Cysteine facilitates the lignocellulolytic response of Trichoderma guizhouense NJAU4742 by indirectly up-regulating membrane sugar transporters
Biotechnology for Biofuels and Bioproducts volume 16, Article number: 159 (2023)
Filamentous fungi possess a rich CAZymes system, which is widely studied and applied in the bio-conversion of plant biomass to alcohol chemicals. Carbon source acquisition is the fundamental driver for CAZymes-producing sustainability and secondary metabolism, therefore, a deeper insight into the regulatory network of sugar transport in filamentous fungi has become urgent.
This study reports an important linkage of sulfur assimilation to lignocellulose response of filamentous fungus. Inorganic sulfur addition facilitated biodegradation of rice straw by Trichoderma guizhouense NJAU4742. Cysteine and glutathione were revealed as major intracellular metabolites responsive to sulfur addition by metabolomics, cysteine content was increased in this process and glutathione increased correspondingly. Two membrane sugar transporter genes, Tgmst1 and Tgmst2, were identified as the critical response genes significantly up-regulated when intracellular cysteine increased. Tgmst1 and Tgmst2 were both positively regulated by the glucose regulation-related protein (GRP), up-regulation of both Tgmst1 and Tggrp can cause a significant increase in intracellular glucose. The transcriptional regulatory function of GRP mainly relied on GSH-induced glutathionylation, and the transcription activating efficiency was positively related to the glutathionylation level, furthermore, DTT-induced deglutathionylation resulted in the down-regulation of downstream genes.
Inorganic sulfur addition induces a rise in intracellular Cys content, and the conversion of cysteine to glutathione caused the increase of glutathionylation level of GRP, which in turn up-regulated Tgmst1 and Tgmst2. Subsequently, the sugar transport efficiency of single cells was improved, which facilitated the maintenance of vigorous CAZymes metabolism and the straw-to-biomass conversion.
The bio-production of biofuels and chemicals from plant biomass is one of the most promising ways to solve the current energy and environmental crisis. Filamentous fungi commonly own abundant lignocellulases systems and complex regulatory networks, which can regulate the lignocellulolytic carbohydrate active enzymes (CAZymes) expression flexibly according to different natural carbon sources . Meanwhile, filamentous fungi play an irreplaceable role in terrestrial ecosystems' carbon cycle and soil microbial ecology's stability by degrading the plant residues . Plant biomass components are mainly composed of cellulose (40–50%), hemicellulose (20–40%), and lignin (20–30%) . Filamentous fungi display a considerable utilization capacity of pentose (C5) and hexose (C6), resulting in the efficient degradation of the complete components of plant biomass. Therefore, they are widely used for agricultural solid waste management, especially for the typical representatives: Trichoderma sp., Aspergillus sp., and Penicillium sp. [4, 5].
The degradation efficiency of filamentous fungi on lignocellulosic materials depends on synergistic work of cellulase, hemicellulase, and auxiliary activity (AA) family proteins [6, 7]. Cellulases are composed of endoglucanases (EG), cellobiohydrolase (CBH), and β-glucosidase (BG), which hydrolyze cellulose to form cellobiose and glucose. In contrast, hemicellulases can hydrolyze polymers of xylose, arabinose, or mannose, such as xylan and glucomannan . Therefore, the activity of these enzymes is an important parameter to evaluate biodegradation efficiency. CAZymes expression level is regulated by elaborate regulatory networks, which mainly contain transcription factor XLNR/ACE2, transcriptional repressor ACE1, CCAAT binding complexes HAP2/3/5, and glucose repression protein CRE1 and AREA . In Trichoderma sp. and Aspergillus sp., XLNR/XYR1/CLR1/CLR2 are slated to regulate the expression of cellulase and hemicellulase genes .
The traditional conversion of biomass into biofuels requires saccharification in early stage and crude sugars fermented by Saccharomyces cerevisiae for alcohol production. Recently, efficient alcohol fermentation by filamentous fungi was successful through metabolic engineering, and the bio-conversion of plant biomass into alcohol chemicals can be achieved in one step . Therefore, exploring a method to improve the filamentous fungi's sugar transport capacity will provide support for the program. The intracellular transport efficiency of lignocellulosic hydrolysates (mainly glucose, xylose, and cellobiose) is the primary driving force for enzyme production sustainability and secondary metabolism. The GPCR/cAMP/PKA signaling cascade is responsible for recognizing extracellular sugar and activating sugar transporters in fungi . Git3/Gpr/HXT are identified as the main glucose sensor, which can sense extracellular glucose and activate adenylate cyclase (AC) through G-protein [13, 14]. AC can convert ATP into cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) is the cAMP receptor, which initiates sugar transporters through phosphorylation , and this process involves protein–protein interactions. Sugar transporter in fungi mainly includes glucose transporter GLT1, xylose transporter XYT1, arabinose transporter LAT1, and xylose and arabinose transporter XAT1 , and all of them are homologous to the major facilitator superfamily proteins (MFS) [17, 18].
Trichoderma guizhouense NJAU4742 is a beneficial fungus isolated from compost, which can secrete various plant-promoting factors such as SWO and IAA and possesses a rich CAZymes system, so it has a high industrial and agricultural value [19,20,21,22]. Here, we report the promotional effects of inorganic sulfides on lignocellulolytic response of NJAU4742, which included an increase in biomass and multiple enzyme activities. Through genetic manipulation, transcriptomic analyses, and protein-DNA interaction studies, we uncovered that sulfur assimilation products inducing the upregulation of sugar transporters were direct contributors and revealed the specific regulation modes. This study provides insights into the carbon source metabolism regulatory network of filamentous fungi and supports for metabolic engineering.
Inorganic sulfide significantly enhanced the lignocellulose utilization ability of NJAU4742
Four adjusted mineral mediums (MM) were set up to evaluate the effect of inorganic sulfur on the lignocellulose utilization ability of NJAU4742: sulfur addition treatment (T1 and T2), T1: (NH4)2SO4 (1 mmol) + KH2PO4 (2 mmol), T2: 2 × T1; No sulfur addition treatment (T3 and T4), T3: (NH4)2HPO4 (1 mmol) + K2HPO4 (1 mmol), T4: 2 × T3, other mineral nutrients and pH were adjusted at a same level, various forms of sulfides and soluble sugar in straw have been removed as much as possible. The growth status of NJAU4742 exhibited variation in different treatments (Fig. 1A). The results indicated that the biomass in different treatments increased with content of sulfur, and the highest value (1.83 × 105 copy g−1) was obtained in T2, and the lowest biomass (0.60 × 105 copy g−1) was found in T3 (Fig. 1B). Filter paper enzyme activity (FPA) can be used to characterize overall CAZymes activity. The FPA of T1 (1.67 U g−1) and T2 (2.05 U g−1) were significantly greater than that of T3 (0.79 U g−1) and T4 (0.99 U g−1) (Fig. 1C), and similar trends were obtained in EG, CBH, and Xylanase activity (Additional file 1: Fig. S5A). The results showed that the enzyme activity and biomass of sulfur treatment (T1 and T2) were generally higher than those of no sulfur addition treatment (T3 and T4), indicating that sulfur may have a potential promoting effect on lignocellulosic response of NJAU4742. The difference between T3 and T4 was not significant, indicating that increasing mineral nutrients without sulfur cannot significantly promote lignocellulose utilization.
To verify this hypothesis, the key sulfur assimilating enzyme ATP sulfatase gene (Tgatps, KEGG ID: K00958, GENE ID: A1A109512.1, NCBI ID: OPB38572.1) was overexpressed in OE-Tgatps (21.04 folds up-regulated, Fig. 1E) and deleted in ∆Tgatps. Concurrently, Tgatps was silenced by constructing the pSilent-Tgatps vector to express RNA-induced silencing complex (RISC) (Fig. 1D; Additional file 1: Fig. S2A). The biomass of OE-Tgatps was significantly higher than that of WT, and deletion or silence of Tgatps caused a sharp reduction in biomass (Fig. 1F). The CAZymes of OE-Tgatps were significantly higher than that of WT, while it declined rapidly both in ∆Tgatps and RNAi-Tgatps relative to WT (Fig. 1G; Additional file 1: Fig. S5B). Besides, the growth of RNAi-Tgatps in PDA medium was not affected but worse in MM + straw, which indicated that the reduction of sulfur assimilation would affect the straw utilization capacity of NJAU4742 and did not affect the basic cellular metabolism in full-nutrient medium.
