CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei

Wang, Lei; Yang, Renfei; Cao, Yanli; Zheng, Fanglin; Meng, Xiangfeng; Zhong, Yaohua; Chen, Guanjun; Zhang, Weixin; Liu, Weifeng

doi:10.3389/fmicb.2019.01700

Cited by 20 publications

(11 citation statements)

References 55 publications

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“…Early studies regarding protein localization have also used heterologous promoters. For example, heterologous promoters tcu1, CBH1, and CaMV-35S were used for investigating subcellular localization of nuclear proteins (CLP1 [35], Rce1 [36], MAT1-2-1 [37], XYR1 [38], and CRE1 [34]) in T. reesei, SNARE proteins (SNCI, SSOI, and SSOII) (29) in T. reesei, and a unique protein RxLR3 in Phytophthora brassicae (65), respectively. Another study about searching strains with anomalous temporal or spatial protein localization also used heterologous promoter xyl or van (66).…”

Section: Discussionmentioning

confidence: 99%

Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei

Pang

Wang

Luo

et al. 2021

mBio

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Trichoderma reesei has 11 putative β-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many β-glucosidases and the distinct role each β-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular β-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei. Four types of cellular distribution patterns were observed for β-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular β-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi. IMPORTANCE Although β-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on β-glucosidase function. This work comprehensively studies all the β-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced β-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b, cel3e, cel3f, cel3h, cel3j, cel3c, or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of β-glucosidases in Trichoderma reesei. Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of β-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.

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Section: Discussionmentioning

confidence: 99%

Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei

Pang

Wang

Luo

et al. 2021

mBio

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“…ChIP assays were performed as previously described (Wang et al, 2019b ; 2021 ). Briefly, the mycelia were incubated in MA medium containing 1% formaldehyde at 30°C for 10 min with shaking before the cross‐linking was quenched via adding 25 ml of 1.25‐M glycine.…”

Section: Methodsmentioning

confidence: 99%

A histone H3K9 methyltransferase Dim5 mediates repression of sorbicillinoid biosynthesis in Trichoderma reesei

Wang

Liu

et al. 2022

Microbial Biotechnology

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Sorbicillinoids (also termed yellow pigment) are derived from either marine or terrestrial fungi, exhibit various biological activities and therefore show potential as commercial products for human or animal health. The cellulolytic filamentous fungus Trichoderma reesei is capable to biosynthesize sorbicillinoids, but the underlying regulatory mechanism is not yet completely clear. Herein, we identified a histone H3 lysine 9 (H3K9) methyltransferase, Dim5, in T. reesei. TrDIM5 deletion caused an impaired vegetative growth as well as conidiation, whereas the ∆Trdim5 strain displayed a remarkable increase in sorbicillinoid production. Post TrDIM5 deletion, the transcription of sorbicillinoid biosynthesis‐related (SOR) genes was significantly upregulated with a more open chromatin structure. Intriguingly, hardly any expression changes occurred amongst those genes located on both flanks of the SOR gene cluster. In addition, the assays provided evidence that H3K9 triple methylation (H3K9me3) modification acted as a repressive marker at the SOR gene cluster and thus directly mediated the repression of sorbicillinoid biosynthesis. Transcription factor Ypr1 activated the SOR gene cluster by antagonizing TrDim5‐mediated repression and therefore contributed to forming a relatively more open local chromatin environment, which further facilitated its binding and SOR gene expression. The results of this study will contribute to understanding the intricate regulatory network in sorbicillinoid biosynthesis and facilitate the endowment of T. reesei with preferred features for sorbicillinoid production by genetic engineering.

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“…To construct the expression cassette for egfp and mCherry under the control of P sor , their encoding regions were amplified from the plasmid P tcu1 - clp1 - gfp -T trpC [ 18 ] and P tcu1 - mCherry - h2b [ 18 ], respectively. Three fragments, including P sor (767 bp) and two terminators T trpC (759 bp) and T cbh2 (1 kb), were then amplified from the genomic DNA isolated from QM9414, respectively.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Bidirectional Promoter from Trichoderma reesei and Its Application in Dual Gene Expression

Yang

et al. 2022

JoF

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The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes’ expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems.

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CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei

Cited by 20 publications

References 55 publications

Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei

Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei

A histone H3K9 methyltransferase Dim5 mediates repression of sorbicillinoid biosynthesis in Trichoderma reesei

Identification of a Bidirectional Promoter from Trichoderma reesei and Its Application in Dual Gene Expression

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