Fig 1.
TrGAL11 interacts with XYR1 in vitro.
(A) Schematic diagram of domain of TrGAL11 and ScGAL11. The KIX domain and glutamine rich region (Q-rich) of each protein are labeled as indicated. (B) Yeast two-hybrid analyses of interactions between XYR1 activation domain (XYR1 AD) and full length or the KIX domain of TrGAL11. Serial dilutions of yeast transformant cells harboring the indicated plasmids were spotted on double dropout medium (DDO, SD/–Leu/–Trp) and quadruple dropout medium (QDO, SD/–Ade/–His/–Leu/–Trp) plates containing 75 ng/mL AbA, respectively, and were allowed to grow at 30°C for 3 days. The p53 plus T was set as a positive control. Significant growth were shown in transformants containing XYR1 AD plus full-length or the KIX domain of TrGAL11 while only very weak growth was observed for control transformants containing pGBKT7 plus pGADT7-TrGAL11 or pGADT7 plus pGBKT7-XYR1 AD. (C) TrGAL11 interacts with XYR1 activation domain (aa 767–860) in a GST pull-down assay in vitro. Recombinant TrGAL11 KIX-6His was incubated with glutathione sepharose 4B beads-coupled GST- XYR1 AD767-860 or GST as a control. TrGAL11 KIX retained on the beads after extensive washing was detected by western blot with anti-His antibody.
Fig 2.
Trgal11 disruption had little effect on T. reesei growth on plates or in liquid medium but reduced its conidia and pigment formation.
(A) Growth of QM9414 and the ΔTrgal11 strains on plates with various carbon sources at a final concentration of 1% (w/v) at 30°C for 3 days. (B) Biomass analysis of QM9414 and a representative ΔTrgal11 in liquid MA medium with 1% glucose as the sole carbon source. The biomass was determined by analyzing the dry weight. (C) Biomass accumulation of QM9414 and ΔTrgal11 after inoculation of equal amounts of pre-cultured mycelia in liquid MA medium with 1% Avicel as the sole carbon source was determined by analyzing the intracellular protein content. (D) Conidiation and pigment formation analyses of QM9414 and the ΔTrgal11 strains on malt extract agar. No statistical difference (t-test, P>0.05) was observed for the growth of these strains.
Fig 3.
Deletion of Trgal11 compromised the fully induced production of cellobiohydrolase and endoglucanase but not of β-glucosidase.
(A-E): Extracellular pNPCase activity (A), CMCase activity (B), filter paper activities (FPAase) (C), protein concentration (D), and pNPGase activity (E) of the culture supernatant from the parental strain QM9414 and three independent ΔTrgal11 transformants cultured on 1% (w/v) Avicel for the indicated time periods. (F) Culture supernatant of QM9414 and the ΔTrgal11 strains on 1% (w/v) Avicel was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. As done with biomass quantification in Fig 2C, equal amounts of the same Avicel culture at the indicated time points after inoculation of pre-cultured mycelia were taken for measuring extracellular activity. Significant differences (t-test, *P<0.05, **P<0.01, ***P<0.001) were detected for the extracellular activities except pNPGase activity between QM9414 and three independent transformants of ΔTrgal11 for the indicated time points after induction.
Fig 4.
Deletion of Trgal11 resulted in a significant decrease in the transcription of cellobiohydrolase and endoglucanase but not β-glucosidase genes.
Transcription of cbh1 (A), eg1 (B), xyr1 (C), bgl1(D), and bgl2 (E) were analyzed by quantitative RT-PCR after induction on 1% (w/v) Avicel. The expression level of the actin 1 gene was used as a reference gene for normalization in all samples. Significant differences (t-test, *P<0.05, **P<0.01) were detected for cbh1, eg1, and xyr1 gene transcription between QM9414 and ΔTrgal11 for the indicated time points after induction. No significant differences (t-test, P>0.05, n.s.) were detected for bgl1 and bgl2 gene transcription between QM9414 and ΔTrgal11 for the indicated time points after induction except bgl1 gene transcription induction for 12 h.
Fig 5.
Overexpression of xyr1 failed to restore the fully induced expression of cellulase genes in the Trgal11 deletion mutant.