The increase of intracellular Cys and Met facilitated the lignocellulolytic response of NJAU4742
Since inorganic sulfur needs to be assimilated into Cysteine (Cys) before it performs its biological function, and Cys is readily converted to Methionine (Met) . Metabolomic analysis (T1 and T3) showed that Cys and Met metabolism was one of the major metabolism that responded to increased extracellular inorganic sulfide, and the Cys and Met content in T1 were significantly higher than that in T3 (Additional file 1: Fig. S7, Additional file 7: Dataset 3: line190 and line339). The intracellular Cys content in different treatments (T1, T2, T3, and T4) was detected, and results showed that exogenous addition of inorganic sulfide could significantly increase intracellular Cys (Additional file 1: Fig. S1A). The cysteine synthetase gene (TgcysK, KEGG ID: K17069, GENE ID: A1A108125.1, NCBI ID: PTB50504.1) and homocysteine methyltransferase (Tghmt, KEGG ID: K00547, GENE ID: A1A102309.1, NCBI ID: KKP02592.1) gene were the key enzyme gene for Cys and Met synthesis, respectively. TgcysK and Tghmt were deleted respectively to obtain the mutants ∆TgcysK and ∆Tghmt, and both exhibited a normal growth on PDA compared to WT (Fig. 2A), which indicated that sulfur assimilation genes (TgcysK and Tghmt) were not indispensable for basic metabolism when nutrients sufficient. Considering that exogenously added Cys and Met could be used as preferred carbon and thus induced carbon catabolite repression (CCR), the intracellular Cys and Met were respectively increased by overexpressing key synthase genes. TgcysK was overexpressed in OE-TgcysK (52.35 folds up-regulated), and Tghmt was up-regulated by 79.75 folds in OE-Tghmt by comparing with WT (Fig. 2C). The intracellular Cys content in OE-TgcysK (26.18 µmol g−1) and intracellular Met content in OE-Tghmt (33.41 µmol g−1) were higher than that in WT (21.48 µmol g−1 and 29.54 µmol g−1), respectively (Fig. 2D). The biomass and CAZymes of OE-TgcysK and OE-Tghmt were all significantly higher than those of WT, while these parameters were lower in ∆TgcysK than in WT and not significantly different in ∆Tghmt and WT. (Fig. 2B, E; Additional file 1: Fig. S5C). These results illustrated that the effect of sulfur assimilation restriction on NJAU4742 growth on straw was not due to a global effect on basic metabolism, and increase of intracellular Cys and Met facilitated the lignocellulolytic response of NJAU4742.
Tgmst1 and Tgmst2 were the key response genes for increased intracellular Cys
To reveal how Cys and Met affected lignocellulolytic response of NJAU4742, the total RNA of OE-TgcysK, ∆TgcysK, OE-Tghmt, ∆Tghmt, and WT were extracted and transcriptomic analysis was conducted to explore the response genes affected by increased intracellular Cys and Met. After reverse transcription and library construction, low-quality raw reads generated in NGS were filtered out. Clean reads were aligned to the genome database of NJAU4742 and the transcripts number in each sample was obtained. Differential transcripts were screened with P-value < 0.05 and FoldChange > 2, and GO and KEGG enrichment analyses of differential transcripts were also performed.
PCA results showed that transcriptome of OE-TgcysK was significantly different from WT, while there was no significant difference between ∆TgcysK, OE-Tghmt, ∆Tghmt, and good repeatability of the experiment (Fig. 3A). The correlation coefficient matrix showed the correlation between different samples, among which ∆TgcysK exhibited the most significant difference to WT (Fig. 3B). Venn showed the numbers of differentially expressed genes in treatments (Fig. 3C). There was no significant difference between transcriptome of OE-Tghmt and ∆Tghmt and have not significantly up/down-regulated functional genes in OE-Tghmt by comparing with WT (Additional file 3:, Additional file 4: Dataset 1B). Thus, Met may not play a key role in the sulfur-promoted lignocellulolytic response, and the phenotype and mechanism of OE-TgcysK and ∆TgcysK were primarily analyzed in subsequent studies.
The differentially expressed genes in OE-TgcysK were mainly ± 4 FoldChanges relative to WT (Fig. 3D). KEGG enrichment showed that the differential genes in OE-TgcysK mainly belonged to amino acid metabolism, carbohydrate metabolism, energy metabolism, and transport metabolism (Fig. 3E), which indicated that the increase of intracellular Cys content would affect these processes. Additionally, GO classification from three categories of cell component (CC), molecular function (MF), and biological process (BP) showed the functional distribution of up-regulated genes in OE-TgcysK (Fig. 3F). The up-regulation folds of TgcysK in transcriptome (10.00 folds) differed from qPCR result (52.35 folds), which might be due to the large gene expression basal values (23,472.7 reads) and fluctuations of transcription level. TgcysK was almost undetectable in ∆TgcysK and only had 0.9 reads (Additional file 3: Dataset 1A).
The highest up-regulated gene was identified as a membrane sugar transporter belonging to the major facilitator superfamily (MFS) and named Tgmst1 (GENE ID: A1A101071.1, NCBI ID: M431DRAFT_463245). Tgmst1 was up-regulated 36.3 folds in OE-TgcysK and down-regulated 3.3 folds in ∆TgcysK. Interestingly, a homofunctional gene named Tgmst2 with a large FoldChange was also up-regulated (11.7 folds) in OE-TgcysK (GENE ID: A1A110058.1, NCBI ID: M431DRAFT_70791). Tgmst2 was also related to transmembrane transport and belonged to MFS (Additional file 3: Dataset 1A). These results signified that an increase in intracellular Cys will induce an up-regulation of sugar transportation-related genes, which implied that Cys may strengthen the lignocellulolytic response of NJAU4742 by activating various sugar transporters.
Up-regulation of MST1 caused an increase in intracellular glucose content. Since Tgmst1 and Tgmst2 belong to the same family and the expression base level and FoldChange of Tgmst1 are much larger than Tgmst2, Tgmst1 was selected as the primary gene for research. The Tgmst1 overexpression strain OE-Tgmst1 (36.44 folds up-regulated) (Fig. 4B) and Tgmst1 deletion mutant ∆Tgmst1 were constructed to reveal the function of MST1 in NJAU4742. The growth of OE-Tgmst1 was better than WT on MM + straw and MM + glucose, while ∆Tgmst1 grew worse than WT (Fig. 4A). FPA of OE-Tgmst1 and ∆Tgmst1 were 2.3 U g−1 and 1.2 U g−1, respectively, and WT was 1.7 U g−1 (Fig. 4C); EG, CBH, and Xylanase activities of OE-Tgmst were significantly higher than that of WT (Additional file 1: Fig. S5D). Similarly, the biomass (2.41 × 105 copy g−1) and intracellular glucose content (58.2 µmol g−1) of OE-Tgmst1 were both higher than that of WT (1.64 × 105 copy g−1, 47.2 µmol g−1). The biomass and intracellular glucose of ∆Tgmst1 (0.89 × 105 copy g−1 and 38.9 µmol g−1) were significantly lower than that of WT, which implied that MST1 is a critical sugar transporter for NJAU4742, and its absence dramatically affects sugar transport capacity, even on the PDA (Fig. 4A, D, E). MST1 also shared high homology with many glucose/xylose/hexose transporters (Additional file 1: Fig. S10). The results showed that the intracellular glucose content of NJAU4742 was positively correlated with the expression level of Tgmst1, which further illustrated that MST1 may be responsible for glucose transportation.
GPCR/cAMP/PKA was a conserved extracellular glucose recognition and signals transduction pathway, and PKA was a conservative protein . The vectors pGADT7-PKA and pGBKT7-MST1 were constructed to perform the yeast two-hybrid assay (Y2H). Yeast transformant grew normally on SD-His-Leu-Trp medium, which suggested that PKA and MST1 have protein–protein interaction. Concurrently, it was almost unable to grow on the SD-Ade-His-Leu-Trp medium, indicating the relatively weak interaction between PKA and MST1 (Fig. 4F). These results obtained above further certify that MST1 was a sugar transporter depending on the activation of PKA.
