Additional file 1 of Cysteine facilitates the lignocellulolytic response of Trichoderma guizhouense NJAU4742 by indirectly up-regulating membrane sugar transporters
Creators: name: Liu, Yang
name: Li, Tuo
name: Zhu, Han
name: Zhou, Yihao
name: Shen, Qirong
name: Liu, Dongyang
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Environmental Sciences not elsewhere classified
subject: Ecology
subject: Science Policy
subject: Biological Sciences not elsewhere classified
subject: Computational Biology

description: Additional file 1: 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&lt; 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 &amp; 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.