • C, 200 rpm.Supernatants of fermentation broth were collected at the culture time of 48-, 72-, 96-, and 120-h.The enzyme activities were measured as previously described [28].Briefly, the filter paper activities (FPA), CMCase activity, and xylanase activity were assayed with Whatman No. 1 filter paper (GE, Hatfield, UK), Sodium carboxymethyl cellulose (CMC-Na), and birch Xylan (Sigma, St. Louis, MO, USA) as the substrates, respectively.The enzyme reactions were performed in 0.2 M NaAc-HAc buffer (pH 4.8) at 50 • C for 60, 30, and 30 min.The reducing sugar was quantified via the 3,5-dinitrosalicylic acid (DNS) method.One enzyme activity unit (U) was defined as the amount of enzyme necessary to generate 1 µmol of glucose or xylose per minute.

Quantitative Reverse Transcription PCR
The WT strain was pre-grown in VSG liquid at 30 • C, 200 rpm for 24 h.Then, 0.3 g filtered mycelia was transferred to 100 mL of VSG liquid freshly prepared for 9 and 24 h to determine the transcription levels of various methyltransferase genes.The WT and the mutants were cultivated according to the method described in section "Determination of enzyme activities" to determine the transcription levels of the cellulolytic gene and transcription factor gene.The mycelia were collected via vacuum filtration and ground to powder in liquid nitrogen.Total RNA was extracted with RNAiso Plus reagent (TaKaRa, Kusatsu, Japan).cDNA synthesis and quantitative reverse transcription-PCR (qRT-PCR) were performed as previously described [28] and q25B-F/q25B-R, were used to amplify Pomtr23C/D/E/F/G/H and Pomtr25A/B, respectively.Primers CBH-QF/CBH-QR, EG-QF/EG-QR, XYN-QF/XYN-QR were used to amplify cellulolytic gene cel7A/cbh1 (gene ID: PDE_07945), cel7B/eg1 (PDE_07929) and xyn10A (PDE_08094).Primers xlnR-QF/xlnR-QR and creA-QF/creA-QR were used to amplify transcription factor gene creA (PDE_03168) and xlnR (PDE_07674), respectively.Primers ACT-QF/ACT-QR were used to amplify housekeeping gene actin (PDE_01092).The copy number of unambiguous transcripts for each gene was normalized to the expression ratio using the transcripts of the actin gene.The primers used for qRT-PCR are listed in Supplementary Table S1.

Subcellular Localization Observation
The hyphae of the Mtr25A-GFP strain were examined using a high-sensitivity laser scanning confocal microscope (ZEISS LSM780) (Carl Zeiss, Oberkochen, Germany).The nuclei were stained in the dark for 15 min by Hoechst 33342 (Sigma, USA).The green fluorescence of Mtr25A-GFP was visualized using excitation light at 488 nm, while Hoechst 33342-stained blue nuclei were visualized using excitation light at 405 nm.

Tandem Affinity Purification and Mass Spectrometry
Detailed steps of tandem affinity purification coupled with mass spectrometry (TAP-MS) were described previously [31].The WT (as the control) and Mtr25A-TAP strains were cultivated in 2 L of VMG liquid at 180 rpm for 48 h at 30 • C. The hyphae were collected, drained, and ground with liquid nitrogen to obtain at least 40 g of powder.Protein lysis buffer (0.9 g NaCl, 1 M Tris-HCl, pH 7.5, 10 mL glycerol, 0.1 mL NP40, and 0.05 mL protease inhibitor per 100 mL) was used to extract the total protein.The Ezview™ Red ANTI-FLAG M2 Affinity Gel (Sigma, USA) was used for the first affinity purification step, added into the protein suspension, and incubated overnight at 4 • C.Then, 500 µL 3× FLAG peptide (with a final concentration of 150 ng/µL) was used to compete with the ANTI-FLAG M2 affinity resin to obtain the first protein elution.The ANTI-HA resin (Thermo Fisher Scientific, Waltham, MA, USA) was used for the second affinity purification step.Finally, 80 µL of 8 M urea was added and incubated with the ANTI-HA resin to obtain the final eluent.The final elution was divided into three parts: one part for Western blot using the ANTI-HA antibody (ABclonal, Wuhan, China), one part for 12.5% SDS-PAGE and silver staining, and one part for LC-MS/MS (APT, Shanghai, China) to determine the putative interacting proteins of bait protein PoMtr25A, respectively.
PoMtr23C/D/E/F/F/G/H and Mtr25A/B are phylogenetically conserved in many filamentous fungi, such as Aspergillus, Neurospora, Trichoderma, Fusarium, Magnaporthe, Beauveria, and Metarhizium (Supplementary Spreadsheet S1).For example, PoMtr23C shares the highest identity of 91.50% with the ortholog in Penicillium ucsense, 44.9% identity with the ortholog in Aspergillus niger, 38.3% identity with the ortholog in Neurospora crassa, and 44.9% identity with the ortholog in T. reesei.PoMtr23D/PoLlmG, the ortholog of AnLlmG, shares the highest identity of 92.9% with the ortholog in P. ucsense, 65.7% identity with the ortholog in A. niger, 40.3% identity with the ortholog in N. crassa, and 48.5% identity with the ortholog in T. reesei.PoMtr25A is somewhat different: its ortholog is not found in N. crassa but is still present in other fungi (Supplementary Spreadsheet S1).
filamentous fungi, such as Aspergillus, Neurospora, Trichoderma, Fusarium, Magnaporthe Beauveria, and Metarhizium (Supplementary Spreadsheet S1).For example, PoMtr23C shares the highest identity of 91.50% with the ortholog in Penicillium ucsense, 44.9% identity with the ortholog in Aspergillus niger, 38.3% identity with the ortholog in Neurospora crassa, and 44.9% identity with the ortholog in T. reesei.PoMtr23D/PoLlmG, the ortholog of AnLlmG, shares the highest identity of 92.9% with the ortholog in P. ucsense 65.7% identity with the ortholog in A. niger, 40.3% identity with the ortholog in N. crassa and 48.5% identity with the ortholog in T. reesei.PoMtr25A is somewhat different: its ortholog is not found in N. crassa but is still present in other fungi (Supplementary Spreadsheet S1).