The optical glucose FRET sensor further verified the function of MST1
The strain TgGluSensor expressing the optical glucose FRET sensor was constructed to monitor the intracellular glucose content in living cells. Since repeated sequences in genome will induce DNA repair, the sequence of the FRET sensor was inserted into the genome while removing the bio-marker (hph, ura3) (Additional file 1: Fig. S2C). The FRET sensor gene was cloned from pDR-GW FLII12Pglu-700μ∆6 (Addgene plasmid #28,002). The fluorescence of FRET sensor comes from cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), both of which were linked via glucose recognition subunit MglB. Glucose binding will change the conformational of MglB, which leads to a change in distance or orientation between CFP and YFP, resulting in a FRET change (Additional file 1: Fig. S2B). The fluorescence intensity ratio of YFP and CFP (FY/FC) could characterize the intracellular glucose content, and the FY/FC value was positively correlated with intracellular glucose content [24, 25].
The strain TgGluSensor was used as original strain and the Tgmst1 overexpression strain TgGluSensor::OE-Tgmst1 was constructed. Meanwhile, the ura3 gene was backfilled into the TgGluSensor genome to obtain the control strain TgGluSensor::CON. TgGluSensor::OE-Tgmst1 and TgGluSensor::CON were cultured on MM + straw (Fig. 5A). The intracellular glucose content of TgGluSensor::OE-Tgmst1 (61.1 µmol g−1) was significantly higher than that of TgGluSensor::CON (48.6 µmol g−1) (Fig. 5D). The fluorescence intensity detected by confocal laser scanning microscope (ZEISS LSM 980), and the FY/FC ratio was counted by ZEN3.4 (Fig. 5B1, 2). The FY of TgGluSensor::OE-Tgmst1 was significantly higher than the FC, while the FY of TgGluSensor::CON was almost the same as the FC (Fig. 5C1, 2). The FY/FC ratio was calculated by counting the pixel luminous intensity. The FY/FC median of TgGluSensor::OE-Tgmst1 and TgGluSensor::CON were1.66 and 0.89 (Fig. 5E) respectively, which indicated that the intracellular glucose concentration of TgGluSensor::OE-Tgmst1 was significantly higher than that of TgGluSensor::CON in living cell. These results further support that MST1 was a sugar transporter with glucose transport capacity.
GRP regulated the transcription of Tgmst1 and Tgmst2 by interacting with their promoters
DNA pull-down assay was performed to confirm the transcription factor of Tgmst1. The promoter region (− 2 kb to − 1 bp) of Tgmst1 was amplified by a biotin-labeled primer (Additional file 1: Fig. S3A) and then combined with streptavidin magnetic beads as the probe (EXP); the fragment without biotin was amplified to be used as control (CON) (Fig. 6A). The probe was incubated with nucleoprotein, and magnetic beads were used to pull down the probe-bound proteins. The precipitated proteins were separated by SDS-PAGE to detect differences between EXP and CON (Fig. 6B). Differential protein strips were digested into polypeptides and identified by LC–MS/MS. Raw data was submitted to the ProteinPilot for NJAU4742 genome database retrieval analysis. With confidence ≥ 0.95 and unique peptides ≥ 1, the numbers of secondary mass spectra peaks of EXP and CON were 431 and 339, and the identified protein numbers of EXP and CON were 116 and 95, while the shared protein numbers were 79 (Fig. 6C). IPR analysis results showed that a high abundance of Hsp70 structural domains were detected in pull-down products (Additional file 1: Fig. S3B), and GO and KEGG enrichment revealed the proteins functions and their associated metabolic pathways (Additional file 1: Fig. S3C, D). The identified proteins were scored according to the signal intensity of mass spectrometry. Proteins with high scores were listed as follows: F-type H+-transporting ATPase subunit β (ATPeF1B, GENE ID: A1A106337.1, NCBI ID: KKP03928.1), glucose regulation-related protein (GRP, GENE ID: A1A105631.1, NCBI ID: KKP00680.1), catalase-peroxidase (KatG, GENE ID: A1A103615.1, NCBI ID: OPB43868.1), F-type H+-transporting ATPase subunit α (ATPeF1A, GENE ID: A1A100553.1, NCBI ID: OPB46749.1), and elongation factor 5α (EF5A, GENE ID: A1A106315.1, NCBI ID: OPB41649.1) (Fig. 6D).
The interactions between these five proteins and the promoter of Tgmst1 were evaluated through yeast one-hybrid assay (Y1H). The vector pAbAi-Tgmst1P was constructed by inserting the Tgmst1 promoter fragment (− 500 to − 1 bp) and it was transformed into Matchmaker Gold Yeast. The transformant (Bait-reporter) cannot grow on SD-Ura containing 400 ng mL−1 aureobasidin A (AbA). Subsequently, the vectors pGADT7-ATPeF1B, pGADT7-GRP, pGADT7-KatG, pGADT7-ATPeF1A, and pGADT7-EF5A were constructed and transformed respectively into bait-reporter. The results indicated that there was a strong interaction between GRP and Tgmst1 promoter, and both KatG and EF5A could weakly interact with Tgmst1 promoter, while ATPeF1A and ATPeF1B barely interacted with Tgmst1 promoter (Fig. 6E).
Furthermore, ChIP-qPCR assay was performed to verify the interaction between GRP and Tgmst1 promoter. The strain GRP-His that 6 × His-tag was added to the C-terminus of GRP was constructed and verified by Western blot (Additional file 1: Fig. S4A). After cross-linking and nucleic acid breaking, GRP-binding DNA fragments were precipitated by a ChIP-class antibody, and the DNA combined by GRP was collected. qPCR was respectively carried out by using the collected DNA of IP, Mock (IgG), and Input as the template to detect the copies of Tgmst1 promoter fragment. The Cts values from primers P-5 and P-6 were significantly different from IP and Mock (IgG) (Additional file 1: Fig. S4B, C1). The qPCR results from primer P-6 were used to count Percent input (Additional file 1: Fig. S4C2) and Fold enrichment (Additional file 1: Fig. S4C3). The Percent input of IP and Mock (IgG) was 17.6% and 0.296%, respectively (Fig. 6F). The Fold enrichment could indicate the specific binding efficiency, and results showed that the enrichment efficiency of GRP for Tgmst1 promoter in IP was 61.6 folds higher than that in Mock (IgG) (Fig. 6G). The amplification range of primer P-6 was -363 bp to -224 bp, which probably contained the binding region of GRP to Tgmst1 promoter.
In addition, we were curious about whether another up-regulated MFS protein gene Tgmst2 was also regulated by GRP protein. The DNA–protein interaction verification between GRP and Tgmst2 promoter (− 500 to − 1 bp) was also conducted through Y1H assay. Surprisingly, GRP and Tgmst2 promotor also exhibited interaction (Fig. 6H), which indicated that the up-regulation of Tgmst2 might also be achieved through GRP.
Tgmst1 and Tgmst2 were positively transcriptional regulated by GRP
The interaction between GRP and Tgmst1 promoter might be accompanied by a specific regulatory relationship. To verify this hypothesis, Tggrp deletion mutant ∆Tggrp and Tggrp overexpression strain OE-Tggrp (45.4 folds up-regulated, Fig. 7B) were constructed. OE-Tggrp grew better on MM + straw than WT, and ∆Tggrp was worse than WT, and they did not differ in growth on PDA. It was noteworthy that the editing of Tggrp affected the growth on MM + glucose, suggesting that Tggrp might be able to influence glucose acquisition (Fig. 7A). The biomass (2.56 × 105 copy g−1) and intracellular glucose content (65.2 µmol g−1) of OE-Tggrp were both higher than WT (1.68 × 105 copy g−1, 54.2 µmol g−1), and these parameters were declined in ∆Tggrp (1.04 × 105 copy g−1 and 46.3 µmol g−1) (Fig. 7C, E). The FPA of OE-Tggrp and ∆Tggrp were 2.24 U g−1 and 1.14 U g−1, respectively, and it was 1.55 U g−1 of WT (Fig. 7D). Similarly, the EG, CBH, and Xylanase activities of OE-Tggrp were significantly higher than that of WT, while these parameters were dropped in ∆Tggrp (Additional file 1: Fig. S5E). The qPCR on Tgmst1 and Tgmst2 was conducted, and the expression level of Tgmst1 and Tgmst2 were up-regulated by 369.1 folds and 151.7 folds respectively in OE-Tggrp (Fig. 7F), which indicated that GRP owned a positive regulation to Tgmst1 and Tgmst2.
