Gang Liu

The thermophilic fungus Myceliophthora thermophila can secrete large amounts of lignocellulolytic enzymes, such as cellulases and xylanases, which are regu

Filamentous fungi produce large quantities of cellulase and xylanase as extracellular enzymes to degrade plant‐derived polysaccharides. This process is controlled by a complex network of transcription factors (TFs). Here, we present the bZIP TF Mthac‐1 exhibiting dual regulatory effects on the production of cellulase and xylanase in Myceliophthora thermophila . The deletion of Mthac‐1 reduced the cellulase and xylanase activities and protein secretion during the early phase of cultivation but enhanced in the middle and late stages of cultivation, compared with the wild‐type (WT) strain. It also led to fungal growth defects, characterised by few hyphal branching and reduced conidiation. Real‐time quantitative reverse transcription PCR (RT‐qPCR) analysis showed that Mthac‐1 dynamically regulates the expression of major cellulase genes. Furthermore, electrophoretic mobility shift assays (EMSAs) demonstrated that Mthac‐1 directly binds to the promoter regions of the β‐glucosidase gene bgl1 ( MYCTH_66804 ), cellobiohydrolase gene cbh1 ( MYCTH_109566 ), endoglucanase gene egl2 ( MYCTH_86753 ), xylanase gene xyn1 ( MYCTH_112050 ) and the regulatory gene xyr1 ( MYCTH_2310145 ), exhibiting higher binding affinity for xyn1 and xyr1 . The comparative transcriptomic analysis indicated that Mthac‐1 also plays an important role in the expression of 26S proteasome‐encoding genes under cellulolytic conditions. This work provides new insights into the regulatory mechanisms underlying cellulase and xylanase gene expression with potential applications in fungal strain engineering in biorefinery industries.

Peacock plant (Calathea orbifolia [Linden] H. A. Kenn.) is grown in China as a valuable houseplant. In September 2024, leaf anthracnose was found on this plant in a field in Shenzhen (22°35'55"N, 113°59'21"E), Guangdong Province, China. The area investigated was ~900 m2 and included ~300 plants. Disease incidence was ~70% of 100 plants. Disease severity per plant was 20 to 60% necrotic lesions on the leaf. The initial symptoms were brown spots with a yellow halo. The spots enlarged, elongated, irregular, 0.5 to 9 × 0.2 to 4 cm, and leaves withered and died. Ten symptomatic leaves were collected for pathogen isolation. Small pieces (5×5 mm2) of lesion margins were surface sterilized in 1% NaClO for 1 min, 70% ethanol for 30 s, rinsed in sterile distilled water, placed on PDA, and incubated at 25°C. After 3 days, fungal colonies were purified on PDA using the single hyphal tip method. Three fungal strains (MBSZU 24-004 to MBSZU 24-006) with similar morphology were obtained from different plant samples with an isolation frequency of 75%. Colonies on PDA were white, 30 to 35 mm in diameter after 3 days at 25°C, then grayish white with cottony mycelia, the reverse pale yellow after 1 week. All strains produced asexual structures. Setae were dark brown, 43.2 to 169.4 × 2.1 to 4.3 µm, a cylindrical base, and an acuminate tip. Conidiophores were septate, hyaline to pale brown, and branched. Conidiogenous cells were hyaline, cylindrical to ampulliform, 10.3 to 32.3 × 2.2 to 4.4 µm. Appressoria were oval to irregular, dark brown to black, 6.2 to 18.9 × 5.7 to 10.1 µm. Conidia were one-celled, hyaline, smooth-walled, cylindrical, ends rounded, guttulate, 9.5 to 20.6 × 3.6 to 6.2 µm (n = 100). Morphologically, all strains resembled the C. gloeosporioides species complex (Weir et al. 2012). The ITS, ACT, CAL, CHS-1, TUB2, GAPDH, and ApMat genes were amplified using primer pairs ITS5/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, T1/T22, GDF1/GDR1, and AMF1/AMR1 (Silva et al. 2012; Weir et al. 2012), respectively. Sequences were deposited in GenBank (ITS: PQ550808 to PQ550810; ACT: PQ558623 to PQ558625; CAL: PQ558629 to PQ558631; CHS-1: PQ558632 to PQ558634; TUB2: PQ558626 to PQ558628; GAPDH: PQ558635 to PQ558637; ApMat: PQ777471 to PQ777473). Maximum likelihood phylogenetic analyses of the combined seven genes identified all strains as C. siamense. To test pathogenicity, both wounded and unwounded healthy leaves were used in this experiment. All leaves were wiped with 0.1% NaClO and then rinsed with sterile water. Conidia suspensions (15 µl of 1 × 106 conidia/ml) of all three strains grown on PDA at 25°C for 2 weeks were placed on all samples using the attached leaf assay. Control leaves were mock-inoculated with sterile distilled water. Each treatment had 10 replicates and was repeated twice. Plants were placed at 25°C and 80 to 85% relative humidity. After 10 days, both wounded and unwounded inoculated leaves exhibited brown lesions with yellow halos, while the control leaves remained asymptomatic. Wounded leaves showed more symptoms than unwounded leaves. Strain MBSZU 24-005 was more virulent than the others. C. siamense was reisolated from the lesions and confirmed by morphology and DNA sequencing, fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on C. orbifolia in China and worldwide. The disease could economically impact commercial growers, necessitating monitoring of its effects on China's peacock plant production.