Various Methyltransferase Genes Display Different Transcriptional Levels
We analyzed the transcriptional levels of these eight methyltransferase genes (Pomtr23B/C/D/E/F/G/H and Pomtr25A/B) using qPCR analysis when the P. oxalicum WT was cultivated in VMG liquid.In order to compare these methyltransferases with the already characterized PoLaeA and PoMtr23B [13,16,18], we also determined the

Various Methyltransferase Genes Display Different Transcriptional Levels
We analyzed the transcriptional levels of these eight methyltransferase genes (Pomtr23B /C/D/E/F/G/H and Pomtr25A/B) using qPCR analysis when the P. oxalicum WT was cultivated in VMG liquid.In order to compare these methyltransferases with the already characterized PoLaeA and PoMtr23B [13,16,18], we also determined the transcriptional levels of genes PolaeA and Pomtr23B.The results showed that PolaeA and Pomtr23B were transcribed at a high level.The transcriptional levels of Pomtr25A and Pomtr23C were the top two highest among the eight methyltransferase genes.Pomtr23E/F/G/H and Pomtr25B were transcribed at low levels (Figure 2A).
PolaeA have the top three highest transcriptional levels, while Pomtr25A's expression is the fourth highest under the starch cellulose plus wheat bran condition (Figure 2E).Both results of qPCR and transcriptional profiling supported that some methyltransferase genes (PolaeA, Pomtr23B/C, and Pomtr25A) are transcribed at high levels, whereas some genes (Pomtr23D/E/F/G/H and Pomtr25B) are transcribed constantly at low levels under different culture conditions.Furthermore, we reviewed the transcriptome data retrieved from the Gene Expression Omnibus (GEO) database of NCBI (https://www.ncbi.nlm.nih.gov/geo/,accessed on 10 October 2022).These data came from the different cultivation conditions when P. oxalicum WT was cultivated on different carbon sources, including glucose (GSE106558) [31], starch (GSE175682) [32], cellulose [28], and cellulose plus wheat bran (GSE 106558) [31].Pomtr23B, Pomtr25A, and Pomtr23C have the top three highest transcriptional levels among the ten methyltransferase genes under the glucose condition (Figure 2B).Pomtr25A, Pomtr23C, and Pomtr23B have the top three highest transcriptional levels under the starch or cellulose condition (Figure 2C,D).Pomtr23B, Pomtr23C, and PolaeA have the top three highest transcriptional levels, while Pomtr25A's expression is the fourth highest under the starch cellulose plus wheat bran condition (Figure 2E).Both results of qPCR and transcriptional profiling supported that some methyltransferase genes (PolaeA, Pomtr23B/C, and Pomtr25A) are transcribed at high levels, whereas some genes (Pomtr23D/E/F/G/H and Pomtr25B) are transcribed constantly at low levels under different culture conditions.

The Differences in Phenotype Caused by the Deletion of Each Methyltransferase Gene Are Not Notable
In order to ascertain the functions of the various methyltransferases, the gene (Pomtr23C /D/E/F/G/H and Pomtr25A/B) deletion mutants (∆mtr23C/D/E/F/G/H and ∆mtr25A/B) were constructed.The mutants were verified using diagnostic PCR and Southern blot analysis.The strategies and the results of the Southern blot are presented in Supplementary Figure S1.
When grown on VMG or VMGly agar, the eight mutants (∆mtr23C/D/E/F/G/H and ∆mtr25A/B) displayed identical colony morphologies to those of the WT.When grown on VMC agar, the seven mutants (∆mtr23C/D/E/F/H and ∆mtr25A/B) showed identical colony morphologies to those of the WT, with the exception that the mutant ∆mtr23G displayed a clearer cellulolytic halo around the colony than the WT, implying a higher cellulolytic enzyme secretion than WT (Figure 3A).As LaeA is a necessary regulator for the conidiation in various filamentous fungi [33], we determined the number of conidia in the mutants to ascertain whether these methyltransferases also contribute to fungal conidiation.Only the ∆mtr25B mutant displayed variation; its conidiation reduced to about 71.1% of the WT (Figure 3B).The absence of PoMtr25B has a minor effect on the production of conidia compared to the reduction in the number of conidia in ∆PolaeA to only ~4% of the WT [13].The colony morphology and conidiation revealed the growth characteristics of the mutants in solid culture.Then, we determined the growth of mutants in VMC liquid culture.WT and the six mutants (∆mtr23C/D/E/F and ∆mtr25A/B) reached their peak biomass at 48 h, whereas ∆mtr25G and ∆mtr25H reached their peak biomass at 24 h.However, the maximum biomass they could reach was similar despite a slight difference in the temporal trend of biomass growth (Figure 3C).