To demonstrate the concordance between protein level of MST1/2 and transcription level of Tgmst1/2, the strains OE-Tggrp::MST1/2-His and WT::MST1/2-His were constructed by adding 6 × His-tag to the C-terminus of MST1/2 based on OE-Tggrp and WT, respectively. The expression level of MST1 and MST2 in OE-Tggrp and WT was determined by Western blot, and β-actin as the internal reference. The results indicated that MST1 and MST2 were respectively up-regulated by 1.7 folds and 1.8 folds in OE-Tggrp (Fig. 7G), which suggested that the protein expression level of MST1 and MST2 was consistent with transcription level in OE-Tggrp.
GRP could affect the intracellular glucose content of NJAU4742
To prove that GRP could affect the intracellular glucose content by regulating the expression of MST1 and MST2, Tggrp overexpression strain TgGluSensor::OE-Tggrp was constructed based on the strain TgGluSensor. After growing on MM + straw at 28 ℃ for 4 days (Fig. 8A), the intracellular glucose concentration of TgGluSensor::OE-Tggrp (63.8 µmol g−1) was significantly higher than that of TgGluSensor::CON (52.3 µmol g−1) (Fig. 8D). Simultaneously, the FY was significantly higher than FC in TgGluSensor::OE-Tggrp, while FY was almost similar to FC in TgGluSensor::CON (Fig. 8B). The statistical results of fluorescence intensity were also consistent with the results of graph (Fig. 8C). The median of FY/FC ratio in TgGluSensor::OE-Tggrp was 1.72, and it was 0.98 in TgGlu::Sensor-CON (Fig. 8E), which indicated that the intracellular glucose concentration of TgGluSensor::OE-Tggrp was higher than that of TgGluSensor::CON in living cell. The results showed that GRP could also promote glucose transportation by up-regulating the expression of Tgmst1 and Tgmst2.
Sulfide addition facilitated GSH synthesis and induced glutathionylation of GRP
Inorganic sulfide assimilation could produce large amounts of primary assimilate Cys (Additional file 1: Fig. S1A), which in turn facilitated the synthesis of glutathione (GSH). Metabolomic analysis (T1 and T3) showed that GSH metabolism was one of major metabolisms that dramatically responded to increased exogenous inorganic sulfide, and the GSH content in T3 was higher than that in T1 (Additional file 1: Fig. S7, Additional file 7: Dataset 3 line5). GSH could induce glutathionylation by forming the disulfide bonds with cysteine residues of proteins . The glutathionylation of transcription factors was beneficial to maintain the structural stability of functional domains and promote the efficiency of DNA binding domain or activation domain, thus increasing the transcriptional activation efficiency .
The intracellular GSH content of WT growing in four treatments (T1, T2, T3, and T4) and the two strains (OE-Tgatps and OE-TgcysK) were determined. The intracellular GSH contents in T1 (12.5 µmol g−1) and T2 (17.3 µmol g−1) were both significantly higher than that in T3 (7.6 µmol g−1) and T4 (8.1 µmol g−1) (Fig. 8F1). The intracellular GSH contents in OE-Tgatps (15.4 µmol g−1) and OE-TgcysK (15.3 µmol g−1) were also significantly higher than that of WT (12.2 µmol g−1) (Fig. 8F2). These results further illustrated that exogenous addition with inorganic sulfide or enhancing the sulfur assimilation would increase the intracellular GSH content.
Glutathionylation is common in Hsp70 family proteins and has positive implications for stabilizing protein structure and promoting substrate catalytic efficiency. Generally, Hsp70 homologs have two individual domains, the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD), which were connected by a flexible linker [28, 29]. GRP, a typical Hsp70 family protein, was also likely to be glutathionylated when intracellular GSH levels were elevated. Commercial anti-GSH antibodies were usually used to evaluate the glutathionylation level. However, the currently available anti-GSH antibody could only test the purified protein . Here, the strain GRP-His was cultured in T1 conditions for protein extraction. GRP was purified by nickel ion magnetic beads for Western blotting. The rabbit polyclonal anti-GSH antibody was primary antibody, and Alexa Flour 488 labeled goat anti-rabbit IgG (H + L) antibody as secondary antibody. The antibody hybridization signal was detected (Additional file 1: Fig. S1B), which indicated that the increase in intracellular GSH caused by inorganic sulfide addition could induce the glutathionylation of GRP.
The expression of MST1 and MST2 positively correlated with glutathionylation level of GRP
Based on the previous results, inorganic sulfide addition could induce glutathionylation of GRP by elevating the intracellular GSH content. Therefore, glutathionylation of GRP might play a central role in the pathway of Cys promoting the up-regulation of Tgmst1/2. Exogenous GSH addition could directly improve the glutathionylation level of some proteins, while dithiothreitol (DTT) could break the disulfide bond and prevent its formation . Since MM + straw was a solid medium, it was not available to show the dosage of GSH and DTT in terms of concentration. Thus, 0.1 mmol of GSH and DTT were added to each plate. GRP was isolated and purified and the concentration was equalized. The hybridization signal intensity of anti-GSH antibody was increased in GSH added treatment (+ GSH, − DTT) and decreased in DTT added treatment (− GSH, + DTT) compared to the control without GSH and DTT addition (− GSH, − DTT) (Fig. 8G1). Notably, since treatments have equal inputs, the blot area was consistent, but there was a significant difference in gray-depth of blot. Gray-depth was dependent on the glutathionylation level of GRP. In this study, the relative glutathionylation level of GRP was used to describe the glutathionylation degree. The grayscale of blots in WB images was counted by the software Image J, and the statistics showed that the relative glutathionylation level of GRP in GSH added treatment (+ GSH, − DTT) was higher than control (− GSH, − DTT), while DTT added treatment (− GSH, + DTT) was lower than control (Fig. 8G2). This result suggested that the glutathionylation level of GRP could be regulated by exogenous GSH or DTT addition.
To investigate the effect of different glutathionylation levels on GRP regulating the expression of MST1 and MST2, the strains WT::MST1-His and WT::MST2-His were cultured on MM + straw added with GSH or DTT, after which the total protein of each treatment was extracted and normalized. The expression level of MST1 and MST2 was quantified by Western blot with β-actin as internal reference. Here, both blot gray-depth and area of MST1 and MST2 were significantly increased in GSH added treatment, while the opposite result was obtained in DTT added treatment (Fig. 8H1). By comparing the internal reference adjusted MST1 and MST2 expression levels of treatments, GSH exhibited a promoting effect on MST1 and MST2 expression while DTT inhibited this process (Fig. 8H2). Previous results have shown that GSH induces glutathionylation while DTT induces deglutathionylation, therefore the results shown in Fig. 8H indicated that the expression level of MST1/2 was positively correlated with glutathionylation level of GRP. Therefore, it was reasonable to extrapolate that Cys promoted MST1/2 expression by indirectly increasing the glutathionylation level of GRP, which in turn increased sugar transport efficiency and ultimately enhanced the lignocellulolytic response of NJAU4742.
The bioconversion of straw to alcohol chemicals usually requires saccharification by microorganisms or industrial lignocellulases followed by fermentation by Saccharomyces cerevisiae [31, 32]. Recombining the fermentation and alcohol tolerance related genes into filamentous fungi is a promising way to achieve a one-step conversion of straw to alcohol or organic acid [33, 34]. Fermentation requires a constant and stable monosaccharides supply, and efficient sugar transport can greatly improve fermentation efficiency. Up-regulation of sugar transport capacity also facilitates efficient cellulase production by microbial cell factories.
Sulfur deposition caused an increase in soil fungi/bacteria ratio, inducing microorganisms to obtain more carbon by increasing the ratio of carbon/nitrogen-acquiring enzymes while decreasing carbon inputs for respiration and increasing carbon utilization efficiency to accommodate carbon limitation . This research enlightened us to find the connection between sulfur assimilation and carbon source utilization pathway. To reveal the response of intracellular metabolites to inorganic sulfur addition, we conducted a metabolomics analysis. Intracellular glutathione and cysteine were revealed to be significantly affected and showed coupled associations with lignocellulolytic response. As the primary sulfur assimilate product, Cys was an important sulfur storage form in cells , which implied to us that Cys was the critical metabolite in response to sulfur addition increase.
