The ubiquitin-conjugating enzyme TrUbc4 contributes to cellulase gene expression

The yeast one-hybrid screen was performed using a cDNA expression library prepared from T. reesei under cellulase-inducing conditions [54]. Since cellobiohydrolase I (CBH1) is the main enzymatic component that is highly induced upon induction, a cbh1 promoter region (–474 to –838 bp) was applied to drive the expression of an Aureobasidin A (AbA) resistance gene AUR1-C for screening for additional transcriptional regulators. Six out of approximately 10⁵ transformants were obtained from the selective plates containing AbA. One clone contained a cDNA sequence encoding a 147‑amino‑acid polypeptide with an ATG start codon and was revealed to correspond to a ubiquitin-conjugating enzyme (E2) belonging to the Ubc4/5 subfamily (hereafter named TrUbc4, GenBank: XP_006968852.1, Tr123773) (S1A Fig). The E2 activity of TrUbc4 was determined by an in vitro Ub conjugation assay, wherein the formation of the TrUbc4-Ub complex as well as the ubiquitin dimer were observed when ATP, E1, Ub, and GST-TrUbc4 were simultaneously available (S1B Fig).

Repeated efforts to obtain the Trubc4 null mutant (ΔTrubc4) to see whether Trubc4 affects the induced cellulase gene expression failed, implicating that Trubc4 is likely an essential gene for fungal growth. We therefore applied an alternative strategy by replacing the Trubc4 promoter with a copper-responsive promoter tcu1, which represses gene expression in the presence of copper while leading to constitutively high expression without addition of copper [40]. While growth of the resultant P_tcu1-Trubc4 strain showed slightly reduced mycelium diameter and density on glucose plate without copper, hardly any growth difference was observed in liquid glucose media regardless of Trubc4 repression or overexpression compared to the control strain QM9414 (S2A and S2B Fig). Analyses of the extracellular (hemi)cellulase activities revealed that repression of Trubc4 exhibited a pronounced reduction (approximately 65%) in pNPC (p-nitrophenyl-β-D-cellobioside), pNPG (p-nitrophenyl-β-D-glucopyranoside), CMC (carboxymethyl cellulose sodium salt) and xylan hydrolytic activities compared to QM9414 (Figs 1A and S2C and S2D). Further quantitative RT-PCR analyses revealed that Trubc4 repression resulted in a significant down-regulation of main cellulase genes (cbh1, eg1, and bgl1) compared with the control strain QM9414 cultured on 1% Avicel (Figs 1B and S2E). Contrary to Trubc4 repression, constitutively enhanced expression of Trubc4 without copper led to hardly any effect on the extracellular cellulase activity of P_tcu1-Trubc4 compared to the control strain QM9414 (Figs 1A and S2C).

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Fig 1. Repression of Trubc4 compromised the induced cellulase gene expression.

(A) Extracellular pNPC hydrolytic activity of P_tcu1-Trubc4, as well as P_tcu1-Trubc4 complemented with the TrUbc4 (ReTrUbc4) or C85A mutant (ReC85A) at the pyr4 locus under the control of the tef1 promoter, cultured on 1% Avicel with or without copper. (B) Quantitative RT-PCR analyses of the transcription of the cbh1 gene in the P_tcu1-Trubc4 strain cultured on 1% Avicel with adding copper to repress Trubc4. (C) Extracellular pNPC hydrolytic activity of the P_tcu1-Trubc4 strain overexpressing xyr1 by the cdna1 promoter (OExyr1-P_tcu1-Trubc4) cultured on 1% Avicel (left) or 1% glucose (right) with added copper to repress Trubc4. Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g001

Since Blast analysis performed using the S. cerevisiae Ubc4 as a query retrieved a total of 19 other putative E2 enzymes in the T. reesei genome (S3A Fig), we individually knocked out these E2 genes in T. reesei. Unlike Trubc4, none except a Ubc8 mutant ΔTr65553 showed a remarkable decrease in the extracellular pNPC hydrolytic activity (S1 Table).

To verify the functional involvement of TrUbc4 as a ubiquitin-conjugating enzyme in regulating T. reesei cellulases, its evolutionarily conserved catalytic cysteine at 85 was substituted for alanine [55], and the C85A mutant was introduced back into P_tcu1-Trubc4 to generate the ReC85A strain. In contrast with the wild-type TrUbc4 complemented ReTrubc4 strain, ReC85A failed to restore cellulase activity when copper was added to repress the endogenous Trubc4 expression (Figs 1A and S2C), indicating that the E2 enzymatic activity of TrUbc4 is essential for its function in regulating cellulase gene expression. Notably, the extracellular pNPC hydrolytic activity of the ReC85A strain was comparable with that of the wild-type strain without addition of copper. This result therefore suggest that even with the expression of C85A mutant TrUbc4, the overexpressed wild-type TrUbc4 is sufficient to maintain the acitivty to ensure the induced cellulase gene expression (Fig 1A).

Since there exists a strict correlation between the transcription level of the critical transcriptional activator gene xyr1 and cellulase genes [40,56], we enhanced xyr1 expression to see whether it could rescue the induction defect due to the downregulation of Trubc4. The resulting strain with xyr1 being expressed from a strong constitutive promoter cdna1 (Tr110879) exhibited a full restoration of cellulase production under either glucose-repressing or Avicel-inducing conditions (Fig 1C). Altogether, these results suggest that TrUbc4 may function in a ubiquitination cascade to modulate Xyr1-mediated cellulase gene expression.

TrUbc4 is a nuclear protein that requires a putative RING E3 complex to influence cellulase gene expression

To determine the subcellular localization of TrUbc4 in T. reesei, green fluorescent protein (GFP) fused to the N-terminus of TrUbc4 was expressed under the control of the tcu1 promoter. The fused GFP did not interfere with TrUbc4 function (S3B Fig), and GFP fluorescence was readily observed in the nucleus that well merged with signals stained by DAPI when the conidia were germinated on glucose (Fig 2A). This nuclear recruitment of TrUbc4 remained unchanged when the conidia were shifted from non-inducing (glucose) to inducing (Avicel) conditions.

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Fig 2. TrUbc4 localizes to the nucleus and interacts with TrRbx1.

(A) Fluorescence microscopy analysis of the subcellular localization of the P_tcu1-gfp-Trubc4 strain cultured on glucose or Avicel conditions. (B) Extracellular pNPC activity of P_tcu1-Trubc4, as well as P_tcu1-Trubc4 complemented with the TrUbc4 (ReUbc4) or F62A/A96D (ReF62A/A96D) mutant at the pyr4 locus under the control of the tef1 promoter, cultured on 1% Avicel with copper. (C) Protein interaction analysis of TrUbc4 or F62A/A96D mutant and TrRbx1 using yeast two-hybrid assay. Yeast cells harboring the indicated combinations of plasmids were plated on DDO lacking leucine and tryptophan or TDO lacking leucine, tryptophan, and histidine but containing 0.1 mM 3- amino-1,2,4-triazole (3AT). The P53/Large T combinations were used as positive. Tenfold serially diluted cell cultures were inoculated on each spot. Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g002

Given that Phe62 and Ala96 in human UBC4 family have been shown to be critical for its specific interaction with RING E3 ligases [57,58], mutations of the corresponding amino acids in TrUbc4 were made and their effect on cellulase gene expression was evaluated. The introduction of the corresponding TrUbc4 mutant (F62A/A96D) into the P_tcu1-Trubc4 strain (ReF62A/A96D) hardly affected the growth (S2B Fig), but only partially rescued the phenotypic defect in cellulase production with Trubc4 repression (Fig 2B). Moreover, a direct weak interaction was detected between TrUbc4 and TrRbx1, which is the only annotated T. reesei homolog (Tr121950) to the RING-finger subunit Rbx1 of the SCF complex (Fig 2C). The interaction was, however, almost abolished with the F62A/A96D double mutations in TrUbc4. Overall, these results strongly suggest that an active TrUbc4-RING E3 complex is involved in controlling the cellulase gene expression in T. reesei.