The Differences in Phenotype Caused by the Deletion of Each Methyltransferase Gene Are Not Notable
In order to ascertain the functions of the various methyltransferases, the gene (Pomtr23C/D/E/F/G/H and Pomtr25A/B) deletion mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) were constructed.The mutants were verified using diagnostic PCR and Southern blot analysis.The strategies and the results of the Southern blot are presented in Supplementary Figure S1.
When grown on VMG or VMGly agar, the eight mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) displayed identical colony morphologies to those of the WT.When grown on VMC agar, the seven mutants (Δmtr23C/D/E/F/H and Δmtr25A/B) showed identical colony morphologies to those of the WT, with the exception that the mutant Δmtr23G displayed a clearer cellulolytic halo around the colony than the WT, implying a higher cellulolytic enzyme secretion than WT (Figure 3A).As LaeA is a necessary regulator for the conidiation in various filamentous fungi [33], we determined the number of conidia in the mutants to ascertain whether these methyltransferases also contribute to fungal conidiation.Only the Δmtr25B mutant displayed variation; its conidiation reduced to about 71.1% of the WT (Figure 3B).The absence of PoMtr25B has a minor effect on the production of conidia compared to the reduction in the number of conidia in ΔPolaeA to only ~4% of the WT [13].The colony morphology and conidiation revealed the growth characteristics of the mutants in solid culture.Then, we determined the growth of mutants in VMC liquid culture.WT and the six mutants (Δmtr23C/D/E/F and Δmtr25A/B) reached their peak biomass at 48 h, whereas Δmtr25G and Δmtr25H reached their peak biomass at 24 h.However, the maximum biomass they could reach was similar despite a slight difference in the temporal trend of biomass growth (Figure 3C).

Various Mutants Have Different Patterns in Cellulolytic Enzyme Production
In addition to the function in regulating fungal conidiation, the LaeA also controls the expression of cellulolytic genes and then affects the synthesis of cellulolytic enzymes in various fungi such as T. reesei [8], M. thermophila [15], and Pleurotus ostreatus [21].P. oxaliucm PoLaeA and PoMtr23B were also reported to be involved in extensively regulating the glycoside hydrolase (GH) gene expression [16,18].Therefore, we determined whether these methyltransferases also contribute to the production of cellulolytic enzymes.
The WT and the mutants were cultivated in the media containing wheat bran and cellulose, an inducible cellulolytic enzyme production medium.Filter paper activity (FPA, indicating total cellulase activity), CMCase activity (indicating β-1,4-endoglucanase activity), and xylanase activity (indicating hemicellulase activity) were determined.Various mutants have different patterns in cellulolytic enzyme production.The FPA of mutants ∆mtr23C/D/G and ∆mtr25A increased compared to the WT.Among them, the ∆mtr23G mutant showed the most significant increase, with its FPA being 2.7, 2.8, 1.6, and 4.1 times higher than that of the WT on days 2, 3, 4, and 5 of culture.The FPA of ∆mtr23E and ∆mtr25B decreased compared to that of the WT.On the fourth day, the FPA of ∆mtr23E and ∆mtr25B were 58.7% and 62.2%, respectively, of those of WT, suggesting that proteins PoMtr23E and PoMtr25B are required in normal cellulolytic enzyme production.The FPA of ∆mtr23F and ∆mtr23H showed a similar pattern to the WT (Figure 4A).

Various Mutants Have Different Patterns in the Expression of the Cellulolytic Enzyme Gene
Since the production of cellulolytic enzymes in fungi is tightly regulated at the transcriptional level, we investigated whether the altered production of cellulolytic enzymes in various mutants was caused by the up-or downregulated expression of the corresponding genes.The transcriptional level of two key cellulase genes, Pocbh1 (PDE_07945) and Poeg1 (PDE_07929), and the key hemicellulase gene Poxyn10A (PDE_08094) in WT and mutants was determined using qPCR.The genes Pocbh1, Poeg1 and Poxyn10A encode cellobiohydrolase, β-1,4-endoglucanase, and β-1,4-xylanase, respectively.Their products are the most abundant cellulase and hemicellulase produced by fungal cells [2].
Compared to the WT, the transcriptional level of the Pocbh1 gene was significantly increased in the Δmtr23C/D/G and Δmtr25A mutants but significantly decreased in the The CMCase activity of mutants ∆mtr23C/D/G and ∆mtr25A increased compared to WT.Among them, the most significant increases were seen in the ∆mtr23G and ∆mtr25A mutants: on days 2, 3, 4, and 5 of culture, the ∆mtr23G mutant had CMCase activity that was 2.3, 1.8, 1.4, and 1.4 times that of WT, and the ∆mtr25A mutant had CMCase activity that was 2.1, 2.0, 1.4, and 1.4 times that of WT.The CMCase activities of ∆mtr23E and ∆mtr25B decreased compared with that of the WT.On the fourth day, the CMCase activities of ∆mtr23E and ∆mtr25B were 37.2% and 38.5% of those of WT, respectively.The CMCase activities of the ∆mtr23F and ∆mtr23H mutants showed similar patterns compared with the WT (Figure 4B).The xylanase activity of mutants ∆mtr23D/G increased, while ∆mtr23E decreased compared to the WT.On the fourth day, the xylanase activity of ∆mtr23D/G increased by 43.6% and 49.9% compared to WT, respectively, while ∆mtr23E decreased to 63.8% of the WT.The xylanase activity of other mutants was not significantly different from the WT (Figure 4C).