The intracellular Cys and Met content were increased by genetic manipulation for transcriptomic analysis. Due to the absence of high-fold differentially expressed genes in the OE-Tghmt transcriptome, we excluded the possibility that Met played a central role in the promotion effect of inorganic sulfides (Additional file 4: Dataset 1B). Tgmst1 and Tgmst2 were the key genes responding to the increased intracellular Cys content. MST1 and MST2 were novel sulfur assimilation-related sugar transporters that belonged to the MFS family, and many monosaccharide transporters share high homology with MST1/2, including xylose transporters, glucose transporters, and hexose transporters (Additional file 1: Fig. S10). In addition, the transport substrates of MST1/2 may not be specific, and the substrate diversity of sugar transporters is universal . For example, the glucose transporter protein HxtB in Aspergillus nidulans also transports xylose . Tr69957 identified by Roberto N. Silva is responsible for transporting mannose, xylose, and cellobiose , and another sugar transporter STP1 is involved in transporting cellobiose and glucose . TrSTR1 is able to transport xylose and arabinose . This implies that this MST1/2 may be able to transport a variety of substrates, including glucose and even cellobiose. Interestingly, Cys could significantly decline the blood sugar . After being fed with Cys for eight weeks, the blood sugar content was also significantly lower than that of control [43, 44]. These results suggest that Cys promotes cellular glucose absorption, which seems similar to our results that Cys can promote cell absorption of extracellular sugar. In summary, with straw as the sole carbon source, the up-regulation of straw hydrolysis products (glucose, xylose, cellobiose, etc.) transport capacity undoubtedly facilitates the maintenance of a vigorous cellular metabolism, especially CAZymes metabolism.
The protein-DNA interaction and regulatory relationship between GRP and Tgmst1 and Tgmst2 have been verified. Transcription factors (TFs) can be functionally divided into two regions: DNA binding domain (BD), which includes helix turn helix (HTH)/helix loop helix (HLH), zinc finger, and basic leucine zipper (bZIP); transcriptional activation domain (AD), which can affect transcription efficiency by directly or indirectly acting on transcription complex [45,46,47]. GRP belongs to the Hsp70 family and has a complete function in transcriptional activation. Hsp70 family proteins are ancient and conserved that were first found to be related to heat shock response . More studies showed the functional diversity of Hsp proteins, and they participated in various physiological activities, including signal transduction, transcriptional activation, apoptosis, transmembrane transport, and DNA repair [49, 50]. Hsp70 proteins and their homologs have two separate domains, named ATPase or nucleotide-binding domain (NBD) and substrate-binding domain (SBD), and the functional diversity of Hsp70 proteins mainly originates from the SBD . GRP possessed BIP-like ATP and nucleotide-binding domain and HTH/HLT-like nucleotide-binding domain, which collaborated to comprise the functionality integrity.
Glutathionylation is a common post-translational modification of proteins caused by sulfur compounds. It is the process of forming mixed disulfide between glutathione and protein cysteine residues, and it is also the protective mechanism of active sulfhydryl group . GSH residue is an active molecule, which may show a variety of conformations in solution or combination with protein. The affinity of anti-GSH antibodies may be reduced significantly owing to the changes in protein conformation or thiolation environment . Currently, available anti-GSH antibodies can be used for purified protein analysis, so GRP should be purified before detection. Glutathionylation has diverse effects on protein function, some studies suggested that glutathionylation inhibits the protein function of c-Jun and NF-kB [53, 54]. However, more reports are consistent with our study. Peroxynitrite can increase SERCA activity by glutathionylation, the presence of GSH is required for the functioning of recombinant SERCA in phospholipid vesicles, and its Ca2+ uptake activity is dependent on peroxynitrite-induced glutathionylation . Our results showed that the up-regulation of glutathionylation of GRP facilitated its transcriptional activation function, which exhibited the promoting effect of glutathionylation on protein function. In addition, glutathionylation of FABP5 at Cys127 promoted its nuclear translocation, glutathionylated FABP5 would enter nucleus and activate transcription of downstream genes . Similar results were obtained in our study, suggesting that glutathionylation promoting protein transcriptional activation efficiency existed.
As a massive element in cells, sulfur has a global impact on cellular metabolism, other assimilates may jointly be involved in this process. Existing studies on the effects of glutathionylation on proteins are not comprehensive. The glutathionylation site and the mechanism of glutathionylation affect the transcriptional activation efficiency of GRP are not elaborated. However, this research still has significance in biomass utilization, the novel sulfur metabolism-regulated sugar transporter proteins provide new targets of molecular cloning for metabolic engineering, and the revealed regulatory pathway of transporters MST1/2 provides the possibility of subsequent gene controllable expression and metabolic modification. During microbial utilization of sugar for alcoholic fermentation or organic acid synthesis, single-cell sugar transport capacity controls the superior limit of fermentation efficiency, and our study may contribute to increasing the superior limit of fermentation efficiency.
This study reported the facilitative effect of inorganic sulfide on lignocellulolytic response of NJAU4742 and elucidated the molecular mechanism of this process (Fig. 9). Cys and GSH were identified as the main differential intracellular metabolites responding to inorganic sulfur addition, and were also closely associated with the lignocellulolytic responses. GSH content was up-regulated due to increased Cys, which induced upregulation of the glutathionylation level of GRP and ultimately upregulated Tgmst1/2 expression. The transcriptional level of the Tgmst1 and Tgmst2 were positively correlated with the glutathionylation level of GRP. MST1 and MST2 were critical proteins that respond to increased intracellular Cys. Up-regulation of MST1 and MST2 enhanced cellular acquisition of extracellular sugar, which facilitated the maintenance of vigorous CAZymes production. In brief, Cys achieved an up-regulation of Tgmst1 and Tgmst2 by indirectly increasing the glutathionylation level of GRP, which in turn promoted the extracellular sugar transport capacity. Efficient carbon source acquisition enhanced the lignocellulolytic response of NJAU4742.
Plasmids, strains, and culture conditions
Plasmids were propagated in Escherichia coli DH5α (Tsingke Biotechnology, China). Bacteria were grown in LB medium (tryptone, 10 g L−1; yeast extract, 5.0 g L−1; NaCl, 5.0 g L−1) at 37 °C. T. guizhouense NJAU4742 (genomic sequence NCBI ID: LVVK00000000.1, Genetic database: https://bioinfo.njau.edu.cn/tgn4742/index.php) was isolated from composting sample and stored in our Lab, growing on PDA/PDB (Oxoid, UK) or MM (Carbon source, 10 g L−1; KH2PO4, 15 g L−1; (NH4)2SO4, 10 g L−1; MgSO4, 0.6 g L−1; FeSO4, 5.0 mg L−1; MnSO4, 1.6 mg L−1; ZnSO4, 1.4 mg L−1; CoCl2, 2.0 mg L−1; CaCl2 1.0 g L−1) at 28 °C. MM + straw was prepared by using liquid MM to adjust the moisture content of straw to 75% for the growth comparison. The Matchmaker Gold Yeast and Y2HGold Yeast (Takara, Japan) were respectively used in yeast one-hybrid (Y1H) and yeast two-hybrid (Y2H), which were grown in YPDA medium (Hopebio, China). SD-Leu-Trp medium was used to screen the positive Y2H transformants, SD-His-Leu-Trp and SD-Ade-His-Leu-Trp medium was used for protein–protein interaction validation. SD-Ura-Leu medium was used to screen the positive Y1H transformants, and SD-Ura-Leu + AbA was used for protein-DNA interaction validation. pAbAi and pGADT7 (Takara, Japan) were used for vector construction in Y1H, while pGBKT7 and pGADT7 (Takara, Japan) were used for Y2H. pDR-GW FLII12Pglu-700μ∆6 (Addgene plasmid #28,002) was used as template to clone the optical intracellular glucose FRET sensor gene. pSilent-1 (Miaolingbio, China) was used to construct the vector to interfere with RNA translation.
CAZymes activity assay
The CAZymes secreted by hyphae were dissolved in NaAc buffer (HAc, 10 mM; NaAc, 100 mM; pH5.5). The EG /Xylanase / FPA activity assay was carried out as follows: 480 μL CMCNa (0.5%, W/V) / 480 μL Xylan (0.5%, W/V) / Whatman filter paper, 500 μL NaAc buffer, and 20 μL crude enzyme solution were mixed, and the reaction system was kept at 50 °C for 20 min, and then 1 mL DNS (NaKC4H4O6, 185 g L−1; C7H4N2O7, 6.3 g L−1; NaOH, 20.1 g L−1; C6H5OH, 5 g L−1; Na2SO3, 5 g L−1) was added to react in boiling water for 10 min, after which OD was determined at 520 nm. CBH activity assay was carried out as follows: 25 μL PNPC (5 mM), 25 μL NaAc buffer, 30 μL ddH2O, and 20 μL crude enzyme solution were mixed, and the system was kept at 50 °C for 20 min, and then 100 μL Na2CO3 (1 M) was added, after which OD was determined at 402 nm. One enzyme activity unit was defined as the amount of enzyme required to liberate 1 µmol glucose or pNP per minute under assayed conditions [57, 58].