Nuclear-localized F-box protein TrFwd1 participates in regulating cellulase gene expression

Given the subcellular localization of TrUbc4, we focused on E3 ligases in T. reesei with putative nuclear localization signals predicted by NLStradamus (http://www.moseslab.csb.utoronto.ca/NLStradamus/) [59]. In silico analysis predicted a total of 103 E3 ligases in T. reesei genome, among which 44 were predicted to contain a putative nuclear localization signal (S2 Table). Homologous recombination-mediated gene knockout or copper-inducible promoter replacement strains was successfully constructed for 11 of the first 13 randomly selected E3 ligase genes. Phenotypic screening was then performed by determining the extracellular pNPC hydrolytic activity on 1% Avicel. This primary screening revealed that the absence of an F-box protein, TrFwd1 (GenBank accession: XP_006966975.1; gene ID: Tr79756), resulted in a phenotype similar to that observed under Trubc4 repression (Fig 3A). TrFwd1 shares 48% identity with N. crassa Fwd1 and contains a conserved F-box domain at the N-terminus along with six WD40 repeats at the C-terminus (S4A Fig), supporting its classification as an integral component of a putative SCF E3 ubiquitin ligase complex. Phylogenetic analysis further indicated that TrFwd1 clusters with orthologs from various other filamentous fungi (S4B Fig).

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Fig 3. Deletion of Trfwd1 compromised the induced cellulase gene expression which was resotred with xyr1 overexpression.

(A) Extracellular pNPC hydrolytic activity of the ΔTrfwd1 strain as well as the deletion strain complemented with GFP-TrFwd1 (P_gpd1-gfp-Trfwd1) or GFP-TrFwd1 without F-box domain (P_gpd1-gfp-Trfwd1-ΔF) cultured on 1% Avicel. (B) Quantitative RT-PCR analyses of the cbh1 gene in the Trfwd1 knockout strain cultured on 1% Avicel. (C) Extracellular pNPC hydrolytic activity of the Trfwd1 knockout strain with xyr1 overexpression driven by tcu1 promoter (OExyr1-ΔTrfwd1) cultured on 1% Avicel (left) or 1% glucose (right). Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g003

While the hyphal growth of ΔTrfwd1 was reduced regarding mycelium diameter and density compared to that of the control strain QM9414 on MM glucose plate, no apparent difference was observed in liquid glucose medium (S5A and S5B Fig). However, unlike repression of Trubc4, ΔTrfwd1 produced hardly any conidia on malt extract medium (S5C Fig). Under cellulase-inducing or xylanase-inducing conditions, the extracellular pNPC, pNPG, CMC and xylan hydrolytic activities in ΔTrfwd1 were decreased by approximately 80% relative to QM9414 (Figs 3A and S6A and S2D). Quantitative RT-PCR analyses further revealed a significant reduction in the transcription of key cellulase genes (cbh1, eg1, and bgl1) in ΔTrfwd1 compared to that of the control strain QM9414 (Figs 3B and S6B). Similar to the repression of Trubc4, Xyr1 overexpression fully restored cellulase production under either glucose-repressing or Avicel-inducing conditions with Trfwd1 deletion (Fig 3C). All the data therefore suggest that TrUbc4 couples with TrFwd1 to target a specific factor by ubiquitination to effect the key transcriptional activator-mediated cellulase gene expression.

Analysis of the subcellular localization of TrFwd1 by expressing an N-terminal GFP-tagged TrFwd1 under the constitutive promoter gpd1 (Tr119735) at Trfwd1 locus in ΔTrfwd1 revealed that GFP-TrFwd1 was fully functional in complementing ΔTrfwd1 regarding defects in conidiation and cellulase production (Figs 3A, S5A, S5B, S5C, and S6A), and that the GFP fluorescence was predominantly localized to the nucleus under both glucose and Avicel conditions (Fig 4A). In contrast with GFP-TrFwd1, a corresponding strain expressing a TrFwd1 lacking the F-box domain (GFP-TrFwd1-ΔF) failed to restore conidiation and exhibited only a marginal recovery (~10%) in extracellular pNPC hydrolytic activity compared to ΔTrfwd1 (Figs 3A, S5A, S5B, S5C, and S6A). Although GFP-TrFwd1-ΔF was similarly nuclear-localized, the detected fluorescence signal was much weaker than the full-length GFP-TrFwd1 (Fig 4A). Western blot analysis of the immunoprecipitation (IP) by GFP-Trap beads demonstrated that while GFP-TrFwd1 was readily detected, GFP-TrFwd1-ΔF was observed at markedly reduced levels (S6C Fig), indicating that the F-box domain is critical for TrFwd1 function, likely because it mediates the proper assembly of TrFwd1 into the SCF ubiquitin ligase complex, and its absence would destabilize the protein in its free-form.

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Fig 4. TrFwd1 localizes to the nucleus and is a component of a putative SCF complex.

(A) Fluorescence microscopy analysis of the cellular localization of P_gpd1-gfp-Trfwd1 and P_gpd1-gfp-Trfwd1-ΔF under glucose or Avicel conditions. (B) Protein interaction analysis of TrFwd1 and TrSkp1, TrRbx1 and TrCul1, TrCul1 and TrSkp1, using yeast two-hybrid assay. Yeast cells harboring the indicated combinations of plasmids were plated on DDO lacking leucine and tryptophan or TDO lacking leucine, tryptophan, and histidine but containing 0.5 mM 3AT. The P53/Large T combinations were used as positive. Tenfold serially diluted cell cultures were inoculated on each spot.

https://doi.org/10.1371/journal.pgen.1012216.g004

Similar results were obtained for a TAP (tandem affinity purification) tagged TrFwd1 or TrFwd1-ΔF fusing with 5 × Myc-6 × His at its N-terminus to avoid any potential interference from GFP (S7A and S7B Fig). Furthermore, a yeast two-hybrid assay revealed that TrFwd1 specifically interacted with the adaptor TrSkp1 (Tr73823) of the SCF complex whereas this interaction was abolished upon deletion of the F-box domain (Fig 4B). Possibility that the instability of the mutant may account for this loss of interaction can not be excluded. Collectively, these results indicate that the F-box domain is critical for both the stability and normal functions of TrFwd1.

To further understand the regulatory influence of TrFwd1 on the induced cellulase biosynthesis, protein interaction studies using a cross-linking-based IP with the Myc-His-tagged TrFwd1 were performed followed by SDS-PAGE and silver staining. While DSP (dithiobis/succinimidylpropionate) crosslinking resulted in protein bands to be predominantly accumulated at the top of the gel, indicating the formation of high-molecular-weight complexes, decrosslinking resulted in the appearance of multiple distinct bands (S7C Fig). Mass spectrometry analysis of the crosslinked protein bands identified TrFwd1 as the most abundant protein in the complex compared to the control immunoprecipitation. Additionally, core SCF components including TrCul1 (Tr55706) and TrSkp1 as well as multiple subunits of the COP9 signalosome such as the catalytic subunit TrCsn5 (Tr62003) that are known to modulate the SCF complex activity, were also detected in the TrFwd1-tagged strain (S3 Table). Further yeast two-hybrid assays confirmed that TrCul1 directly interacted with both TrSkp1 and TrRbx1, supporting the presence of an integral SCF complex harboring TrFwd1 (Fig 4B).

To assess the functional involvement of the identified key subunits of the putative SCF complex in T. reesei cellulase gene expression, we constructed the promoter replacement strains for Trcul1, Trskp1, and Trrbx1 with the tcu1 promoter, respectively, along with a knockout mutant of Trcsn5. Whereas repressing Trskp1 and Trcul1 but not Trrbx1 significantly impaired strain growth, hardly any growth defect was observed with their overexpression in the absence of copper for all the indicated strains except that Trcul1 overexpression reduced the growth on malt extract plates. Deletion of Trcsn5 had no observable effect on growth (S8A and S8B Fig). Extracellular pNPC hydrolytic activity under cellulase-inducing conditions decreased by approximately 75% with Trcul1 and Trskp1 repression as well as deletion of Trcsn5, but by only 20% when repressing Trrbx1 (S8C Fig). These findings underscore the important regulatory roles of a putative nuclear T. reesei SCF complex in fungal growth and cellulase gene expression.

Cre1 is identified as a direct interacting substrate for TrFwd1

To investigate whether the altered transcription of xyr1 or other relevant transcriptional factors contributes to the observed changes in cellulase gene expression with Trubc4 repression or Trfwd1 deletion, quantitative RT-PCR analyses were performed and revealed that the transcript levels of either xyr1 or the other main repressor genes including cre1 and ace1 remained largely unchanged under cellulase-inducing conditions (S9A Fig), suggesting that the putative ubiquitination cascade may not act by altering the transcription of these core regulators, but rather target one of them to modulate its function.