Various Mutants Have Different Patterns in the Expression of the Cellulolytic Enzyme Gene
Since the production of cellulolytic enzymes in fungi is tightly regulated at the transcriptional level, we investigated whether the altered production of cellulolytic enzymes in various mutants was caused by the up-or downregulated expression of the corresponding genes.The transcriptional level of two key cellulase genes, Pocbh1 (PDE_07945) and Poeg1 (PDE_07929), and the key hemicellulase gene Poxyn10A (PDE_08094) in WT and mutants was determined using qPCR.The genes Pocbh1, Poeg1 and Poxyn10A encode cellobiohydrolase, β-1,4-endoglucanase, and β-1,4-xylanase, respectively.Their products are the most abundant cellulase and hemicellulase produced by fungal cells [2].
Compared to the WT, the transcriptional level of the Pocbh1 gene was significantly increased in the ∆mtr23C/D/G and ∆mtr25A mutants but significantly decreased in the ∆mtr23E and ∆mtr25B mutants and remained essentially unchanged in the ∆mtr23F and ∆mtr25H mutants.For example, at the 24th hour of cultivation, the transcriptional levels of the Pocbh1 gene in ∆mtr23C/D/G and ∆mtr25A mutants were 85.7, 22.0, 241.4,and 28.6 times higher than those in the WT, respectively.The transcriptional levels of the Pocbh1 gene in the ∆mtr23E and ∆mtr25B mutants reduced to 9.6% and 11.9% of the WT, respectively (Figure 5A).Compared to Pocbh1 and Poeg1, of which transcriptional levels varied dramatically in several mutants, the expression change of Poxyn10A in the corresponding mutants was less pronounced.At the 24th hour of cultivation, the expression levels of the Poxyn10A gene in Δmtr23C/D/G mutants were 2.8, 4.0, and 11.7 times higher than those in the wild strain, respectively.The transcriptional level of the Poxyn10A gene in the Δmtr23E mutant reduced to 25.7% of the WT.The transcriptional levels of the Poxyn10A gene remained essentially unchanged in the Δmtr23E/F/H and Δmtr25A mutants (Figure 5C).The results generally support the determination of cellulolytic enzyme production in Figure 4; the higher the transcription of cellulolytic genes, the higher cellulolytic enzyme production, and vice versa.

Some Methyltransferases Affected the Expression of Transcription Factors
Cellulolytic gene transcription is regulated by the cooperative action of numerous transcription factors (TFs), including activators and repressors.The activator XlnR and repressor CreA are key dose-dependent regulators of cellulolytic gene transcription [4,28].We assessed the transcriptional levels of PoxlnR and PocreA to determine whether the variations in cellulolytic gene expression in each mutant were affected by the transcriptional levels of PoXlnR and PoCreA.
Only the Δmtr23D and Δmtr23G mutants displayed elevated PoxlnR gene expression; these mutants showed 6.2-and 15.3-fold increases in PoxlnR gene expression compared to WT.The PoxlnR gene expression levels in other mutants were similar to the WT (Figure Poeg1 gene expression changes in each mutant follow a pattern similar to how Pocbh1 gene expression changes in each mutant.Compared to the WT, the expression of the Poeg1 gene was significantly increased in the ∆mtr23C/D/G and ∆mtr25A mutants but significantly decreased in the ∆mtr23E and ∆mtr25B mutants and remained essentially unchanged in the ∆mtr23F and ∆mtr25H mutants.For example, at the 24th hour of cultivation, the transcriptional levels of the Poeg1 gene in ∆mtr23C/D/G and ∆mtr25A mutants were 5.1, 19.3, 97.0, and 12.8 times higher than those in the WT, respectively.The transcriptional levels of the Poeg1 gene in the ∆mtr23E and ∆mtr25B mutants reduced to 14.0% and 8.1% of the WT, respectively (Figure 5B).
Compared to Pocbh1 and Poeg1, of which transcriptional levels varied dramatically in several mutants, the expression change of Poxyn10A in the corresponding mutants was less pronounced.At the 24th hour of cultivation, the expression levels of the Poxyn10A gene in ∆mtr23C/D/G mutants were 2.8, 4.0, and 11.7 times higher than those in the wild strain, respectively.The transcriptional level of the Poxyn10A gene in the ∆mtr23E mutant reduced to 25.7% of the WT.The transcriptional levels of the Poxyn10A gene remained essentially unchanged in the ∆mtr23E/F/H and ∆mtr25A mutants (Figure 5C).The results generally support the determination of cellulolytic enzyme production in Figure 4; the higher the transcription of cellulolytic genes, the higher cellulolytic enzyme production, and vice versa.

