Additional file 1 of Cellular ATP redistribution achieved by deleting Tgparp improves lignocellulose utilization of Trichoderma under heat stress
Creators: name: Li, Tuo
name: Liu, Yang
name: Zhu, Han
name: Cao, Linhua
name: Zhou, Yihao
name: Liu, Dongyang
name: Shen, Qirong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: Marine Biology
subject: Computational Biology

description: Additional file 1: Fig. S1. Growth and straw utilization efficiency of T. guizhouense by using rice straw as sole carbon sources at different temperatures. (a) Growth of T. guizhouense on plates at different temperatures using rice straw as sole carbon sources. (b) Scanning electron micrographs of the substrates including CK (raw material), T28 and T37, respectively. Fig. S2. Quality control of SWATH analysis results. (a) Experimental design to compare the extracellular proteins extracted from T. guizhouense at different temperatures. (b) The distribution of proteins' unique peptides number achieved the data analysis quality. (c) The length of identified peptides. (d) Classification of the SWATH-quantified proteins according to the biological function. (e) Distribution of proteins ratio of each sample. (f) Correlation of the quantitative values for all the identified proteins between replicated injections. Fig. S3. Model of the critical proteins for the lignocellulose utilization based on the SWATH and PPI analysis. PPI analysis predicted five proteins were considered critical during the lignocellulose utilization process. (a) The PPI network was constructed based on the STRING database between different treatments. (b) Proposed model of T. guizhouense in the lignocellulose utilization at different temperature. By compared with T28, four downregulated proteins including Nonribosomal peptide synthetases (NRPS protein, OPB39084), delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH, OPB43669), aldehyde dehydrogenase (NAD+) (ALDH, KKP01310), and ADPRase (OPB41268) and one up-regulated protein (PARP, OPB37503) were deemed as pivotal in T37. NRPS was the modular protein that produced peptide antibiotics and siderophores. P5CDH preferentially used NAD+ as a coenzyme to transform glutamate semialdehyde into glutamate. ALDH, a nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent enzyme, was involved in detoxification, biosynthesis, antioxidant functions, and structural and regulatory mechanisms. ADPRase and PARP were also involved in the metastasis of ADPR. Intracellular free ADP‐ribose was a highly reactive and potentially toxic molecule. Activation of ADPRase or inhibition of PARP could relieve ER stress by increasing cellular ATP levels. AMP and ribose 5‐phosphate (Ribose 5‐P) might be beneficial for nucleotide recycling, resulting in the suppression of the over-consumption of NAD+ and ATP. Fig. S4. Knockout or over-expression of different functional genes in T. guizhouense. (a) Schematic diagram for genes disruption through double crossover recombination. (b) Schematic diagram for gene over-expression. (c) Diagram of construction principle for preparing the mutants of lignocellulases-GFP fusion strains. HA1 and HA2 were used as two arms of homologous recombination and the HygB gene was used as the biomarker for screening. (d) Analysis of expression level of Tgadprase gene relative to Tef in wt and OE-Tgadprase strain determined by qPCR. Data were calculated from three biological replicates. Error bars represent ± SDs. *** P &lt; 0.001. A P-value &lt; 0.05 is regarded as statistically significant. The expression value was normalized to wt. (e–f) Verification of ΔTgparp (left) and ΔTgire (right) by PCR to verify homologous recombination and whether gene exists. (g) Growth of mutants (ΔTgire and ΔTgireΔTgparp) inoculated on PDA at 28 °C (top row) and rice straw medium at 37 °C (bottom row), respectively. (h) Verification of ΔTgireΔTgparp by PCR to verify homologous recombination and whether gene exists. (i) Verification of recombinant strains with GFP labeling by PCR using two relevant primer pairs of E-egl (E-cbh, E-blg, and E-xyl) and E-GFP to verify homologous recombination. Fig. S5. Southern blot analysis of gene Tgparp. The genomic DNA was digested with the restriction enzymes and hybridized with the probes amplified by primers listed in Table. S2. Lane M: DNA marker; Lane 1: wt-1; Lane 2: ΔTgparp-1; Lane 3: positive plasmid-1; Lane 4: wt-2; Lane 5: ΔTgparp-2; Lane 6: Positive plasmid-2. Fig. S6. The profile of carbon utilization using Biolog FF Microplates at 28 °C. Three independent biological experiments