RNA expression analysis using reverse transcription qPCR requires reference genes for accurate data normalization. However, some reference genes can show significant variability in specific biological samples, raising concerns about the reliability of this method. This study assessed the expression levels of 12 reference genes in Thermothelomyces fergusii during spore and mycelium cultivation under different carbon source media (avicel, cellobiose, and glucose) using RT-qPCR. T. fergusii is a thermophilic fungus known for its abundance of cellulase and industrial applications. Quantifying fungal gene expression using the RT-qPCR method is essential for investigating the molecular biology of T. fergusii. The stability of 12 reference genes in T. fergusii was evaluated using RefFinder, geNorm, NormFinder, BestKeeper, and delta Ct techniques. The robustness of the chosen reference gene was verified using the LPMO AA9-4 gene. Analysis of the different methods showed variations in the results of reference genes under different sugar treatments. Consistently, EF1, GAPDH, and 18 S-3 were identified as reliable reference genes by the geometric mean of ranking values from Pearson correlation coefficient (r) analysis of pooled data, ranking values of RefFinder in pooled data, and NormFinder of pooled data. However, BT-3, ACT-3, and 18 S-2 exhibited inconsistency as reference genes. These results emphasize the importance of verifying reference genes in specific experimental models and provide valuable recommendations for selecting reliable reference genes in RT-qPCR. These findings have broad applicability for gene expression investigations involving T. fergusii.

Xylanases are crucial for the breakdown of hemicellulose, enabling the conversion of lignocellulosic biomass into fermentable sugars for biofuels and other industrial applications. For the first time, we investigated the biochemical and genetic characteristics of 22 xylanase genes from Thermothelomyces fergusii within glycoside hydrolase (GH) families GH10, GH11, and GH43. Xylanase genes structural diversity clustered the phylogenetic tree into GH10, GH11, GH43-I, and GH43-II groups. Structural analysis revealed that all TfGH10 and TfGH11 genes contained conserved GH domains, with CBM1 present in TfGH10-5 and TfGH11-4. Secondary domains, including CBM35, CBM42, and CBM91, were found in the GH43 gene family. The presence of key glutamic (Glu) and aspartic (Asp) residues in active sites is essential for substrate binding and catalysis. RT-qPCR analysis revealed substrate-dependent gene expression, with peak upregulation on day three in beechwood xylan (BWX) cultures and day two in corncob xylan (CCX) and rice straw (RS) cultures. Consistent with these findings, enzymatic assays demonstrated the highest xylanase activity in BWX-induced cultures, followed by RS and CCX, underscoring the differential regulation of these enzymes in response to distinct hemicellulosic substrates. These findings provide valuable insights into the structural, functional, and regulatory mechanisms of T. fergusii xylanases, facilitating their industrial application.