Some Methyltransferases Affected the Expression of Transcription Factors
Cellulolytic gene transcription is regulated by the cooperative action of numerous transcription factors (TFs), including activators and repressors.The activator XlnR and repressor CreA are key dose-dependent regulators of cellulolytic gene transcription [4,28].We assessed the transcriptional levels of PoxlnR and PocreA to determine whether the variations in cellulolytic gene expression in each mutant were affected by the transcriptional levels of PoXlnR and PoCreA.
Only the ∆mtr23D and ∆mtr23G mutants displayed elevated PoxlnR gene expression; these mutants showed 6.2-and 15.3-fold increases in PoxlnR gene expression compared to WT.The PoxlnR gene expression levels in other mutants were similar to the WT (Figure 6A).The PocreA gene expression increased in the ∆mtr23G mutant, about 2.6 times higher than in the WT.In the ∆mtr25A mutant, the PocreA gene expression level decreased to 23.7% of WT.PocreA expression levels in other mutants were similar to WT (Figure 6B).

Preliminary Characterization of the Protein PoMtr25A
Excluding the previously characterized PoLaeA and PoMtr23B [13,16,18], among the remaining eight genes (Pomtr23C/D/E/F/G/H and Pomtr25A/B), the transcription level of Pomtr25A gene is the highest under four of the five detection conditions (i.e., qPCR, transcriptome analysis under glucose, starch, and cellulose) (Figure 2A-D), and the second highest under one condition (transcriptome analysis under cellulose plus wheat bran) (Figure 2E).Even though that high transcriptional level does not necessarily indicate a gene's functional importance, PoLaeA and PoMtr23B, which have high transcriptional levels, do play significant regulatory roles.Therefore, we further characterized PoMtr25A, which has a high transcriptional level, including its subcellular localization and identification of its putative interacting proteins.
We fused the PoMtr25A-coding sequence with the GFP sequence and introduced it to the WT to obtain the strain Mtr25A-GFP.The overlap of green fluorescence (green dots) and nuclear staining (blue dots) on the merged image indicated the nuclear localization of PoMtr25A in P. oxalicum (Figure 7A).
In A. nidulans, AnLaeA and the two LLMs AnLlmF and VipC (VipC is AnLlmB) have respective interactions with VeA, a velvet domain protein, and play a role in regulating fungal development and secondary metabolism via the interaction [10].Therefore, the TAP coupled MS (TAP-MS) method was used to identify PoMtr25A putative interacting

Preliminary Characterization of the Protein PoMtr25A
Excluding the previously characterized PoLaeA and PoMtr23B [13,16,18], among the remaining eight genes (Pomtr23C/D/E/F/G/H and Pomtr25A/B), the transcription level of Pomtr25A gene is the highest under four of the five detection conditions (i.e., qPCR, transcriptome analysis under glucose, starch, and cellulose) (Figure 2A-D), and the second highest under one condition (transcriptome analysis under cellulose plus wheat bran) (Figure 2E).Even though that high transcriptional level does not necessarily indicate a gene's functional importance, PoLaeA and PoMtr23B, which have high transcriptional levels, do play significant regulatory roles.Therefore, we further characterized PoMtr25A, which has a high transcriptional level, including its subcellular localization and identification of its putative interacting proteins.
We fused the PoMtr25A-coding sequence with the GFP sequence and introduced it to the WT to obtain the strain Mtr25A-GFP.The overlap of green fluorescence (green dots) and nuclear staining (blue dots) on the merged image indicated the nuclear localization of PoMtr25A in P. oxalicum (Figure 7A).

