Additional file 1 of Increasing NADPH impairs fungal H2O2 resistance by perturbing transcriptional regulation of peroxiredoxin
Creators: name: Li, Jingyi
name: Sun, Yanwei
name: Liu, Feiyun
name: Zhou, Yao
name: Yan, Yunfeng
name: Zhou, Zhemin
name: Wang, Ping
name: Zhou, Shengmin
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Plant Biology
subject: Computational Biology

description: Additional file 1: Table S1. A. nidulans strains used in this study. Table S2. Primers used in this study. Fig. S1. CRISPR/Cas9-mediated disruptions of hapC gene in A. nidulans. A Schematic diagram of gene disruption by in vitro assembled Cas9/sgRNA and donor DNAs. B Gene replacement of the target gene loci via the homology-directed repair pathway. Left, Schematic diagram of locus changes of target genes before and after gene replacement. Right, Confirmation of gene disruptions by PCR using the indicated primer pairs. hapC disruptant was verified with primer pairs AN4034-uF/pyrG-check-5′-R (lane 1), AN4034-dR/pyrG-check-3′-R (lane 2), respectively; primer pair hapC-check-F/hapC-check-R was used to amplify AN4034 ORF in the parent (lane 3) and disruptant (lane 4) strains. M, marker. Primers are listed in Table S2. Fig. S2. Constructions of promoter substitution strains of A. nidulans. A–D Conditional promoter replacement strategy for homologous recombinant strains nP.gsdA (A), gP.gsdA (B), gP.gndA (C), gP.prxA (D) and validation of the corresponding recombinations by PCR with the indicated primers (right). The isolated nP.gsdA transformant was verified with primer pairs AN2981-uF/pyrG-check-5′-R (lane 1), AN2981-dR/pyrG-check-3′-R (lane 2), respectively; Primer pair AN2981-uF/AN2981-dR was used to amplify corresponding regions in the transformant (lane 3) and parent (lane 4) strains. M, marker. The similar methods were performed to validate the resultant transformants of gP.gsdA and gP.gndA. For verifying the indicated recombination in gP.prxA, primer pair M13-R/pyroA-check-3′-F was used to amplify corresponding regions in the transformant (lane 3) and parent (lane 4) strains. Primers are listed in Table S2. Fig. S3. Construction and phenotype analysis of GFP-tagged NapA strain and GFP-tagged PrxA strain. A and C Schematic diagram of locus changes of napA gene (A) and prxA gene (C) before and after the gene replacements (left) and the corresponding confirmation using PCR (right). Primer pair napA-5′-F/napA-3′ UTR-R was used to amplify the corresponding region in the isolated transformant (lane 1) and the parent (lane 2) strains. M, marker. Primers are listed in Table S2. B GFP-tagged NapA strain (N_Gfp) shows similar H2O2 resistance to the control strain (WT_argB). Conidia (1 × 105) from both strains were spotted on MM plates with or without 2 mM H2O2 and incubated at 37 °C for 2 days. D GFP-tagged GFP strain (P_Gfp) recovered the H2O2 resistance and similar to the control strain (WT_argB). Conidia (1 × 105) from both strains were spotted on MM plates with or without 1 mM H2O2 and incubated at 37 °C for 2 days. Fig. S4. Construction of Flag-tagged HapC strain and phenotype analysis of variants of hapC mutant under unstressed conditions. A The recombinant plasmid pUC19-pyroA-hapC-Flag was used to transform to ∆hapC to construct H_Flag strain. The indicated recombinant transformant was isolated and further confirmed by PCR with the primer pair M13-R/pyroA-check-3′-F (right, lane 1). Lane 2 showed the corresponding result of the control strain. Primer are listed in Table S2. B Conidia (1 × 105) from the control (WT_argB), ∆hapC, nP.gsdA/∆hapC and HapC::Flag fusion protein-expressing strains H_Flag, and nP.gsdA/H_Flag were spotted on