Given the well-documented implication of various components involved in putative ubiquitination and de-ubiquitination targeting CreA to control CCR in Aspergillus [49,50,52,53], we set out to focus on T. reesei Cre1. A yeast two-hybrid assay was first employed to test possible physical interactions between TrFwd1 and several important transcriptional factors including Xyr1, Cre1, or Ace1. Whereas TrFwd1 did not seem to interact with Xyr1 or Ace1, a specific interaction was observed with Cre1 (Figs 5A, S9B and S9C), suggesting that Cre1 is a potential ubiquitination substrate of the identified SCF^{TrUbc4-TrFwd1} complex.

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Fig 5. Cre1 is a regulatory target of the TrUbc4-TrFwd1-mediated ubiquitination cascade.

(A) Protein interaction analysis of TrFwd1 and Cre1 using yeast two-hybrid assay. Yeast cells harboring the indicated combinations of plasmids were plated on TDO lacking leucine, tryptophan, and histidine, and supplemented with 0.5 mM 3-AT. The P53/Large T combinations were used as positive. Tenfold serially diluted cell cultures were inoculated on each spot. (B) Extracellular pNPC hydrolytic activity of QM9414, the ΔTrfwd1 or the Trubc4-repressed strains with their endogenous cre1 replaced by a tcu1 promoter driving cre1-gfp (P_tcu1-cre1-gfp, ΔTrfwd1-P_tcu1-cre1-gfp, and P_tcu1-Trubc4-P_tcu1-cre1-gfp) cultured on 1% Avicel, with or without copper irons to control the expression of cre1-gfp. Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g005

To elucidate the regulatory role of TrFwd1 targeting Cre1 in T. reesei, we constructed a C-terminal GFP-tagged Cre1 strain (P_tcu1-cre1-gfp) under the control of tcu1 promoter by replacing the endogenous cre1 locus. Growth assays revealed that whereas overexpression of cre1 without copper exerted no discernible impact, repressing cre1 expression with copper significantly impaired both mycelial expansion and conidiation (S10A Fig), a phenotype similar to the knockout of cre1 [60]. Unlike the effect on growth, Cre1 overexpression resulted in an approximately 70% reduction in the extracellular cellulase activity relative to QM9414 whereas cre1 repression led to a significant increase (60%) in cellulase activity (Fig 5B). These results verified that Cre1-GFP was equally functional in mediating cellulase gene repression in T. reesei. When Trfwd1 was simultaneously deleted in P_tcu1-cre1-gfp, the obtained ΔTrfwd1-P_tcu1-cre1-gfp strain exhibited a further reduction in extracellular cellulase activity with Cre1 overexpression upon induction by 1% Avicel, implicating that the absence of TrFwd1 exacerbates the repressive effect of Cre1. In contrast, cre1 repression fully restored the cellulase activity to the control level, which was even 60% higher at 96 h of induction. Similar phenotypic characteristics regarding the induced cellulase production were observed when both the expression of cre1 and Trubc4 were controlled by the tcu1 promoter. Notably, the simultaneous repression of cre1 and Trubc4 with copper resulted in a significant recovery of the enzymatic activity up to 80% of the control level. Together these data suggest that TrUbc4-TrFwd1-mediated ubiquitination likely functions to antagonize Cre1-mediated repression.

To test the effect of the putative de-ubiquitination pathway on Cre1, we investigated the role of T. reesei Cre2, a deubiquitinating enzyme homolog of CreB that is thought to antagonize the ubiquintination of CreA [37,50,51]. Deletion of cre2 in QM9414 resulted in an approximately 20% increase in extracellular pNPC hydrolytic activity (S10B Fig). Of note, the absence of cre2 in ΔTrfwd1 partially restored extracellular pNPC hydrolytic activity, reaching approximately 60% of that in QM9414. Altogether, these results indicate that there exists a strong genetic interaction between TrFwd1 and Cre2 ubiquitylating modification of Cre1 to tightly control the induced cellulase gene expression in T. reesei.

TrUbc4 and TrFwd1 mediate Cre1 ubiquitination that is required for CCR relief

To investigate whether Cre1 was indeed ubiquitinylated by an SCF complex integrating TrFwd1, mycelial lysates from the P_tcu1-cre1-gfp and ΔTrFwd1-P_tcu1-cre1-gfp strains cultured for 2 h of 1% Avicel induction were immunoprecipitated with GFP-Trap beads followed by western blot analysis using anti-GFP and anti-ubiquitin antibodies, respectively. The results revealed that Cre1 was apparently ubiquitylated and this ubiquination was markedly reduced in the absence of Trfwd1 (Fig 6A). To check the role of TrUbc4 on Cre1 ubiquitination, we fused GFP tag directly at the C-terminus of the endogenous cre1 in QM9414 (generating P_cre1-cre1-gfp) and P_tcu1-Trubc4 (generating P_tcu1-Trubc4-P_cre1-cre1-gfp), respectively (S11A and S11B Fig). Similar reduction in the ubiquitination of Cre1 was observed with Trubc4 repression (Fig 6B), implicating that TrUbc4 likely functions together with TrFwd1 in mediating Cre1 ubiquitination.

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Fig 6. Ubiquitination of Cre1 mediated by TrUbc4/TrFwd1 and the nuclear accumulation of Cre1 had no effect on its function.

(A) Western blot analysis of Cre1-GFP (predicted molecular weight 71 kDa) was performed on immunoprecipitates obtained from mycelial proteins of P_tcu1-cre1-gfp and ΔTrfwd1-P_tcu1-cre1-gfp strains cultured on 1% Avicel for 2 h. The blots were subsequently probed with an anti-ubiquitin antibody and an anti-GFP antibody, respectively. (B) Western blot analysis of Cre1-GFP (predicted molecular weight 71 kDa) was performed on immunoprecipitates obtained from mycelial proteins of P_cre1-cre1-gfp and P_tcu1-Trubc4-P_cre1-cre1-gfp strains cultured on 1% Avicel for 2 h with copper being added to repress Trubc4. The blots were subsequently probed with an anti-ubiquitin antibody and an anti-GFP antibody, respectively. (C) Fluorescence microscopy observation of the subcellular localization of the P_tcu1-cre1-gfp::cre1-gfp, P_tcu1-cre1-gfp::mcre1-gfp, ΔTrfwd1-P_tcu1-cre1-gfp::cre1-gfp and ΔTrfwd1-P_tcu1-cre1-gfp::mcre1-gfp strains upon transition from 1% glucose to 1% Avicel conditions with copper being added to repress the endogenous cre1. (D) Extracellular pNPC hydrolytic activity of the P_tcu1-cre1-gfp::cre1-gfp, P_tcu1-cre1-gfp::mcre1-gfp, ΔTrfwd1-P_tcu1-cre1-gfp::cre1-gfp and ΔTrfwd1-P_tcu1-cre1-gfp::mcre1-gfp strains cultured on 1% Avicel with copper being added to repress the endogenous cre1. (E) Western blot analysis of Cre1-GFP and mCre1-GFP was performed on immunoprecipitates obtained from mycelial proteins of P_tcu1-cre1-gfp::cre1-gfp and P_tcu1-cre1-gfp::mcre1-gfp strains cultured on 1% Avicel for 2 h with copper being added to repress the endogenous cre1. The blots were subsequently probed with an anti-ubiquitin antibody and an anti-GFP antibody, respectively. Data are represented as mean ± SD. ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g006

Previous studies have suggested a scenario wherein the ubiquitination of CreA may directly affect its function by promoting its nuclear export to the cytoplasm followed by its degradation [47]. To exclude the possibility that nuclear export is required for the relief of Cre1-mediated CCR, we sought to examine whether forced nuclear retention of Cre1 by blocking its nuclear export influenced cellulase gene expression. A putative nuclear export signal (NES) sequence analogous to the A. oryzae CreA were identified in Cre1 (S12A Fig). Simultaneous mutations of two highly conserved residues (L338 and L346) to alanine in NES of A oryzae CreA have been reported to result in its substantial nuclear retention and significantly extended half-life even under maltose-inducing conditions [61]. The corresponding mutations were first introduced in Cre1 (mCre1-gfp), which was then integrated at the pyr4 locus of the P_tcu1-cre1-gfp and ΔTrfwd1-P_tcu1-cre1-gfp strains, respectivley, to generate P_tcu1-cre1-gfp::mcre1-gfp and ΔTrfwd1-P_tcu1-cre1-gfp::mcre1-gfp expressing mCre1 from the cre1 promoter. Corresponding strains expressing WT Cre1 were similarly constructed to obtain P_tcu1-cre1-gfp::cre1-gfp and ΔTrfwd1-P_tcu1-cre1-gfp::cre1-gfp, respectively, as controls. While there was hardly any difference in the growth between the mutant and WT Cre1 control strains on either glucose or malt extract media plates with addition of copper ion to repress the endogenous cre1 expression (S12B Fig), GFP fluorescence displayed a consistent nuclear localization of both WT Cre1 and mCre1 in T. reesei conidia germinated on either glucose or Avicel with copper to repress the endogenous cre1 (S12C Fig). Further analysis of potential dynamic changes in subcellular localization after mycelia transfer from glucose to Avicel revealed that the nuclear fluorescence signal noticeably diminished for Cre1-GFP after 8 h of Avicel induction and became marginally detectable by 12 h. In contrast, mCre1-GFP maintained strong nuclear fluorescence throughout the induction period regardless of the presence or absence of TrFwd1 (Fig 6C).