Discussion
LaeA/Lae1 and LaeA-like methyltransferases (LLMs) play essential roles in fungal growth, development, virulence, and the production of secondary metabolite and cellulolytic enzymes in various fungi [9][10][11][12][13][17][18][19].As P. oxaliucm is a typical fungus that produces cellulolytic enzymes, we focused on the functions of methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) in cellulolytic enzyme gene expression.In different methyltransferase gene deletion mutants, higher cellulolytic gene transcription resulted in a higher production of cellulolytic enzymes, and vice versa.The results were expected because cellulolytic enzyme production in fungi is always regulated at a In A. nidulans, AnLaeA and the two LLMs AnLlmF and VipC (VipC is AnLlmB) have respective interactions with VeA, a velvet domain protein, and play a role in regulating fungal development and secondary metabolism via the interaction [10].Therefore, the TAP coupled MS (TAP-MS) method was used to identify PoMtr25A putative interacting proteins.The final elution of TAP was divided into three parts: one part for Western blot verification, one part for 12.5% SDS-PAGE and silver staining, and one part was assayed via LC-MS/MS to determine the putative interacting proteins of the PoMtr25A bait, respectively.Western blot analysis indicated the existence of the PoMtr25A bait (Figure 7B).One specific band was found between the control (WT) and Mtr25A-TAP from the gel of SDS-PAGE (Figure 7C).The band (Figure 7C, Black arrow, approximately 32~35 kDa) was cut from the gel and identified via LC/MS-MS as PoMtr25A (theoretical MW: 32.1 kDa).The third part of the eluent was analyzed via LC-MS/MS to identify the PoMtr25A bait and its putative interacting proteins.The proteins in Mtr25A-TAP triplicates but not in any controls are listed in Supplementary Spreadsheet S2.The proteins were ranked by their exponentially modified protein abundance index (emPAI) [33], which was used to estimate the absolute protein amount.
Among the proteins identified via LC-MS/MS, it is expected that PoMtr25A is the top protein according to emPAI.In addition to PoMtr25A itself as the bait, PDE_02916, an ortholog of Saccharomyces cerevisiae 60S ribosomal protein L20 (66.7% identity) exhibited the highest emPAI.PDE_01194, an ortholog of Schizosaccharomyces pombe glutathione Stransferase Gst2 (40.2% identity), and PDE_09363, an ortholog of catalytic subunit SNF2 (56.7% identity) of S. cerevisiae SWI/SNF chromatin-remodeling complex exhibited the second and the third-ranking emPAI, respectively.Other proteins were only detected in one of the Mtr25A-TAP triplicates, so they were not considered credible.