MM plate without H2O2 and incubated at 37 °C for 2 days. Fig. S5. Reversible inhibition of NADPH to G6PD activity. A SDS-PAGE (12%) analysis of purified recombinant AnG6PD expressed by E. coli. M, maker. Lane 1, purified AnG6PD. B Activity of recombinant G6PD samples was estimated by generation of NADPH using a UV–Vis spectrophotometer at 340 nm. G6PD activity was measured in a 1 ml reaction mixture containing 5 mM glucose-6-phosphate, 0.3 mM NADP+, and 5 µM G6PD; 30 µM NADPH was added to the reaction mixture to evaluate the inhibition effects on G6PD activity. Further addition of 20 µM flavohemoglobin (Fhb1), and 50 µM NO donor MAHMA NONOate was used to consume NADPH for inhibition relief of G6PD activity. Fig. S6. Downregulation of A. nidulans gsdA is detrimental to fungal growth and oxidative stress resistance. A Survival rates of WT (WT_pyrG) and nP.gsdA on MM plates using ammonium tartrate as nitrogen source under oxidative stress conditions. Fresh conidia (1 × 108) of both strains were spread on MM plates containing the indicated concentrations of H2O2. Colonies were counted after a 48-h incubation, and survival rate are expressed as percentages of the CFU for strains incubated without H2O2. B–C G6PD activities and the relative NADPH/NADP+ ratio in WT and NH4+-repressed nP.gsdA strains before and after treatment of H2O2. Both strains were cultivated in MM liquid media using ammonium tartrate as the nitrogen source for 16 h, and then treated with the indicated concentrations of H2O2 for 30 min. (mean ± SD; n = 3, *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001; n.s., not significant, one-way ANOVA.) Fig. S7. Replacement of gsdA native promoter with gpdA promoter also perturbed NADPH rhythm and impaired fungal resistance to H2O2. A–B Relative expression levels of gsdA (A) and NADPH/NADP+ ratios (B) in WT (WT_pyrG), nP.gsdA and gP.gsdA (replacing gsdA promoter with gpdA promoter in WT) strains. All strains were precultivated in liquid NO3–-MM for 16 h and then exposed to 1 mM H2O2 for 30 min. The WT level of gsdA was set to 1, and the levels of gsdA in other strains were normalized to this. C Comparison of the H2O2 resistance of WT (WT_pyrG), nP.gsdA and gP.gsdAstrains. (mean ± SD; n = 3, *P &lt; 0.05, **P &lt; 0.001, t-test.) Fig. S8. Enhancing NADPH by overexpression of gndA impairs fungal resistance to H2O2. A–B Relative expression levels of gndA (A) and NADPH/NADP+ ratios (B) in WT (WT_pyrG) and gP.gndA (replacing gndA promoter with gpdA promoter in WT) strains. All strains were precultivated in liquid NO3–-MM for 16 h and then exposed to 1 mM H2O2 for 30 min. The WT level of gndA was set to 1. (C) Comparison of the H2O2 resistance of WT (WT_pyrG) and gP.gsdA strains. (mean ± SD; n = 3, *P &lt; 0.05, **P &lt; 0.001, t-test.) Fig. S9. Intracellular H2O2 accumulation accounts for the growth retardation of nP.gsdA strain under oxidative stress conditions. Growth comparison of WT (WT_pyrG) and nP.gsdA strains under oxidative stresses or unstressed conditions. Conidia (1 × 105) from both strains were spotted and cultivated for 2 days on NO3–-MM plates supplied with or without 10 mM NAC and 1 mM H2O2 as indicated by “+” and “−”, respectively. Fig. S10. Quantitative analysis of relative levels of intracellular HapC. Total contents of intracellular HapC levels of WT and nP.gsdA strains at different periods are shown by the intensity of bands on denaturing and reducing PAGE. HapC levels were normalized to actin contents calculated by the same method for the further quantitative comparison. Each value represents the mean ± SD of triplicate determinations (mean ± SD; *P &lt; 0.05, **P &lt; 0.01, one-way ANOVA).