Further determination of the extracellular cellulase activity of the P_tcu1-cre1-gfp::mcre1-gfp strain revealed that the persistent nuclear retention of Cre1 had no apparent effect on the incuded cellulase biosynthesis compared with the similarly expressed WT Cre1 in Ptcu1-cre1-gfp::cre1-gfp (Fig 6D). However, this induced cellulase gene expression with accumulated mCre1-GFP still required TrFwd1 since its absence displayed a 50% reduction in the extracellular hydrolytic activity. Analysis of Cre1 ubiquitination showed no significant difference in the ubiquitination smear intensity between mCre1-GFP and Cre1-GFP (Fig 6E), implicating that the nuclear-accumulated mutant Cre1 is still capable of being ubiquitinated. Taken together, these results implicate that while ubiquitination of Cre1 may facilitate its nuclear export and subsequent degradation, its export form the nucleus may not be a prerequisite for CCR relief since enhanced nuclear accumulation of Cre1 did not deteriorate Cre1-mediated repression with functional TrFwd1.

Identification of Cre1 K361 as a major site for targeted ubiquitination by the SCF-TrFwd1 complex

Cre1 contains 12 lysine (K) residues as potential sites of ubiquitination. To pin down the exact site that is ubiquitylated by the TrUbc4-TrFwd1 enzyme cascade, we individually or combinatorially substituted these lysine for arginine and introduced the respective point mutant as well as WT Cre1 into P_tcu1-cre1-gfp. All the obtained strains would express the mutant Cre1 from the pyr4 locus under the control of the cre1 promoter while its endogenous cre1 was repressed with copper. Comparing the extracellular pNPC hydrolytic activity revealed that while most mutants just behaved like P_tcu1-cre1-gfp::Cre1 and displayed intermediate cellulase activities between the control and the cre1 repressed strain, P_tcu1-cre1-gfp::K361R/K369R and P_tcu1-cre1-gfp::K361R mutants stood out with significantly compromised cellulase activities that were only 50%-60% of the control level (Fig 7A), indicating an important role of K361 in Cre1 modulating the induced cellulase gene expression. Of note, unlike the growth of the cre1 repressed control strain, P_tcu1-cre1-gfp::K361R/K369R and P_tcu1-cre1-gfp::K361R mutants hardly constrained mycelial growth and exhibited comparable colony expansion to P_tcu1-cre1-gfp::Cre1 on solid glucose and malt extract media supplemented with copper (S13A Fig), indicating the targeted mutations specifically affect cellulase production while maintaining other functions of Cre1 in supporting hyphal growth and conidiation.

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Fig 7. Cre1 K361 represents a key site for ubiquitination to regulate its control over cellulase gene expression.

(A) Extracellular pNPC hydrolytic activity cultured on 1% Avicel with copper being added to repress the endogenous cre1 while allowing only the expression of Cre1 mutants. Data were from two replicates. (B) Extracellular pNPC hydrolytic activity of the ReK361R strain with xyr1 overexpression driven by tcu1 promoter (OExyr1-ReK361R) cultured on 1% Avicel (left) or 1% glucose (right). (C) Extracellular pNPC hydrolytic activity of the Δcre1 deletion strain, the corresponding strains complemented with wild-type Cre1 (ReCre1) or the K361R mutant (ReK361R), and a C-terminal GFP-tagged version (ReCre1-GFP and ReK361R-GFP) upon 1% Avicel. (D) Western blot analysis of Cre1-GFP was performed on immunoprecipitates obtained from mycelial proteins of ReCre1-GFP and ReK361R-GFP strains cultured on 1% Avicel for 2 h. The blots were subsequently probed with an anti-ubiquitin antibody and an anti-GFP antibody, respectively. Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g007

To verify the important role of K361 by avoiding any interference from the leaky expression of the endogenous cre1 resulting from the tcu1 promoter, the K361R mutant was introduced into a cre1 knockout strain. Although Δcre1 showed consistent phenotypic defects regarding impaired colony expansion and conidiation with the cre1 repressed strain (S13B Fig), the complete absence of Cre1 only exhibited a much moderate increase in cellulase production during an early induction period (48 h) (Fig 7B). Complementation of Δcre1 with either the WT or the K361R mutant Cre1 almost fully corrected the growth defect (S13B Fig). However, unlike ReCre1, ReK361R failed to initiate an efficient cellulase and xylanase biosynthesis on induction and resulted in a significant reduction in an extracellular pNPC and xylan hydrolytic activity (Figs 7B and S2D). These results are consistent with the phenotypic characteristics obtained in the endogenous cre1 repressed background, confirming the critical role of K361 in Cre1 function. Similar to deletion of Trfwd1 or repression of Trubc4, overexpression of Xyr1 fully restored cellulase biosynthesis in the ReK361R mutant (Fig 7C).

To further investigate whether K361R mutation affects the ubiquitination status of Cre1, GFP-tagged Cre1 (ReCre1-GFP) and K361R (ReK361R-GFP) were expressed in Δcre1, respectively. Both strains behaved almost the same as their untagged counterparts regarding growth and cellulase production (Figs 7C and S13B). The moderate increase in the extracellular cellulase activity in ReK361R-GFP compared with ReK361R suggests a potential interference from GFP with Cre1 function [46]. Notwithstanding this, IP followed by western blot analysis showed significantly reduced ubiquitin modification of K361R-GFP compared to the Cre1-GFP control (Fig 7D), a phenotypic defect that exactly coincides with that caused by the absence of TrUbc4 or TrFwd1. It should be noted that K361R-GFP modification does not seem to impact Cre1 degradation despite the reduced ubiquitination. Altogether these results support the idea that K361 represents a critical site for Cre1 ubiquitination that is dependent on the TrUbc4-TrFwd1 pathway which plays an important role in derepressing Cre1 to facilitate the successful induced cellulase gene expression.

Ubiquitination of Cre1 diminishes its occupancy on cellulase gene promoters and relieves its antagonizing effect on Xyr1’s binding

Considering that enhancing nuclear accumulation of Cre1 did not deepen CCR, we wondered whether TrFwd1-mediated ubiquitination of Cre1 is able to modulate its binding to target gene promoters. Quite a few Cre1 DNA-binding motifs (5’-SYGGRG-3’) were distributed along the three main cellulase gene promoters (cbh1, eg1, and bgl1), alternating with Xyr1 binding sites (5’-GGCWWW-3’) that are especially highly enriched in the cbh1 promoter (Fig 8A). To gain an overview of Cre1 recruitment to the various cellulase gene promoters, chromatin immunoprecipitation (ChIP)-qPCR assays were performed to investigate their occupancy by Cre1 with Cre1-GFP expressed in QM9414, P_tcu1-Trubc4 and ΔTrfwd1, respectively, after induction with 1% Avicel (S11A and S11B Fig). As shown in Figs 8B and 8C, a significantly higher enrichment of Cre1 was observed on the respective promoter regions either in the absence of Trfwd1 or with repression of Trubc4 compared to the control strain.

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Fig 8. Trubc4 repression or Trfwd1 deletion enhanced Cre1 recruitment while reducing Xyr1 occupancy at cellulase gene promoters by ChIP analyses.