Discussion
LaeA/Lae1 and LaeA-like methyltransferases (LLMs) play essential roles in fungal growth, development, virulence, and the production of secondary metabolite and cellulolytic enzymes in various fungi [9][10][11][12][13][17][18][19].As P. oxaliucm is a typical fungus that produces cellulolytic enzymes, we focused on the functions of methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) in cellulolytic enzyme gene expression.In different methyltransferase gene deletion mutants, higher cellulolytic gene transcription resulted in a higher production of cellulolytic enzymes, and vice versa.The results were expected because cellulolytic enzyme production in fungi is always regulated at a transcriptional level.However, there may be differences in the mechanisms by which these methyltransferases regulate the transcription of cellulolytic enzyme genes.
The eight mutants displayed identical colony morphologies to those of the WT when they were cultivated on VMG or VMGly agar (Figure 3A).The results showed that the normal metabolism of glucose and glycerol in cells was unaffected by the lack of each protein; glucose and glycerol can be used by cells in a normal manner.∆mtr23G was the only mutant that displayed a clearer cellulolytic halo around the colony than did the WT.This result is consistent with the highest FPA and CMCase results shown by ∆mtr23G in liquid culture (Figure 4A,B).Interestingly, despite having high FPA in liquid culture, the ∆mtr23C and ∆mtr23D mutants did not exhibit cellulolytic halo around the colony.This result may be due to the different enzyme production patterns of fungi grown under liquid and solid culture conditions.For example, glucoamylase A and xylanase G2 secreted by Aspergillus oryzae are specifically produced in liquid culture while rarely detected in solid culture [34].The ability of ∆mtr23C and ∆mtr23D to synthesize significant amounts of cellulolytic enzyme in solid culture is unknown.
Four mutants (∆mtr23C/D/G and ∆mtr25A) showed a significant increase in cellulolytic gene expression compared to the WT.For the ∆mtr25D mutant, the high transcription level of PoxlnR gene should be one reason for the increased transcription of its cellulolytic enzyme gene, as TF XlnR is not only the key transcriptional activator of cellulolytic and xylanolytic genes in P. oxalicum [28] but its orthologs have also been reported positively correlating with the production of cellulase and xylanase in various fungi, such as T. reesei, N. crassa and A. niger [4,35].For the ∆mtr25A mutant, the downregulation of the PocreA gene may be the main reason for the upregulation of cellulolytic gene transcription since CreA, a TF involved in carbon catabolite repression (CCR), is a crucial repressor of cellulolytic enzyme gene [36].The low transcriptional levels or reduced activity of CreA (via mutation/disruption/deletion) can activate the expression of extracellular glycoside hydrolase (e.g., cellulases, xylanases, and amylases) genes in various fungi [37][38][39][40].
The production of cellulolytic enzymes was highest in the ∆mtr25G, the only mutant with a cellulolytic halo surrounding the fungal colony.Interestingly, the transcription levels of activator XlnR and repressor CreA genes in the ∆mtr25G mutant were significantly increased compared to WT.The dose-controlled "seesaw model" could explain the result, in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.In other words, the expression of a cellulolytic gene is dependent on the presence of the TFs and severely dependent on the TFs dose effects of transcript abundances [28].The increase (~15.3 folds) in the transcriptional level of the activator XlnR gene was significantly higher than that (~2.6 folds) of the repressor CreA gene in the ∆mtr25G mutant; the activation effect dominates.
Another interesting discovery is that, after four days of culture, the FPA of the ∆mtr23G mutant remained high, while the FPA of the other seven mutants declined, indicating the degradation of cellulolytic enzyme.This result could be because the patterns of autolytic enzyme production vary among mutants.With the extension of cultivation time, nutrition starvation-induced fungal autolysis is associated with increased autolytic enzymes such as proteases, glucanases, and chitinases.The degradation of protein products was often observed during this active process of self-digestion [41,42].It has been found that different LLMs have different roles in regulating protease and chitinase production.Both ∆PolaeA and ∆mtr23B deletion mutants showed a decrease in protease production; while the δPolaeA mutant displayed decreased chitinase production, the ∆mtr23B mutant displayed higher chitinase production [16,18].Therefore, it is possible that the ∆mtr23G mutant, more than other mutant strains, delays or reduces protease synthesis, which delays or decreases cellulase degradation.
Neither the ∆mtr23C mutant with high cellulolytic gene expression nor the mutants (∆mtr23E and ∆mtr25B) with low cellulolytic gene expression exhibited significant differences in the expression levels of the PoxlnR and PocreA genes compared with the WT.Evidently, PoMtr23C, PoMtr23E, and PoMtr25B do not regulate cellulolytic genes through TF PoXlnR and PoCreA.Except for XlnR and CreA, many other TFs intricately regulate the expression of the cellulolytic gene.In P. oxalium, along with XlnR and CreA, TFs ClrB and AraR can positively regulate the expression of cellulolytic genes directly, while TF AmyR negatively does so in an indirect way [28,30,43].More TFs, including activators Ace2/3/4, BglR, Clr1/4, Hac1, Vib1, and repressors Ace1 and Rce1, have been discovered to be linked to the transcription of cellulolytic genes in various fungi [4,35].The absence of LaeA can lead to changes in the abundance of multiple TFs.For example, the lack of Lae1 in M. thermophila caused a significant reduction in the expression of Cre1, Clr-1, Hac1, and Vib-1 and an increase in the expression of AmyR and Ace1 [15].According to transcriptome study, P. oxalicum PoLaeA and PoMtr23B affect the expression of 21.3% (2138 of 10,021 genes) and 14.1% (1415 of 10,021 genes) of the genome, respectively [13,18].Lae1/LaeA also affect the expression of 11.7% (1069 of 9123 genes) and 9.8% (943 of 9626 genes) of the genome in T. reesei and A. fumigatus, respectively [44,45].In addition to TFs, other regulators, such as cellodextrin transporters and heterotrimeric G-protein [4,46,47], which are also linked to the transcription of cellulase genes, are present among the genes regulated by LaeA and LLms.Most orthologs of these regulators have been found in the genome of P. oxalicum.Therefore, PoMtr23C, PoMtr23E, and PoMtr25B may affect TFs other than XlnR and CreA or other regulators to affect the transcription of cellulolytic genes.
The proteins that interact with LaeA and LLMs must also be considered.A. nidulans AnLaeA and AnLlmF interact directly with VeA, a fungus-specific velvet domain protein [17,48].Vel1, the T. reesei orthologue of A. nidulans VeA, is a positive regulator of the cellulolytic gene.The deletion of the vel1 gene completely impaired the expression of cellulases, xylanases, and the transcription activator Xyr1 (the ortholog of XlnR) on a cellulase-inducing medium [49].The vel1 overexpression enhanced the production of cellulases and xylanases [50]. A. nidulans VeA interacts with at least four methyltransferases, including AnLaeA, AnLlmF, and the methyltransferase heterodimers VipC-VapB (VipC is LlmB), but not all methyltransferases interact with VeA [10].P. oxaliucm PoLaeA interacted with PoVeA, whereas PoMtr23B did not [18].PoVeA was not discovered in the PoMtr25A interacting proteins discovered by TAP-MS in this study either.Interestingly, SNF2, the catalytic subunit of the SWI/SNF chromatin-remodeling complex, was found among the putative proteins interacting with PoMtr25A.The SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin-remodeling complex critical in coordinating chromatin architecture and gene expression [51].T. reesei activator Xyr1 recruits the SWI/SNF complex to remodel chromatin at cellulase gene promoters, activating cellulase gene expression [52].Therefore, PoMtr25A may affect not only the transcription of CreA but also transcription by recruiting SWI/SNF complexes that affect chromatin structure and thereby regulate the transcription of target genes.However, as the putative interacting proteins identified via TAP-MS might include those that indirectly interact with PoMtr25A as mediated via other proteins, actual physical interactions between PoMtr23B and SNF2 need to be further explored in order that the mechanism underlying the regulation of specific gene expression via Mtr23B can be explained.
For the mutants ∆mtr23F and ∆mtr23H, neither fungal growth, spore production, nor cellulase production were found to be significantly different from the WT.Similarly, the absence of the AnLlmD (the ortholog of PoMtr23F) and AnLlmI (the ortholog of PoMtr23H) in A. nidulans did not lead to any noticeable phenotypic changes [17].Additionally, considering that their transcriptional levels were constantly low under several conditions, we preferred that Pomtr23F and Pomtr23H might be redundant genes in the genome.
This study showed that different methyltransferases have different effects on the expression patterns of cellulolytic genes from different mechanisms, indicating their functional differentiation.According to previous studies, different LLMs demonstrated significant differentiation via various mechanisms in their effects on fungal growth, development, and SM formation, in addition to controlling the expression of cellulolytic genes.The absence of P. oxalicum PoLaeA and PoMtr23B both alter colony morphology.However, the underlying mechanisms are inconsistent: PoMtr23B primarily regulates conidial pigment formation, whereas PoLaeA is crucial for conidiation but has little impact on conidial pigment formation [18]. A. nidulans AnLaeA, localized in the nucleus, positively regulates sterigmatocystin production [9], whereas AnLlmF, located in the cytoplasm, negatively regulates sterigmatocystin production via mediating the subcellular localization of VeA [17].Three LLMs from the P. ostreatus, designated PoLaeA1, PoLaeA2, and PoLaeA3, also demonstrated functional differentiation.Silencing PoLaeA1 reduced the intracellular polysaccharide, whereas silencing PoLaeA2 or PoLaeA3 did not.Silencing PoLaeA2 and PoLaeA3 decreased cellulase production by interfering with intracellular Ca 2+ signaling, whereas silencing PoLaeA1 did not [21].Therefore, future research on LLMs may contribute to the broader understanding of the molecular mechanisms involved in fungal growth, development, synthesis of secondary metabolites, and cellulolytic enzyme production.