(A-C): Extracellular pNPCase activity (A), FPAase activity (B), and pNPGase activity (C) of the culture supernatant from OExyr1, QM9414 and two independent OEX_ΔTrgal11 transformants on 1% (w/v) Avicel for the indicated time periods. Significant differences (t-test, *P<0.05, **P<0.01, ***P<0.001) were detected for the pNPCase activity, filter paper activities (FPAase) between OEX_ΔTrgal11 and OExyr1 or between OEX_ΔTrgal11 and QM9414. No significant difference (t-test, P>0.05; n.s.) was detected for the pNPGase activity. (D) SDS-PAGE analysis of the culture supernatant from the OExyr1, QM9414 and OEX_ΔTrgal11 strains cultured on 1% (w/v) Avicel. Coomassie Brilliant Blue was used for gel staining. (E-H): Transcription of cbh1 (E), eg1 (F), bgl1 (G), and xyr1 (H) were analyzed by quantitative RT-PCR after induction on 1% (w/v) Avicel. The expression level of the actin gene was used as a reference gene for normalization in all samples. Significant differences (t-test, *P<0.05, **P<0.01, ***P<0.001) were detected for the transcription of cbh1, eg1, and xyr1 between OEX_ΔTrgal11 and OExyr1 or between OEX_ΔTrgal11 and QM9414. No significant difference (t-test, P>0.05; n.s.) was detected for bgl1 transcription between OEX_ΔTrgal11 and OExyr1 or between OEX_ΔTrgal11 and QM9414.
Fig 6.
TrGAL11 was recruited to cellulase gene promoters in an XYR1-dependent manner.
(A-E): ChIP assay for TrGAL11 binding to the cbh1 (A), cbh2 (B), eg1 (C), bgl1 (D), and actin (E) promoters in the OEX _Trgal11-proA strain after induction for 9 h and 24 h on 1% Avicel with or without adding exogenous copper. QM9414 was set as a negative control strain. IgG-agarose beads was used to immunoprecipitate TrGAL11-proA fusion protein bound to promoters. Significant differences (t-test, *P<0.05, **P<0.01, ***P<0.001) were detected for TrGAL11 binding to cellulase gene promoters when XYR1 was overexpressed without copper compared to with copper or QM9414 after Avicel induction. No significant difference (t-test, P>0.05, n.s.) was detected for TrGAL11 binding to the actin promoter regardless of the presence or absence of copper. (F) Semi-quantitative PCR products amplified from the above promoters with the immunoprecipitated DNA and resolved by agarose electrophoresis. -Cu2+, +Cu2+ and QM denoted samples from the OEX_Trgal11-proA strain cultured without and with copper as well as QM9414, respectively. (G) An overview of TrGAL11 occupancy over the cbh1 promoter after Avicel induction for 9 h. The analyzed regions include cbh1-ORF (418 to 603), Pcbh1-250 (-179 to -355), Pcbh1-500 (-460 to -559), Pcbh1-800 (-664 to -905), Pcbh1-1000 (-952 to -1149), Pcbh1-1400 (-1286 to -1427), Pcbh1-1700 (-1603 to -1840), and Pcbh1-2100 (-2112 to -2283). The numbers within brackets are the nucleotide position relative to the start codon ATG.
Fig 7.
Trgal11 deletion resulted in a significantly higher XYR1 occupancy on cellulase gene promoters.
(A-E): ChIP assay of XYR1 binding to cbh1(A), cbh2 (B), eg1(C), bgl1(D), and bgl2 (E) promoters of QM9414 and the ΔTrgal11 mutant after induction for 6 h and 9 h on 1% Avicel. Anti-XYR1 antibody was used to immunoprecipitate XYR1 bound to all detected cellulase gene promoters. Significant differences (t-test, *P<0.05, ** P<0.01) were detected for XYR1 occupancy on cellulase gene promoters between QM9414 and ΔTrgal11 mutant after Avicel induction for 6 h and 9 h. (F) An overview of XYR1 occupancy over the cbh1 promoter analyzed by ChIP after Avicel induction for 9 h. The analyzed regions are defined as in Fig 6G.
Fig 8.
TrGAL11 plays a critical role in RNA Pol II recruitment on the core-promoter of cbh/eg genes but not of bgl genes.
ChIP assay of RNA Pol II occupancy on the core promoters (TATA box) of the cbh1 (A), eg1 (B), bgl1 (C), and bgl2 (D) genes of QM9414 and ΔTrgal11 under 1% Avicel induction for 6 h and 9 h. Anti-Rpb1 CTD antibody was used to immunoprecipitate Rpb1 bound to cellulase gene core promoters. Significant differences (t-test, *P<0.05, **P<0.01) were detected for RNA Pol II occupancy on cbh1 and eg1 core promoters after induction for 6 h and 9 h but no significant differences (t-test, P>0.05, n.s.) were detected on bgl1 and bgl2 core promoters. (E) Semi-quantitative PCR products amplified from the above core promoter regions from the immunoprecipitated DNA and resolved by agarose electrophoresis.
Fig 9.
A schematic model of how Mediator is recruited by XYR1 to participate in activating cellulase gene expression in T. reesei.
Once expressed upon induction, XYR1 binds to the upstream binding sites (XBS) in cellulase gene promoters and recruits the Mediator complex by interacting with the tail module subunit TrGAL11 to further facilitate the recruitment of the general transcription machinery including RNA Pol II to cbh and eg genes.