(A) Schematic diagram of the predicted binding sites for Xyr1 and Cre1 on cellulase gene promoters. The numbers are the nucleotide positions relative to the start codon ATG. (B) ChIP analyses of Cre1 occupancy on cbh1, eg1, bgl1, and actin promoters in Trfwd1-deleted strain (ΔTrfwd1-P_cre1-cre1-gfp) and Trubc4-repressed strain (P_tcu1-Trubc4-P_cre1-cre1-gfp) supplemented with copper. The analyzed promoter regions includes P_cbh1 (-604 to -796), P_eg1 (-162 to -323), and P_bgl1 (-1817 to -2123), respectively. (C) ChIP analyses to provide an overview of Cre1 occupancy over the cbh1 promoter in Trfwd1-deleted strain (ΔTrfwd1-P_cre1-cre1-gfp) and Trubc4-repressed strain (P_tcu1-Trubc4-P_cre1-cre1-gfp) supplemented with copper. The analyzed regions include cbh1-p1 (-46 to -255), cbh1-p2 (-604 to -796), cbh1-p3 (–923 to -1088), and cbh1-p4 (-1325 to -1533). (D) ChIP analyses of Xyr1 occupancy on cbh1, eg1, bgl1, and actin promoters in Trfwd1-deleted strain (ΔTrfwd1-P_cre1-cre1-gfp) and Trubc4-repressed strain (P_tcu1-Trubc4-P_cre1-cre1-gfp) supplemented with copper. The analyzed promoter regions includes P_cbh1 (-698 to -867), P_eg1 (-162 to -323), and P_bgl1 (-1817 to -2123), respectively. (E) ChIP analyses to provide an overview of Xyr1 occupancy over the cbh1 promoter in Trfwd1-deleted strain (ΔTrfwd1-P_cre1-cre1-gfp) and Trubc4-repressed strain (P_tcu1-Trubc4-P_cre1-cre1-gfp) supplemented with copper. The analyzed regions include cbh1-p1 (-46 to -255), cbh1-p2 (-235 to -418), cbh1-p3 (-399 to -579), cbh1-p4 (–698 to -867), cbh1-p5 (–923 to -1088), and cbh1-p6 (–1325 to -1533). The numbers within brackets are the nucleotide position relative to the start codon ATG. No significant difference (t test P > 0.05 [n.s.]) was observed for Cre1 or Xyr1 occupancy on cellulase gene or actin promoters. All the mycelia mentioned above were collected after induction on 1% Avicel for 2 h. Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g008

To investigate whether the increased Cre1 occupancy interferes with the binding of the major transcriptional activator Xyr1, similar ChIP assays with an Xyr1-specific antibody were performed. In contrast with Cre1, whereas a relatively higher recruitment of Xyr1 occurred on cellulase gene promoters upon 1% Avicel induction, the enrichment signals for Xyr1 were significantly reduced with compromised ubiquitination of Cre1 (Figs 8D and 8E). As expected, no significant enrichment of Cre1 or Xyr1 was detected on the actin promoter. Altogether these data suggest that the identified TrUbc4–TrFwd1 pathway as well as Cre1 K361 are involved in modulating the promoter binding ability of Cre1, which is critical for relieving its interference with the efficient Xyr1 binding to activate cellulase gene expression.

To further corroborate the effect of ubiquitination on Cre1 occupancy on cellulase gene promoters, ReK361R-GFP mutant was checked for its binding to relevant promoters by ChIP assays. The results revealed that ReK361R-GFP was significantly more enriched on the corresponding promoter regions compared to the Cre1-GFP control upon 1% Avicel induction (Figs 9A and 9B). Similar to what was observed in P_tcu1-Trubc4 and ΔTrfwd1, the increase in Cre1 occupancy was accompanied by a concomitant reduction in Xyr1 recruitment at the cbh1, eg1, and bgl1 promoters in the ReK361R-GFP strain (Figs 9C and 9D). Although a direct competition between binding of these important transcription factors can not be deduced, these findings implicate that K361R mutation may impair the ubiquitin modification of Cre1 which leads to its more persistent binding to DNA, but consequently impedes Xyr1 access to the cellulase gene promoters.

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Fig 9. Cre1-K361R increased Cre1 recruitment while reducing Xyr1 occupancy at cellulase gene promoters.

(A) ChIP analyses of Cre1 occupancy on cbh1, eg1, bgl1, and actin promoters in the ReK361R-GFP strain. The analyzed promoter regions includes P_cbh1 (-604 to -796), P_eg1 (-162 to -323), and P_bgl1 (-1817 to -2123), respectively. (B) ChIP analyses to provide an overview of Cre1 occupancy over the cbh1 promoter in the ReK361R-GFP strain. The analyzed regions include cbh1-p1 (-46 to -255), cbh1-p2 (-604 to -796), cbh1-p3 (–923 to -1088), and cbh1-p4 (-1325 to -1533). (C) ChIP analyses of Xyr1 occupancy on cbh1, eg1, bgl1, and actin promoters in the ReK361R-GFP strain. The analyzed promoter regions includes P_cbh1 (-698 to -867), P_eg1 (-162 to -323), and P_bgl1 (-1817 to -2123), respectively. (D) ChIP analyses to provide an overview of Xyr1 occupancy over the cbh1 promoter in the ReK361R-GFP strain. The analyzed regions include cbh1-p1 (-46 to -255), cbh1-p2 (-235 to -418), cbh1-p3 (-399 to -579), cbh1-p4 (–698 to -867), cbh1-p5 (–923 to -1088), and cbh1-p6 (–1325 to -1533). The numbers within brackets are the nucleotide position relative to the start codon ATG. No significant difference (t test P > 0.05 [n.s.]) was observed for Cre1 or Xyr1 occupancy on cellulase gene or actin promoters. All the mycelia mentioned above were collected after induction on 1% Avicel for 2 h. Data are represented as mean ± SD. ^nsP > 0.05, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

https://doi.org/10.1371/journal.pgen.1012216.g009

Strains and culture conditions

T. reesei QM9414 (ATCC 26921) and Δpyr4, in which the uridine trophic marker gene was deleted in QM9414, were used throughout this work as control and parental strains, respectively. All T. reesei strains were maintained on malt extract agar (30 g/L malt extract, 1.5% Agar). For transcription and cellulase production analysis, T. reesei strains were pre-grown in 1 L Erlenmeyer flasks on a rotary shaker (200 rpm) at 30°C in 250 mL Mandels-Andreotti (MA) medium with 1% glycerol as the carbon source for 48 h. Mycelia were harvested by filtration and washed twice with medium without carbon source. Equal amounts of mycelia were then transferred to a fresh medium without peptone containing 1% Avicel or other carbon sources as indicated, and incubation was continued for the indicated time period. MA medium was prepared as follows: Na₂HPO₄·12H₂O 17.907 g/L, KH₂PO₄ 2 g/L, (NH₄)₂SO₄ 1.4 g/L, Urea 0.3 g/L, Tween-80 0.5 ml/L, Carbon source 1% (w/v). The solution was brought to 1 L with deionized water with pH being adjusted to 5.0 with anhydrous citric acid. The medium was finally sterilized. MgSO₄·7H₂O 0.6 g/L, CaCl₂ 0.6 g/L, and 1 × trace elements were added. Uridine (2.4 g/L) was supplemented when required. Based on the provided formula for the 1000 × trace element stock solution (0.5 g FeSO₄·7H₂O, 0.16 g MnSO₄·H₂O, 0.14 g ZnSO₄·7H₂O, and 0.2 g CoCl₂·2H₂O per 100 mL). For pre‑culture with glycerol, peptone (2.0 g/L) was added to MA. Minimal Medium (MM) used for vegetative growth assay was prepared as follows: 20 g glucose, 5 g (NH₄)₂SO₄, and 15 g KH₂PO₄ per liter. The pH was adjusted to 5.5 using NaOH. After sterilization, the following sterile stock solutions were added per liter of medium, 4 mL of 250 × MgSO₄ stock solution (0.61 M), 4 mL of 250 × CaCl₂ stock solution (1.35 M), and 1 ml of filter-sterilized 1000 × trace element stock solution. All T. reesei strains are listed in S4 Table.

Escherichia coli DH5α was used for plasmids construction and E. coli BL21 (DE3) was used as a host for the production of recombinant proteins. Both strains were cultured on Luria-Bertani medium (1% yeast extract, 2% peptone and 2% NaCl) with a rotary shaker (200 rpm) at 37°C.