Conclusions
The biological roles of eight LaeA-like proteins were investigated in P. oxalicum.The absence of each protein has different patterns in cellulolytic gene transcription and cellulolytic enzyme production.The underlying mechanisms are also different.This study can improve our knowledge of the role of the LaeA-like protein in fungal development, growth, and cellulolytic enzyme production.

Figure 1 .
Figure 1.Domain architecture and phylogenetic analysis of P. oxalicum LaeA and putative methyltransferases.(A) Domain architecture analysis.The maps were constructed with equa proportions of the respective sequences according to the results of SMART and Pfam analyses.(B Phylogenetic analysis.The tree is drawn to scale, with branch lengths in the same units as those o the evolutionary distances used to infer the phylogenetic tree.Blue fonts represent the proteins from P. oxalicum.Green fonts represent the proteins from A. nidulans.

Figure 1 .
Figure 1.Domain architecture and phylogenetic analysis of P. oxalicum LaeA and putative methyltransferases.(A) Domain architecture analysis.The maps were constructed with equal proportions of the respective sequences according to the results of SMART and Pfam analyses.(B) Phylogenetic analysis.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.Blue fonts represent the proteins from P. oxalicum.Green fonts represent the proteins from A. nidulans.

Figure 2 .
Figure 2. Transcriptional levels of ten methyltransferase genes.The results via RT-qPCR (A), and the results of transcriptional profiling when P. oxalicum was cultivated on different carbon sources,

Figure 3 .
Figure 3. Phenotypic analysis of WT and the mutants.(A) Colony morphology of four-day-old cultures on Vogel's medium agar with 2% glucose (VMG), 2% glycerol (VMGly), or 1% ball-milled microcrystalline cellulose (VMC) at 30 • C. (B) Levels of conidiation on VMG agar.Plates were incubated at 30 • C for 5 days, and 0.5 mm diameter colony agar plugs in triplicate were sampled for each strain.The number of conidia was counted with a hemocytometer.(C) The biomass determination.The error bars indicate standard deviations.** p < 0.01.

Figure 4 .
Figure 4. Cellulolytic activity of WT and various mutants.The strains were cultivated in liquid Vogel's salts supplemented with 1% wheat bran and 1% microcrystalline cellulose as carbon resources to induce cellulolytic enzyme synthesis.(A) FPA, (B) CMCase activity, (C) xylanase activity.

Figure 4 .
Figure 4. Cellulolytic activity of WT and various mutants.The strains were cultivated in liquid Vogel's salts supplemented with 1% wheat bran and 1% microcrystalline cellulose as carbon resources to induce cellulolytic enzyme synthesis.(A) FPA, (B) CMCase activity, (C) xylanase activity.

Figure 7 .
Figure 7. Assay of subcellular localization and TAP for PoMtr25A.(A) Subcellular localization of PoMtr25A.The top, green fluorescence (green dots); the second from the top, white light; the third from the top, nucleus staining using Hoechst 33342 (blue dots); the bottom, merged, white dots indicate the location that showed green fluorescence and nucleus staining and the overlap of green dots and blue dots on the merged image.Western blot analysis (B) and silver staining (C) of TAPtagged PoMtr25A protein together with putative-associated proteins after two steps of affinity purification.The specific band between the control (WT) and Mtr25A-TAP was cut from the gel and identified as PoMtr25A (Black arrow, approximately 32~35 kDa, theoretical MW: 32.1 kDa).

Figure 7 .
Figure 7. Assay of subcellular localization and TAP for PoMtr25A.(A) Subcellular localization of PoMtr25A.The top, green fluorescence (green dots); the second from the top, white light; the third from the top, nucleus staining using Hoechst 33342 (blue dots); the bottom, merged, white dots indicate the location that showed green fluorescence and nucleus staining and the overlap of green dots and blue dots on the merged image.Western blot analysis (B) and silver staining (C) of TAP-tagged PoMtr25A protein together with putative-associated proteins after two steps of affinity purification.The specific band between the control (WT) and Mtr25A-TAP was cut from the gel and identified as PoMtr25A (Black arrow, approximately 32~35 kDa, theoretical MW: 32.1 kDa).