Intracellular Cys, Met, and GSH content assay
Hyphae were washed several times with deionized water and then rapidly frozen with liquid nitrogen and milled. The homogenate was dissolved in the extraction solution (mass/volume of extracting solution = 1 / 5) and 8000 × g centrifuged at 4 °C for 10 min, and the supernatant was kept on ice for subsequent tests. Cysteine/Methionine Assay Kit (Abcam, UK) and Glutathione content detection Kit (Boxbio, China) were used to determine the content of intracellular Cys, Met, and GSH.
The hyphae and medium (straw) were fully mixed, and 10.0 g sample was accurately weighed. The samples were rapidly frozen with liquid nitrogen and milled. Hyphae DNA was extracted by the PowerSoil Pro Kit (QIAGEN, Germany) according to the manufacturer's instructions and then dissolved with the same volume of ddH2O. Copies of DNA solution: 4.5 × 1010 copy μL−1 (2000 bp, 100 ng μL−1), dilute 10 folds successively, and the Cts of each treatment were determined by qPCR. The standard curve equation is Y = (34.3 − X)/3.2, R2 = 0.999, Y = log10(copies of template), X = Average of Cts.
Gene knockout and overexpression in NJAU4742
The homologous arms were obtained through a PCR kit (TaKaRa, Japan). hygromycin phosphotransferase gene (hph) and the uridine synthetase gene (ura3) were the biomarkers. The functional fragment structure of knockout and overexpression is “upstream-hph/ura3-downstream” and “upstream-hph/ura3-promoter-gene” respectively. 100 μL spores (1 × 108 spores mL−1) were coated on PDA covered with cellophane, the newly germinated spores were lysed by Solution A (Sorbitol, 6 M; KH2PO4, 0.5 M; pH was adjusted to 5.6 by KOH) containing fungal cell-wall lyase (SIGMA, US) and protoplasts.
Protoplasts, 400 μL Solution B (Sorbitol, 10 M; CaCl2·2H2O, 0.5 M; 5 mM pH 7.5 Tris–HCl), 100 μL PEG solution (PEG6000, 25%, W/V; CaCl2·2H2O, 0.5 M; 5 mM 7.5 Tris–HCl), and 20 μL functional DNA (200 ng μL−1) were mixed gently, and then incubated on ice for 20 min, after which 2 mL PEG solution and 3 mL Solution B was gradually added. The mixture was uniformly coated on sucrose PDA (PDA, 39 g L−1; sucrose,1 M), cultured at 28 °C for about 18 h, and then covered with Hygromycin PDA (PDA, 39 g L−1; Hygromycin B, 0.2 g L−1) on coated plate. The transformants were verified by PCR, and the correct transformant need to be verified again after passage .
The 6 × His-tag labeled strains were cultured on medium at 28 °C for 3 days, and then total protein was extracted by RIPA lysis buffer (Beyotime, China). The protein was transferred to the nitrocellulose membrane after SDS-PAGE, and then blocked with TBST (Solarbio, China) containing 5.0% non-fat milk. After being incubated with mouse monoclonal 6 × His-tag antibody (Beyotime, China) at 4 °C overnight, the membrane was incubated with Goat-anti-Mouse IgG (H + L) Alexa Fluor 680 labeled antibody (ThermoFisher, US) at normal temperature for 2 h. Finally, the blot was detected by Protein Immunoblotter (Bio-Rad, US).
pSilent-1 is a shuttle plasmid with hygromycin and ampicillin resistance gene, and it has two polyclonal sites on both sides of the Magnaporthe grisea cutinase gene intron [60, 61]. Using the One Step Cloning Kit (Vazyme Biotech, China), the sense chain of target gene was inserted into polyclonal sites, and the other with the anti-sense chain was inserted into polyclonal sites. The vector can express the stem-loop structure RNA that could be recognized and cut by Dicer to form a Silencing-complex (RISC). RISC could specifically identify and cut the RNA transcribed by the target gene  (Additional file 1: Fig. S1a). According to the method of transformation mentioned above, the correct vector was transformed and verified by PCR.
Construction of the glucose FRET sensor functional strain
The FRET sensor gene was cloned from pDR-GW FLII12Pglu-700μ∆6, and the hph gene was cloned from pSilent-1, while other DNA fragments were cloned from the NJAU4742 genome. The above cloned gene fragments were fused by fusion PCR into a long DNA fragment with gene expression function, which is structured as “ura3-hph-ura3 upstream-promoter-sensor-ura3 downstream”. The fragments were transferred into the protoplasts by using the transformation method mentioned above (PEG/CaCl2 transformation method), The ura3 and ura3 downstream act as homology arms and recombine homologously with the NJAU4742 genome. Since the inserted fragment contains ura3 upstream, this results in the presence of repetitive sequences in the inserted fragment and the genome, which induces DNA repair and results in the loss of the fragment between the repetitive sequences. Taking advantage of this feature, we can knock out the ura3 gene while inserting the FRET sensor gene into the genome, and hph gene will be lost along, so that we can obtain a ura3-deficient FRET sensor strain for subsequent gene editing on this strain. The ura3 gene could express nucleoside lactide-5'-monophosphate decarboxylase, resulting in 5-Fluoroorotic acid being converted to a toxic form (5-fluorouracil). Therefore, 5-Fluoroorotic acid (1 mg mL−1) was used to screen the ura3 deletion transformant (Additional file 1: Fig. S1C). The target transformants were obtained by PCR laser scanning microscope verification (LSM 980, ZEISS, Germany), Setting up dual-channel simultaneous detection of CFP and YFP fluorescence intensity, YFP and CFP are excited at 500 nm and 430 nm respectively, and emission wavelength of YFP and CFP at 525 nm and 470 nm respectively; The transformant hyphae was incubated in a glucose gradient solution, and the fluorescence intensity of CFP and YFP were detected respectively, and an increase in FYFP/FCFP with gradient indicating the glucose FRET sensor could work successfully in NJAU4742.
Yeast one-hybrid assay
By using the Ultra One Step Cloning Kit (Vazyme Biotech, China), the promoter region of Tgmst1/2 was inserted into the polyclonal site of pAbAi, and the CDS of GRP was inserted into the polyclonal site of pGADT7. Based on PFG/LiAc transformation method, 100μL Gold Yeast, 50 μL NaCl (0.9%, W/V), 5 μL DTT (1 M), 500 μL PEG Mix (45 mL 50% PEG3350; 5 mL 1 M LiAc; 500 μL 1 M Tris–HCl and 100 μL 0.5 M EDTA were mixes) and 1 μg pAbAi-TgmstP linearized by BstBI were mixed gently and incubated at 200 rpm and 30 °C for 30 min, and then 20 μL DMSO was added. The prepared system was heat shocked at 42 °C for 15 min and then kept on ice for 5 min. After resuscitation, the yeast was coated on SD-Ura solid medium (Takara, Japan) and cultured at 30 °C for about 2–3 Days. The transformant (Bait-reporter) was verified by PCR. 10 μL transformant solution (OD600 = 0.8) was dropped onto SD-Ura medium containing AbA (400 ng mL−1) for self-activation verification. The pGADT7-GRP was transformed into the Bait-reporter and verified by PCR. Similarly, 10 μL transformant solution was dropped onto SD-Ura-Leu medium containing AbA (400 ng mL−1), and then cultured at 30 °C for 3 days, until the colony formed.
Yeast two-hybrid assay
By using the Ultra One Step Cloning Kit (Vazyme Biotech, China), the CDS of TgpkA was inserted into the polyclonal site of pGADT7, and the CDS of Tgmst1 was inserted into pGBKT7. According to the PFG/LiAc transformation method, pGBKT7-MST1/2 and pGADT7-EV were transformed into Y2H Gold Yeast. The transformants were verified by PCR and sequencing. 10 μL transformant solution (OD600 = 0.8) was dropped onto SD-His-Trp solid medium (Takara, Japan), and then cultured at 30 °C for self-activation verification. pGADT7-PKA was transformed into the previous transformant and verified by PCR. 10 μL transformant solution was dropped onto SD-His-Leu-Trp solid medium (Takara, Japan), and then cultured at 30 °C for about 4 days until colony formed.