S. cerevisiae strain Y187 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, gal80Δ, met^–, URA3::GAL1UAS–Gal1_TATA–LacZ MEL1) was used as the host for one-hybrid screening. S. cerevisiae strain Y2HGold (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1_UAS-Gal1_TATA-His3, GAL2_UAS-Gal2_TATA-Ade2 URA3::MEL1_UAS-Mel1_TATA AUR1-C MEL1) were used for yeast two-hybrid assay. Yeast cells were routinely cultivated at 30°C in YPD medium (1% yeast extract, 2% peptone and 2% glucose). Yeast transformants were cultivated in synthetic complete (SC) medium with appropriate amino acids used for auxotroph selection. All Y2H assay were performed according to the manufacturer’s manual (Clontech-TaKaRa Bio).

cDNA library and plasmids for yeast-based screen

For preparation of the cDNA library, strain QM9414 mycelia induced on 1% Avicel for 3 h were harvested and mRNAs were extracted using the TRIzol reagent (Invitrogen). A cDNA library was constructed by ligating the reverse transcribed cDNA fragments into the pGADT7 plasmid according to the Matchmaker two-hybrid system manual (Clontech-TaKaRa Bio) [54]. Ten micrograms of T. reesei cDNA library DNA was then transformed into the Y187 strain. The bait plasmid used for the yeast one-hybrid screen was constructed by amplifying the AbA (Aureobasidin A) resistance gene AUR1-C from S. cerevisiae Y2HGold (Clontech-TaKaRa Bio) genomic DNA and inserting it into pRS304 that had been digested with XhoI/NotI. The S. cerevisiae gal1 core promoter was then amplified from S. cerevisiae genomic DNA and inserted upstream of the AUR1-C gene within the ApaI and XhoI sites to obtain the pRS1 plasmid [54]. The 365 bp cbh1 promoter region (-474 to -838 bp) was finally amplified from T. reesei genomic DNA which was ligated into the pRS1 plasmid digested with KpnI and ApaI to obtain the pRS3 bait plasmid. pRS3 was transformed into Y187 after being linearzied with LguI to obtain the bait yeast strain, which could not grow on plates with 100 ng/mL AbA, indicating that the cbh1 promoter (-474 to -838 bp) could not drive reporter gene expression by itself.

The yeast two-hybrid assay utilized pGADT7 and pGBKT7 (Clontech-TaKaRa Bio) as cloning vectors, which were digested using NdeI and EcoRI to clone the respective target genes. The indicated genes encoding proteins for interaction assay were amplified from T. reesei cDNA, subsequently digested and ligated into the above plasmids before being transformed into Y2HGold strain.

Yeast transformation was performed using a commercial yeast transformation kit (Clontech-TaKaRa Bio). Briefly, yeast cells were cultured on YPD to a mid-log phase (OD600 = 0.4-0.5), which were then collected and washed twice with sterile water, once with LA buffer (100 mM LiAc, 10 mM Tris–HCl, 1 mM EDTA, pH 7.5), and finally resuspended in 600 μL LA buffer on ice to prepare competent cells. For transformation, 20 μg denatured carrier DNA that was boiled for 5–10 min and chilled on ice, and 5 μg plasmid were mixed with 600 μL competent cells, followed by addition of 2.5 mL LAP buffer (LA buffer supplemented with 40% PEG-3350). After a 45-min incubation at 30 °C with gentle mixing every 15 min, 160 μL DMSO was added. The mixture was heat-shocked at 42 °C for 25 min with intermittent mixing, then cooled on ice for 2 min. To enhance transformation efficiency, cells were finally recovered in 3 mL YPD Plus medium (30 °C, 200 rpm, 90 min), washed once with 4 mL 0.9% NaCl, and resuspended in 4 mL 0.9% NaCl. Transformants were selected on SC plates with appropriate amino acids used for auxotroph selection. The transformant colonies were picked and the harbored plasmids were verified by DNA sequencing after retransformation.

Plasmids and recombinant T. reesei strains construction

All gene knockouts in T. reesei were performed by transforming the indicated strains with a gene knockout cassette obtained via the double-joint (fusion) PCR method [72]. Briefly, the upstream homologous arm (~2 kb) of the target gene, a selectable marker gene such as the uridine auxotrophic marker gene pyr4 or the hygromycin B resistance gene hph, and the downstream homologous arm (~2 kb) were PCR fused together to generate the final knockout cassette.

All complemented gene expression in T. reesei involved constructing the gene expression cassette into pUC19 [73], followed by linearizing the plasmid with an appropriate restriction enzyme that avoids cutting the expression cassette. Plasmid construction was performed using the one-step T5 exonuclease DNA assembly (TEDA) protocol [74]. All primers used were listed in S5 Table.

To construct a plasmid for conditionally repressing Trubc4 expression under the control of the copper-responsive promoter P_tcu1, a 2.2 kb coding region starting from the start codon ATG was amplified from genomic DNA of T. reesei QM9414, and inserted into the AscI site of the pMDP_tcu1-pyr4 plasmid [73]. Subsequently, the resulting plasmid was digested with HindIII and ligated with a 2.0 kb DNA fragment corresponding to the upstream non-coding region of Trubc4. The plasmid was then linearized with ScaI before being transformed into the the Δpyr4 and the OExyr1 [75] strains to obtain P_tcu1-Trubc4 and OExyr1-P_tcu1-Trubc4, respectively.

For P_tcu1-controlled expression of GFP-TrUbc4 at the endoenous Trubc4 locus in T. reesei, the coding sequence of Trubc4 was amplified from the pGADT7-TrUbc4 plasmid, and 1.5 kb downstream of Trubc4 was amplified from genomic DNA of T. reesei QM9414, The resulting PCR products were fused and digested with AscI and SpeI, and then ligated into pMDP_tcu1-gfp-T_trpC [76]. Subsequently, the resulting plasmid was digested with HindIII and ligated with a 2.0 kb DNA fragment corresponding to the upstream non-coding region of Trubc4. The resulting plasmids were linearized with SspI before being transformed into the Δpyr4 strain to obtain the P_tcu1-gfp-Trubc4 strain.

To construct strains with targeted expression at the pyr4 locus, DNA fragments corresponding to approximately 1.8 kb of upstream and 1.4 kb of downstream non-coding regions of the pyr4 gene were first amplified from genomic DNA of QM9414. These fragments were then fused the T_trpC terminator and the hph selectable marker via overlapping PCR. The final PCR product with the up- and downstream homologous arms flanking the the T_trpC terminator and hph was ligated into the SmaI site of pUC19 to obtain the pUC19-pyr4::T_trpC-hph plasmid.

To achieve the complemented expression of Trubc4 at the pyr4 locus, the Trubc4 coding sequence and an 1.4 kb tef1 promoter were amplified from pGADT7-TrUbc4 and QM9414 genomic DNA, respectively, followed by PCR fusion. The fused fragment was subsequently ligated into the SgsI site of the pUC19-pyr4::T_trpC-hph plasmid to make Trubc4 under the control of P_tef1. Similarly, the C85A and F62A/A96D TrUbc4 mutant expression cassettes were constructed using the same strategy. The resulting plasmids were linearized with SmaI and transformed into the P_tcu1-Trubc4 strain to obtain the ReUbc4, ReC85A, and ReF62A/A96D strains, respectively.

To express the N-terminal GFP or Myc-His tagged TrFwd1 at the Trfwd1 locus in T. reesei, a two-step assembly strategy was employed. First, a fusion DNA fragment was generated by PCR comprising sequentially the 0.8 kb constitutive P_gpd1 promoter amplified from QM9414 genomic DNA, the GFP or 5 × Myc-6 × His coding sequence, the full-length TrFwd1 or its ΔF-box mutant coding sequence, and an 1.6 kb downstream non-coding region of Trfwd1. The obtained DNA fragment was ligated into the BamHI/HindIII sites of pUC19. Second, a DNA fragment containing an upstream non-coding region of Trfwd1 and the pyr4 gene were amplified from QM9414 genomic DNA and fused together with PCR, which was then ligated into the EcoRI site of the intermediate plasmid from step one. The final plasmid was linearized with Eco105I and transformed into the ΔTrfwd1 strain to generate the complemented strains including P_gpd1-gfp-Trfwd1, P_gpd1-gfp-Trfwd1-ΔF, P_gpd1-myc-his-Trfwd1, and P_gpd1-myc-his-Trfwd1-ΔF, respectively.