were performed and the data were collected. Error bars represent ± SDs. Fig. S7. Transcriptional level of representative lignocellulase genes in wt and ΔTgparp at 37 °C. Data were calculated from three biological replicates. Error bars represent ± SDs. * P &lt; 0.05, ** P &lt; 0.01. A P-value &lt; 0.05 was regarded as statistically significant. The expression values are normalized to wt. Endoglucanase genes: 1–5 (A1A106567.1, A1A101831.1, A1A107632.1, A1A104288.1, and A1A110611.1); cellobiohydrolase genes: 6–9 (A1A104298.1, A1A108865.1, A1A104556.1, and A1A102650.1); glucosidase genes: 10–13 (A1A110026.1, A1A105994.1, A1A107778.1, and A1A111035.1); hemicellulase and auxiliary hydrolyzing genes: 14–19 (A1A111258.1, A1A111547.1, A1A102817.1, A1A112191.1, A1A105029.1, and A1A100991.1). Fig. S8. The ATP level between wt and ΔTgparp with the gradual increase of tempeature (from 28 °C to 37 °C). Three independent biological experiments were performed and data was collected. Error bars represent ± SDs. ** P &lt; 0.01, ** P &lt; 0.001. A P-value &lt; 0.05 was regarded as statistically significant. Fig. S9. Growth of T. guizhouense inoculated at 37 °C for 48 h when different concentrations of exogenous ATP-Na2 were added. Fig. S10. Construction and growth of strains with the ATP sensor. (a) Schematic of the ATP sensor. (b) Equally harvested biomass of strains wt-ATP and ΔTgparp-ATP were inoculated on PDA at 28 °C (left) and rice straw medium at 37 °C (right), respectively. (c) Verification of the strains wt-ATP and ΔTgparp-ATP through PCR by using two relevant primer pairs of E-ATP-F and E-ATP-R to verify the ATP sensor fragment, and E3 was used to verify the existence of gene Tgparp. Fig. S11. Flow cytometry dot plot of the protoplasts containing the lignocellulases-GFP at different treatments under heat stress. The lignocellulases-GFP fusion strains contained egl-gfp (a), cbh-gfp (b), bgl-gfp (c), and xyl-gfp (d); the dot plot of wt is represented in blue, while ΔTgparp are depicted in red. Fig. S12. Growth condition and lignocellulose utilization efficiency of RNAi-Tgparp under heat stress by knocking down of Tgparp through the RNA interference technology. (a) Schematic diagram for the generation of RNAi-Tgparp. MCS1: Sense strand of gene Tgparp, MCS2: Antisense strand of gene Tgparp. (b) Relative expression of gene Tgparp between wt, RNAi-Tgparp-1, and RNAi-Tgparp-2. The expression value is normalized to wt. (c) Biomass of wt, RNAi-Tgparp-1 and RNAi-Tgparp-2 inoculated on PDA (top row) and rice straw medium (bottom row), respectively. (d) Comparison of the lignocellulolytic activities including FPA, EG, CBH, and XYL between wt, RNAi-Tgparp-1, and RNAi-Tgparp-2 under heat stress. (e) Relative expression of genes related to DNA repair between wt, ΔTgparp and RNAi-Tgparp-1. Data were calculated from three biological replicates. Error bars represent ± SDs. ** P &lt; 0.01, *** P &lt; 0.001. A P-value &lt; 0.05 is regarded as statistically significant. Fig. S13. Construction and growth of strains with NAD+ sensor. (a) Schematic of the ATP sensor. (b) The growth condition of wt with mCherry-cpYFP / mCherry-FiNad and ΔTgparp with mCherry-cpYFP / mCherry-FiNad inoculated on PDA at 28 °C (top row) and rice straw medium at 37 °C (bottom row), respectively. (c) Verification of the strains that successfully expressed the mCherry-cpYFP or mCherry-FiNad in wt and ΔTgparp. Fig. S14. Confocal imaging and fluorescence intensity analysis of NAD+ in T. guizhouense. (a-b) Confocal imaging analysis of hyphae from wt-mCherry-cpYFP (left) and ΔTgparp-mCherry-cpYFP (right) under heat stress, bar = 3 μm. (c-d) Confocal imaging analysis of hyphae from wt-mCherry-FiNad (left) and ΔTgparp-mCherry-FiNad (right) under heat stress, bar = 3 μm. (e–h) Fluorescence intensity of the two channels (mCherry and cpYFP) detected in different strains. (i-l) Green/Red ratios plotted for single pixels of the mycelium with the mCherry-cpYFP or mCherry-FiNad in wt and ΔTgparp. Blue numbers represent the average of all values. (m–n) Green/Red ratio of mCherry-cpYFP or mCherry-FiNad in wt and ΔTgparp from more mycelia repeats. The box plot means the median values of ratios. (o) Ratio of mCherry-FiNad/mCherry-cpYFP between wt and ΔTgparp according to the results in graphs m and n. (p) Biochemical analysis of cellular NAD+ content under heat stress between wt and ΔTgparp. Table S1. SWATH quantifies proteins of Trichoderma guizhouense NJAU4742 at different temperatures using straw as the only carbon source. Table S2. PCR primers used in this study.