The DNA fragment “Tggrp upstream-hph-promoter-Tggrp-6 × His-Tggrp downstream” was transformed and verified by sequencing and Western blot. Hyphae were immersed in Cross-linking buffer (Sucrose, 0.4 M; pH 8.0 Tris–HCl, 10 mM; EDTA, 1.0 mM; FMSF, 1 mM; Formaldehyde, 1%, V/V) for 25 min, and then terminated by 10 × Glycine (Beyotime, China). Subsequently, the cross-linked hyphae were fully milled and the genome was fragmented to 200–1000 bp. 1.8 mL ChIP Diffusion Buffer (Beyotime, China) and 1 μg 6 × His-tag ChIP-class antibody (Abcam, UK) were added into 200 μL supernatant, and incubated at 4 °C overnight. Afterward, 60 μL Protein A/G beads were added into the ChIP system and incubated at 4 °C for 4 to 5 h. The supernatant was removed and the sediment was cleaned by Low Salt Immune Complex Wash buffer, High Salt Immune Complex Wash buffer, LiCl Immune Complex Wash buffer, and TE buffer (Beyotime, China) in turn. Finally, 500 μL Elution Buffer (SDS, 1%, W/V; NaHCO3, 0.1 M) was added to elute the Protein-DNA complex on Protein A/G beads and the nucleic acid was collected after de-crosslink.
The collected nucleic acid of IP, Mock (IgG), and Input was diluted to the same concentration. By using TB Green Premix Ex Taq Kit (Takara, Japan), qPCR was performed by multiple primers for the Tgmst promoter. Percent Input (% Input) and Fold Enrichment statistical methods were performed. Input is defined as 1.0%., and the Input dilution factor (IDF = 6.644) was subtracted. The formulas were as follows : IDF = log2100 = 6.644 (1.0% Input); Adjusted input = Raw Ct – IDF; %Input IP/Mock = 100 × 2 ^ (Adjusted Input-Ct IP/Mock) × 100%; Fold enrichment = 2 ^ [ (Ct Mock) – (Ct IP)].
DNA pull-down assay
The promoter region of Tgmst was amplified with biotin-labeled primers, and the product was used as a probe. Nucleoprotein was extracted by CelLytic™ PN Isolation/Extraction Kit (Sigma-Aldrich, US). 1 µg DNA probe and 70 µg nucleoprotein were premixed, and the pull-down system was incubated at 4 °C overnight. Streptavidin magnetic beads were added into the pull-down system, and incubated at 4 °C for 1 h. the protein was pulled down by magnetic frame and used for SDS-PAGE. Differential bands were removed and decolored. DTT (10 mM) and IAM (55 mM) were added in turn for reduction and alkylation; 500 μL acetonitrile was added and vacuum dried for 5 min; 0.01 μg μL−1 trypsin was added and ice bath for 30 min; 300 μL extract solution was added and sonicated for 10 min, and eventually the peptide samples (supernatant) was collected. The peptide samples were dissolved in 2% acetonitrile / 0.1% formic acid and analyzed by Triple TOF 5600 plus mass spectrometer coupled with the Eksigent nanoLC system (AB Sciex, USA). The original files were submitted to ProteinPilot (AB Sciex, USA) for protein identification, the Paragon algorithm was used to compare the T. guizhouense NJAU4742 proteome database (https://bioinfo.njau.edu.cn/tgn4742/index.php).
Glutathionylation detection assay
The GRP-His labeled strain was cultured on straw medium at 28 °C for 3 Days, then total protein was extracted by RIPA lysis buffer (Beyotime, China) after the hyphae were fully milled. The nickel ion magnetic bead was washed twice with PBS (Beyotime, China) and diluted by PBS to 50% (W/V). 100 μL nickel ion magnetic bead was added to 1 mL protein and incubated at 4 °C for 10 min, then GRP was isolated by magnetic separator. the purified GRP was diluted by PBS (Containing 1 mM PMSF) to 1 μg μL−1. The reaction system was heated for 10 min and performed SDS-PAGE. According to the Western blot method, the rabbit anti-GSH antibody (Cloud-clone, China) was used to specifically identify the GSH residues .
Total RNA was extracted by mirVana miRNA Isolation Kit (Ambion, US). RNA integrity was evaluated by Agilent 2100 Bioanalyzer (Agilent, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to subsequent analysis. The libraries were constructed by using mRNA LTSample Prep Kit (Illumina, USA). Then these libraries were sequenced by the Illumina sequencing platform and 125 bp / 150 bp paired-end reads were generated. FPKM value of each gene was calculated, and the read counts of each gene were obtained by htseq-count. DEGs were identified by using DESeq (2012) R package functions estimateSizeFactors and nbinomTest. P value < 0.05 and FoldChange > 2 or FoldChange < 0.5 were set as thresholds for significantly differential expression . Hierarchical cluster analysis of DEGs was performed to explore gene expression patterns . GO enrichment and KEGG pathway enrichment analysis of DEGs was performed by R based on hypergeometric distribution. The alternatively splicing analysis of differentially regulated transcripts, isoforms, or exons was performed by ASprofile. SNP and INDEL were called by using samtools and bcftools, then the effects of variants on genes were annotated and predicted by snpeff .
Fresh WT spores were incubated in T1 and T3 treatments mentioned above at 28 °C for 5 days, and each treatment was set up with 6 biological repeats. The hyphae were fully milled and dissolved by extraction buffer (Methanol/acetonitrile/water = 2/2/1, V/V), and centrifuged at 10,000 × g for 2 min. The supernatant was filtered through 0.25 µm filters. The substances in samples were analyzed by liquid chromatography-mass spectrometry (LC–MS/MS). The raw data were logarithmically transformed and centered by using SIMCA (V16.0.2, Umea, Sweden) and then analyzed by automatic modeling. Subsequently, UV formatting and OPLS-DA modeling analysis were performed for principal component analysis. The adjusted data were compared with the metabolite database and combined with univariate statistical analysis (UVA) and multivariate statistical analysis (MVA). Finally, a series of bioinformatics analyses were performed to visualize the biological functions of differential metabolites.
Availability of data and materials
The referenced data can be obtained from the corresponding article and NCBI. Some of the original data in this study are represented in the supplementary material, and some data are being published.
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Thanks to Addgene for providing the plasmid pDR-GW FLII12Pglu-700μ∆6.
This work was supported by the National Natural Science Foundation of China (NSFC, 32172680 and 31972513), and Nanjing Agricultural University (XUEKEN2023039).