To construct plasmids for replacing the Trcul1, Trrbx1, and Trskp1 promoters with the P_tcu1 promoter, P_tcu1 and the pyr4 gene were first amplified from QM9414 genomic DNA. These two DNA fragments were subsequently PCR fused together and ligated into BamHI/HindIII-digested pUC19 to generate the pUC19-P_tcu1-pyr4 plasmid. Plasmids for promoter replacement was further constructed using a two‑step cloning strategy. DNA fragments corresponding to the respective coding sequences and 1.5 kb downstream non-coding regions were firstly amplified from QM9414 genomic DNA. The resulting DNA fragments were individually ligated into the HindIII-digested pUC19-P_tcu1-pyr4 plasmid. The obtained intermediate plasmids were then digested with SgsI and the corresponding 1.5 kb to 1.9 kb upstream non-coding homologous regions of each target gene were inserted to generate the final targeting plasmids. Each plasmid was linearized with SspI and transformed into the Δpyr4 strain to obtain the P_tcu1‑Trcul1, P_tcu1‑Trrbx1, and P_tcu1‑Trskp1 strains, respectively.

To achieve the expression of cre1-gfp under the control of the P_tcu1 promoter, the respective plasmids were constructed using a two-step cloning strategy. The cre1 gene along with approximately 1.7 kb of its downstream non-coding region was first amplified from QM9414 genomic DNA and PCR fused with gfp. The obtained DNA fragment was then inserted into the HindIII site of pUC19-P_tcu1-pyr4. Subsequently, an 1.6 kb upstream non-coding region of cre1 was inserted into the SgsI site of the above intermediate plasmid to obtain pUC19- P_tcu1-cre1-gfp plasmid, which was then linearized with Eco105I before being transformed into the Δpyr4 and the ΔTrfwd1 strains to obtain the P_tcu1-cre1-gfp, ΔTrfwd1-P_tcu1-cre1-gfp, respectively. By substituting the hph marker for the pyr4 selection marker in pUC19-P_tcu1-pyr4, the similarly modified targeting plasmid was linearized with Eco105I and transformed into the P_tcu1-Trubc4 strain to obtain the P_tcu1-Trubc4-P_tcu1-cre1-gfp strain.

For targeted integration of the cre1-gfp expression cassette to substitute for the endogenous cre1 locus, a fusion DNA fragment was first assembled by PCR comprising the following six sequential components, an 1.6 kb of its upstream non-coding region including the cre1 promoter, the cre1 coding sequence, the gfp gene, the T_trpC terminator, the hph marker gene, and an 1.7 kb of the cre1 downstream non-coding region. The assembled DNA fragment was then ligated into the EcoRI-digested pUC19 plasmid to obtain the P_cre1-cre1-gfp plasmid and the resulting plasmid was linearized with LguI before being transformed into Δpyr4, ΔTrfwd1 and P_tcu1-Trubc4 to obtain the P_cre1-cre1-gfp, ΔTrfwd1-P_cre1-cre1-gfp, and P_tcu1-Trubc4-P_cre1-cre1-gfp strains, respectively.

To generate strains expressing cre1 or cre1-gfp under the control of its own promoter at the pyr4 locus, the expression cassette comprising the 1.6 kb cre1 promoter and the cre1 coding sequence as well as the gfp gene (if applicable) was first amplified directly from the previously constructed pUC19-P_cre1-cre1-gfp plasmid and fused together. The resultant DNA fragment was subsequently ligated into the SgsI-linearized pUC19-pyr4::T_trpC-hph plasmid. Following linearization with SmaI, the construct was transformed into the P_tcu1-cre1-gfp or Δcre1 strain. Strains expressing point mutant Cre1 were generated using a similarly strategy.

Transformation of T. reesei was performed using PEG-mediated uptake of DNA by protoplast [77]. Briefly, fresh conidia spores of T. reesei were inoculated into 100 mL of MM medium and cultured at 30°C with shaking (200 rpm) for 20–24 h to obtain young hyphae suitable for enzymatic digestion. Mycelia were harvested via filtration through a G2 sintered glass funnel and washed three times with SK solution (1.0 M sorbitol, 9.1 mM KH₂PO₄, 1.3 mM K₂HPO₄) to remove residual medium. The washed mycelia were resuspended in 8 mL of SK solution and incubated with 0.04 g each of snailase (Klontech, Amresco) and lywallzyme (GDMCC, Guangdong) at 30°C for 2-3 h with gentle agitation. Protoplast formation was monitored microscopically before being filtered through a G1 sintered glass funnel and protoplasts were collected by centrifugation at 4,000 × g for 10 min at 4°C, washed twice with 4 mL of STC solution (1.0 M sorbitol, 75 mM CaCl₂, 34 mM NaCl, 10 mM Tris-HCl, pH 7.5), and finally resuspended in 100 µL of STC. For transformation, plasmid DNA (≥10 µg) was added to the protoplast suspension, followed by the addition of 25 µL of PTC solution (60% PEG4000, 75 mM CaCl₂, 10 mM Tris-HCl, pH 7.5). The mixture was incubated on ice for 20 min followed by addition of 500 µL of PTC. The reaction mixture was gently mixed and allowed to stand at room temperature for 5 min. Subsequently, 1 mL of STC solution was added, and 200–300 µL aliquots were spread onto regeneration agar plates (MM medium supplemented with 1 M sorbitol). Transformants were selected on minimal medium either for uridine prototroph or for resistance to hygromycin (120 µg/mL). Anchored PCR were used to verify the correct integration events.

Observation of mycelium by fluorescence microscopy

T. reesei conidia spores were inoculated in the MM medium containing 1% glucose for 24 h at 30°C. Mycelia were used directly for microscopic observation or transferred to MA medium containing 1% Avicel induction for different time. After incubation, germlings were fixed on the coverslips using methanol and then stained with 100 μg/ml of DAPI (40,6-diamidino-2-phenylindole dihydrochloride; Beyotime C1002) solution for 5 min. The fluorescence of the mycelium of the recombinant strains was detected with a Nikon Eclipse 80i fluorescence microscope (Nikon, Melville, NY, USA), and images were captured and processed with the NIS-ELEMENTS AR software program.

The vegetative growth assay

To assay vegetative growth, equal amount of growing mycelia were inoculated on MM agar plates containing glucose or on malt extract agar plates, and incubated at 30°C for 3 days.

For determination of the growth in liquid culture, equal amount of mycelia were transferred to fresh MA with 1% (w/v) glucose as the sole carbon source. At the indicated time points, mycelia cultured on glucose were filtrated on filter paper, dried at 80°C for 48 h, and then weighed.

Enzyme activity measurements

Cellobiohydrolase and β-glucosidase activities were determined by measuring the amount of p-nitrophenol, using p-nitrophenyl-D-cellobioside (pNPC; Sigma) and p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma), respectively, as the substrates. The assays of cellulase activity were carried out in 200 μL of reaction mixture containing 40 μL of culture supernatant and 40 μL of the respective substrate plus 80 μl of 50 mM sodium acetate buffer (pH 4.8) with incubation at 50°C for 30 min. The reaction was stopped by addition of 40 μL of 10% Na₂CO₃ (w/v). The amount of p-nitrophenyl is determined by measuring the absorbance at 420 nm. One unit (U) of pNPC or pNPG activity is defined as the amount of enzyme releasing 1 μmoL of p-nitrophenyl per minute.

CMC (carboxymethyl cellulose sodium salt, Sigma) hydrolytic activity was determined by measuring the released glucose using as the substrate. Briefly, the assay was performed in 120 μL of reaction mixture including 60 μL of 50 mM sodium acetate buffer (pH 4.8) and 60 μL of diluted culture supernatant, and the mixture was then incubated at 50°C for 30 min. The reducing sugar released in the mixture was determined by the 3, 5-dinitrosalicylic acid method with glucose as the standard. One unit (U) of CMC hydrolytic activity is defined as the amount of enzyme releasing 1 μmoL of reducing sugar per minute.