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: Fig. S1. Cys determination and glutathionylation detection of GRP. (A) Intracellular Cys content in different sulfate-content treatments, wild-type NJAU4742 was grown in the treatments at 28 °C for 5 days. (B) The strain GRP-His was incubated under high sulfur conditions, and glutathionylation of GRP was detected. Rabbit anti-GSH antibody was used as the primary antibody and the hybridization signal could be detected. Fig. S2. The schematic diagram of the construction principle of RNAi, FRET sensor, and ura3 deficient strains. (A) The vector construction method and working principle of RNA interference of Tgatps. The green and blue fragments respectively express Sense and Antisense chain, and the two fragments are linked by Magnaporthe grisea cutinase gene intron (yellow), TrpC Promoter (TrpC P) and TrpC terminator (TrpC T) to start and stop the transcription of the functional fragment, and Hygromycin resistance gene (hph) was used as the biomarker. It could eventually express a stem-loop RNA, which would be recognized and cut by Dicer, and form an RNA interference silencing complex (RISC). (B) Diagram of structure and working principle of the Optical intracellular glucose FRET sensor. The glucose-binding protein subunit MglB connects CFP (blue) and YFP (yellow). The color and length of the wavy line (red is excitation light) represented the fluorescent category and intensity, respectively. Glucose binding MglB would change the relative spatial position of CFP and YFP, then lead to the change of FRET, and finally cause the change of FY/FC. (C) Diagram of the ura3 deficiency strain construction principle that expressed the glucose FRET sensor. Make the target DNA fragment (the top one) replace ura3 and ura3 downstream (2kb) through homologous arm recognition. It made two repetitive sequences appear in the near region of the genome, which would cause the DNA repair mechanism to cause DNA (ura3 and hph) loss between repetitive sequences. 5-FOA itself was non-toxic to NJAU4742 cells, ura3 could express nucleoside lactide-5’-monophosphate decarboxylase, which would be transformed into a toxic form (5-fluorouracil) so that cells couldn’t grow. 5-FOA (1 mg mL-1) was used to screen correct transformants. Fig. S3. The IPR statistic, KEGG, and GO enrichment of pull-down products. (A) Schematic diagram of the biotin-primer amplification region, the bait sequence was amplified from the -2000bp to -1bp region of Tgmst. (B) Results of structural domain annotation of all identified proteins in the EXP group; among them, a higher percentage of Hsp70 protein domains were detected. (C) KEGG Pathway annotation results for all identified proteins in the EXP group. (D) Gene ontology annotation results for all identified proteins in the EXP group. Fig. S4. The Protein-DNA interaction between GRP and Tgmst1 promoter was verified by ChIP-qPCR. (A) Western blot of GRP labeled by His-tag was added, and three repeats were used to verify the successful addition of His-tag. (B) qPCR primers for ChIP products. (C) Statistical process of ChIP-qPCR; (C1) The specific recognition site of these primers was within 1200 bp upstream of the Tgmst1 promoter, and their amplification products are about 200bp and different from each other. The Cts detected by P-5 and P-6 were significantly different between IP and Mock, and the most significant difference was detected by P-6, which amplified the region from -363 bp to -224 bp. (C2) The percentage of Tgmst1 promoter fragments in the immunoprecipitated DNA of IP and Mock was calculated. input (1%) was adjusted to 100% and its Ct was 16, and the Tgmst1 promoter region in IP accounted for 17.6% of the precipitated DNA fragments by equation 2^ (16 - Ct), while it was 0.296% in Mock (IgG), indicating that GRP has significant DNA-protein interactions with the Tgmst promoter region. (C3) The precipitation efficiency of IP on the Tgmst1 promoter fragment was 61.6 folds higher than that of Mock (IgG), indicating a strong interaction between GRP and the Tgmst1 promoter region, with the interaction region ranging from -363 bp to -224 bp. Fig. S5. EG, CBH, and Xylanase activity of different treatments and strains. (A1, 2, 3) The EG, CBH, and Xylanase activities of NJAU4742 in different sulfate-content MM+straw, grown at 28 °C for 5 days. (B1, 2, 3) The EG, CBH, and Xylanase activities of WT, OE-Tgatps, ∆Tgatps, and RNAi-Tgatps on MM+straw, strains were grown at 28 °C for 5 days. (C1, 2, 3) The EG, CBH, and Xylanase activities of WT, OE-TgcysK, ∆TgcysK, OE-Tghmt, and ∆TgcysK on MM+straw, strains were grown at 28 °C for 5 days. (D1, 2, 3) The EG, CBH, and Xylanase activities of WT, OE-Tgmst, and ∆Tgmst, on MM+straw, strains were grown at 28 °C for 5 days. (E1, 2, 3) The EG, CBH, and Xylanase activities of WT, OE-Tggrp, ∆Tggrp on MM+straw, strains were grown for 5 days at 28 °C. Bars represent mean ± SEM, with n = 3 biologically independent experiments; red dots resemble values from individual experiments. ANOVA was conducted in (A), and there was a significant effect of inorganic sulfide on the EG, CBH, and Xylanase activities at the P< 0.05 level for the 4 treatments, post hoc comparisons using the Tukey's HSD test indicated that the mean score for the EG, CBH, Xylanase activities of T2 were significantly greater than that of T1; and T1 was significantly greater than T3. However, T4 did not significantly differ from T3. Student’s t-testing was conducted in (B, C, D, E), *significant difference to WT at two-tailed P = 0.017 (B1, OE-Tgatps), 0.031 (B3, OE-Tgatps); **significant difference to WT at two-tailed P = 0.0018 (B1, RNAi-Tgatps), 0.0080 (B2, OE-Tgatps) ***significant difference to WT at two-tailed P = 0.0000020 (B1, ∆Tgatps), 0.00079 (B2, ∆Tgatps), 0.00054 (B2, RNAi-Tgatps), 0.000014 (B3, ∆Tgatps), 0.00073 (B3, RNAi-Tgatps); *significant difference to WT at two-tailed P = 0.034 (C2, OE-Tghmt); **significant difference to WT at two-tailed P = 0.0018 (C1, OE-Tghmt), 0.0052 (C3, OE-TgcysK), 0.0054 (C3, ∆TgcysK); ***significant difference to WT at two-tailed P = 0.00029 (C1, OE-TgcysK), 0.0000060 (C1, ∆TgcysK), 0.000042 (C2, OE-TgcysK), 0.000017 (C2, ∆TgcysK); no significant difference to WT at two-tailed P = 0.480633 (C1, ∆Tghmt), 0.61 (C2, ∆Tghmt), 0.15 (C3, OE-Tghmt), 0.60 (C3, ∆Tghmt); **significant difference to WT at two-tailed P = 0.0025 (D1, OE-Tgmst), 0.0028 (D1, ∆Tgmst), 0.0084 (D2, OE-Tgmst), 0.0024 (D2, ∆Tgmst), 0.0076 (D3, OE-Tgmst); no significant difference to WT at two-tailed P = 0.070 (D3, ∆Tgmst); *significant difference to WT at two-tailed P = 0.022 (E1, OE-Tggrp), 0.018 (E1, ∆Tggrp), 0.030 (E2, OE-Tggrp), 0.012 (E3, OE-Tggrp); **significant difference to WT at two-tailed P = 0.0049 (E2, ∆Tggrp), 0.0055 (E3, ∆Tggrp). Fig. S6. Original images of Y1H, Y2H, and Western blot analysis results. (A) Original image of yeast one-hybrid; the growth on SD-Ura-Leu + AbA medium of the yeast transformants that pABAi-Tgmst1P and pGADT7-ATPeF1B / pGADT7-GRP / pGADT7-KatG / pGADT7-ATPeF1A / pGADT7-EF5A / pGADT7-EV were co-transformed. (B) Original image of yeast one-hybrid; the growth on SD-Ura-Leu + AbA medium of the yeast transformants that pABAi-TgmstP and pGADT7-GRP / pGADT7-EV were co-transformed. (C) Original images of yeast two-hybrid, (c1) The growth on SD-His-Leu-Trp medium of yeast transformant with pGADT7-PKA and pGBKT7-MST1 were co-transformed; (c2) The growth on SD-Ade-His-Leu-Trp medium of yeast transformant with pGADT7-PKA and pGBKT7-MST1 were co-transformed; (c3) The growth on SD-His-Leu-Trp medium of yeast transformant with pGADT7-EV and pGBKT7-MST1 were co-transformed. (D) Original images of Western blot; first incubated with mouse anti-His-tag primary antibody, washed with TBST, and then incubated with mouse primary β-actin antibody, and finally incubated with secondary antibody. FoldChange of the expression levels of MST1 and MST2 in OE-Tggrp relative to WT, with β-actin as an internal reference. (E) Original images of Western blot; effect of different glutathionylation levels of GRP on the expression level of MST1 and MST2, with β -actin as an internal reference. Fig. S7. The metabolic pathway of NJAU4742 affected by exogenous inorganic sulfide addition. The area of the square indicates the size of the influence factor of the pathway in the topological analysis, the larger the size the larger the influence factor; the color of the square indicates the -ln P-value of the enrichment analysis, and the darker the color the smaller the P-value. The treemap results displayed the mainly changed metabolic pathways in T2 relative to T1, Glutathione metabolism and Cysteine & methionine metabolism were significantly affected under the conditions of exogenous inorganic sulfide addition, which was in accordance with our previous assay, suggesting that increased the addition of inorganic sulfide favored cysteine and glutathione synthesis. In addition, mainly some amino acid synthesis pathways, such as alanine, aspartate, glutamate, valine, leucine, etc., responded to the increase of exogenous inorganic sulfide content. The pyruvate metabolism, a key precursor for cysteine production, was likewise significantly affected under this condition. Fig. S8. LC-MS/MS and database comparison showed the intracellular metabolites of NJAU4742 affected by exogenous inorganic sulfide addition.
The differentially expressed gene of OE-TgcysK and ∆TgcysK relative to WT.
The differentially expressed gene of OE-Tghmt and ∆Tghmt relative to WT relative to WT.
The Pull-down protein identified in the control (CON) group.
The Pull-down protein identified in the experiment (EXP) group.
The mass spectrometry identification result of intracellular substances of hyphae grown under T1 and T3 treatment conditions.
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Liu, Y., Li, T., Zhu, H. et al. Cysteine facilitates the lignocellulolytic response of Trichoderma guizhouense NJAU4742 by indirectly up-regulating membrane sugar transporters. Biotechnol Biofuels 16, 159 (2023). https://doi.org/10.1186/s13068-023-02418-9