Xylanase activities were determined by measuring the amount of released xylose using xylan as substrate. Briefly, a reaction mixture containing 60 μL of diluted culture supernatant and 60 μL of beechwood xylan (5 g/L) dissolved in 50 mM sodium acetate buffer (pH 4.8) was incubated at 50˚C for 15 min. The reducing sugar released in the mixture was determined using DNS method with xylose as the standard. One unit of enzyme activity was defined as the amount of enzyme capable of releasing 1 μmol of xylose per minute

Quantitative RT-PCR (qRT-PCR)

Total RNAs were extracted using Trizol reagent (Sangon Biotech, Shanghai, China) and purified using the TURBO DNA-free kit (Ambion, Austin, TX, USA) to eliminate genomic DNA contamination according to the manufacturer’s instructions. Reverse transcription was performed using HiScript Q RT SuperMix for quantitative PCR (qPCR) (Vazyme, Nanjing, China) according to the user protocol. Quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a Roche LightCycler 96 thermocycler (Roche). Gene expression was analyzed using the comparative ΔΔCT method [78], whereby the target gene’s threshold cycle (CT) value was first normalized to the endogenous control actin within each sample (ΔCT = CT_target – CT_actin), subsequently compared to a calibrator sample to calculate ΔΔCT (ΔΔCT = ΔCT_sample – ΔCT_control), and finally converted to a relative fold-change using the formula 2^^(–ΔΔCT).

DSP Cross-linking and Immunoprecipitation (IP)

The cross-linking procedure for detecting the potential SCF complex in T. reesei was performed as follows. Conidia spores were inoculated in the MM medium containing 1% glucose at 30°C for 36 h and the mycelia were transferred to MA medium containing 1% Avicel for a 24-h induction, which were then harvested and washed twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM, pH 7.4). The collected cells were resuspended in PBS buffer containing 1 mM DSP (dithiobis/succinimidylpropionate) and incubated at 30 °C with shaking at 200 rpm for 1 h to allow protein cross-linking. The reaction was subsequently quenched by adding glycine to a final concentration of 50 mM, followed by incubation at room temperature for 15 min with periodic mixing. Finally, the mycelia were washed with PBS buffer, collected by vacuum filtration, snap-frozen in liquid nitrogen, and stored at –80 °C or processed immediately for subsequent experiments.

To perform IP for detection of SCF after crosslinking treatment, the above frozen mycelis were completely ground to a fine powder and then resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 1 mM EDTA, 5% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 40 μM MG132, 20 mM NEM, and a protease inhibitor cocktail). The intracellular protein concentration was determined using the BCA protein assay method with bovine serum albumin (BSA) as the standard. Equal amounts of protein were then used for immunoprecipitation (IP) with ChromoTek GFP-Trap or Myc-Trap Agarose (Proteintech, Wuhan, China) according to the manufacturer’s instructions. Briefly, 60 µL of GFP-Trap or Myc-Trap beads were aliquoted and equilibrated twice with 1200 µL of dilution buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) with centrifugation at 5000 g for 2 min each time. Subsequently, an equal amount of mycelial lysate (concentration-normalized and diluted with dilution buffer as needed) was mixed with the equilibrated beads and incubated with rotation at 4 °C for 3 h. After centrifugation at 5000 g for 2 min, the beads were then washed sequentially with 1.2 mL of dilution buffer containing 20% glycerol, 1 M NaCl, 4 M urea, and 1% Triton X‑100 at 4 °C for 5 min on a rotator. The beads were finally washed with 1.2 mL dilution buffer, and bound proteins were eluted twice with an equal volume of 0.2 M glycine (pH 2.5). The eluate was immediately neutralized with 1 M Tris buffer.

To analyze the ubiquitination of Cre1, conidia spores were inoculated in the MM medium containing 1% glucose at 30°C for 36 h and the mycelia were transferred to MA medium containing 1% Avicel for a 2-h induction. Preparation of total cellular extract, immunoprecipiation with GFP-Trap beads and elution were essentially the same as described above.

Western blot

Total cellular extract or protein elute from IP was resolved by SDS-PAGE followed by western blotting with specific antibodies for detection. The following antibodies were used, a GFP monoclonal antibody (B-2, catalog #SC-9996, Santa Cruz Biotechnology), a monoclonal anti-ubiquitin antibody (EPR8830, catalog #ab134953, Abcam), and a Myc monoclonal antibody (60003–2-Ig, Proteintech, Wuhan, China).

Chromatin immunoprecipitation (ChIP) analysis

ChIP assay was essentailly as described previously [79]. Briefly, the mycelia were fixed in minimal medium containing 1% formaldehyde for 10 min at 30°C, 200 rpm and then quenched cross-linking by adding 25 mL 1.25 M glycine (pH 7.5) for 5 min [80]. The cross-linked mycelia were ground and resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, protease inhibitors). Crude lysate were further sonicated to obtain an average DNA fragment size of ~500 bp fragments. For each immunoprecipitation assay, 1 ml protein (2 mg/mL) was used and each sample was pre-cleared with protein A/G at 4°C for 5 h, protein A/G pre-coated with 1 mg/mL BSA and 1 mg/mL denatured fish sperm DNA. And then the chromatin immunoprecipitation was performed using 2 μL anti-Xyr1 antibody at 4°C for 5 h followed by with the same amount of Protein A/G for per IP at 4°C for 5 h. The polyclonal anti-Xyr1 antibody was custom-made by immunizing the rabbit with a KHL-conjugated peptide (aa 106–119) of the DNA binding domain of Xyr1. Alternatively, chromatin immunoprecipitation was carried out using ChromoTek GFP-Trap Agarose (Proteintech, Wuhan, China). Following immunoprecipitation and extensive sequential washing steps with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl, pH 8.0), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 20 mM Tris–HCl, 500 mM NaCl, pH 8.0), and LNDET buffer (0.25 M LiCl, 1% NP40, 1 mM EDTA, 10 mM Tris–HCl, pH 8.0), the immunoprecipiated DNA was eluted with elution buffer (100 mM Tris-HCl, pH 7.8, 10 mM EDTA, 1% SDS, 10 mM NaHCO_3, 100 mM NaCl) at 65 °C for 5 hours. The eluted material was then treated with proteinase K at 45 °C for 1 hour. DNA was recovered by phenol-chloroform extraction and ethanol precipitation. The final DNA concentration was determined by absolute quantification using quantitative PCR (qPCR). ChIP–qPCR data were normalized to the corresponding input DNA (incubated with protein G/A beads), and data are presented as percentage of input DNA. ChIP efficiency was calculated as 2^−ΔCt × 100%, ΔCt = Ct_Sample−(Ct_{Input,adjusted}). An excel file containing the original numerical data for ChIP-qPCR were included as S6 Table.

Recombinant protein production in E. coli

For the expression of TrUbc4 in E. coli, the DNA fragments coding for TrUbc4 were amplified from QM9414 cDNA and ligated into the pGEX4T-1 plas after digestion with NdeI and XhoI to obtain pGEX4T-1-TrUbc4. Then DNA sequencing was performed to confirm. The indicated expression constructs were transformed into CaCl₂-treated competent E. coli BL21 (DE3) cells [81]. E. coli strains with the indicated expression constructs were grown at 37°C until they reached an OD600 of 0.5–0.6. IPTG (iso propyl-b-D-thiogalactopyranoside) was added at a final con centration of 1 mM followed by continued incubation for 16 h at 20°C. The induced proteins were purified with Glutathione Sepharose 4B (GE Healthcare), essentially according to the manu facturer’s instructions. The fusion proteins were eluted using 50 mM Tris-HCl (pH 8.0), 10 mM glutathione. All of the protein preparations were stored at −80°C in the presence of 20% (v/v) glycerol.

In vitro ubiquitination assay

Reactions containing 125 nM human E1 activating enzyme UBA1 (abcam, ab207988) and 1 μM E2 conjugating enzyme TrUbc4 were incubated with 1 μg ubiquitin (abcam, ab80760) in ubiquitination buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 0.5 mM DTT, 2 mM ATP) in a reaction volume of 20 μL for different time at room temperature. Samples were stopped with 5 × SDS-PAGE loading buffer to stop the reaction and proceed for western blot with anti-Ub antibody.

Sequence analysis

Amino acid sequences were obtained from the NCBI database and sequence alignment was performed using ClustalW. Phylogenetic analysis was carried out with MEGA using the neighbor-joining method with 2000 bootstraps [82].

Statistical analysis

Graphs were produced using Prism (GraphPad Software) showing all individual data points, with each point representing one biological replicate. Figures were assembled in Adobe Illustrator. Statistical significance was determined by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test from three biological replicates. Data are presented as the mean ± SD of these replicates.