Peacock plant (Calathea orbifolia (Linden) H.A.Kenn.) is cultivated as a valuable houseplant in China. In September 2024, leaf blight was found on this plant in a field located in Shenzhen (22°35'55"N, 113°59'21"E), Guangdong Province, China. The area investigated was ~900 m2. The disease incidence was ~70% of 100 plants. Initial symptoms of brown spots appeared on leaves, gradually becoming enlarged, elongated, 0.5 to 10 × 0.2 to 4.5 cm, irregular, dark brown, with a yellow halo, and leaves blighted and desiccated. Ten symptomatic leaves were collected for pathogen isolation. Leaf pieces (5×5 mm2) of lesion margins were surface disinfected with 1% NaClO for 1 min, 70% ethanol for 30 s, rinsed in sterile distilled water, placed on PDA, and incubated at 25°C. After 3 days, fungal colonies were purified by a single hyphal tip method on PDA. Three isolates (MBSZU 24-001 to MBSZU 24-003) with similar morphology were obtained. Colonies on PDA were 48 to 54 mm in diameter after 1 week at 25°C, then gray, flat with entire edges, aerial mycelia dense, and dark in the center and gray at the margin on the reverse side after 2 weeks. Conidiophores were olivaceous-brown, cylindrical, septate, unbranched, up to 230 × 3.4 to 5 µm. Conidiogenous cells were integrated, straight to curved, proliferating sympodial, 25 to 60 × 4.5 to 7 µm. Conidia were straight to slightly curved, ellipsoid, pale olivaceous brown to dark olivaceous brown, 2-6-distoseptate, with a distinct basal hilum, 26.9 to 70 × 11.4 to 21.6 µm (n = 50). Morphological characteristics of all isolates resembled the Exserohilum (Hernández-Restrepo et al. 2018). The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (tef1-α) genes were amplified using primer pairs ITS5/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1999), and EF1-983F/EF1-2218R (Rehner and Buckley 2005), respectively. Sequences were deposited in GenBank (ITS: PQ498876 to PQ498878; GAPDH: PQ509760 to PQ509762; tef1-α: PQ509763 to PQ509765). In the BLAST analysis, the ITS, GAPDH, and tef1-α sequences showed 99.50% (599 bp out of 602 bp), 98.89% (536 bp out of 542 bp), and 99.78% (926 bp out of 928 bp), respectively, to E. rostratum (CBS 325.87). Maximum likelihood and Bayesian inference phylogenetic analyses of the concatenated three genes identified all isolates as E. rostratum. To test pathogenicity, both wounded and unwounded healthy leaves were used in this experiment. All leaves were wiped with 0.1% NaClO and then rinsed three times with sterile water. Conidia suspensions (15 µl of 1 × 106 conidia/ml) of each isolate grown on PDA at 25°C for 3 weeks were placed on all samples using the attached leaf assay. Control leaves were mock-inoculated with sterile distilled water. Ten replications were conducted for each treatment and repeated twice. Plants were incubated at 25°C with 80 to 85% relative humidity. After 10 days, all inoculated leaves had brown lesions with yellow halos, while control leaves remained asymptomatic. E. rostratum was consistently reisolated from the lesions on PDA to complete Koch's postulates. Prior to this study, E. rostratum was identified as a causal agent of leaf blight on Calathea picturata in China (Taiwan, Chern et al. 2011). This is the first report of leaf blight caused by E. rostratum on C. orbifolia in China and worldwide. These findings will provide insights for epidemiological investigations and future approaches to managing this disease.

Trichoderma reesei holds immense promise for large-scale protein production, rendering it an excellent subject for deeper exploration using genetic engineering methods to achieve a comprehensive grasp of its cellular physiology. Understanding the genetic factors governing its intrinsic regulatory network is crucial, as lacking this knowledge could impede the expression of target genes. Prior and ongoing studies have concentrated on advancing new expression systems grounded in synthetic biology principles. These methodologies involve utilizing established potent promoters or engineered variations. Genomic and transcriptomic analyses have played a pivotal role in identifying robust promoters and expression systems, including light-responsive, copper-inducible, L-methionine-inducible, and Tet-On systems, among others. This chapter seeks to highlight various research endeavors focusing on tunable and constitutive promoters, the impact of different promoters on both native and foreign protein expression, the discovery of fresh promoters, and strategies conducive to future research aimed at refining and enhancing protein expression in T. reesei. Characterizing new promoters and adopting innovative expression systems hold the potential to significantly expand the molecular toolkit accessible for genetically engineering T. reesei strains. For instance, modifying potent inducible promoters such as Pcbh1 by replacing transcriptional repressors (cre1, ace1) with activators (xyr1, ace2, ace3, hap2/3/5) and integrating synthetic expression systems can result in increased production of crucial enzymes such as endoglucanases (EGLs), β-glucosidases (BGLs), and cellobiohydrolases (CBHs). Similarly, robust constitutive promoters such as Pcdna1 can be converted into synthetic hybrid promoters by incorporating activation elements from potent inducible promoters, facilitating cellulase induction and expression even under repressive conditions. Nevertheless, further efforts are necessary to uncover innovative promoters and devise novel expression strategies to enhance the production of desired proteins on an industrial scale.

Cellobiose dehydrogenase (CDH) catalyzes the oxidation of cello‐oligosaccharides and transfers electrons to oxygen or other electron receptors, such as lytic polysaccharide oxygenases (LPMOs). Cellobiose dehydrogenase has the potential to be used as an electron donor for LPMOs. Therefore, it is important to obtain CDHs under reaction conditions that are compatible with those of cellulases and LPMOs. Here, a CDH gene from Myceliophthora thermophila (MtCDH‐A) was constitutively expressed in Trichoderma reesei, and its ability to donate electrons to the LPMO was validated. MtCDH‐A has an optimal pH of 6.0 and maintains relatively high activity over a wide pH range. The optimal temperature was 60°C, and it remained stable at 50°C for a long time. These enzymatic properties were compatible with the catalytic conditions of fungal LPMOs and cellulases. When MtCDH‐A was used as an electron donor, the synergistic effect of LPMO and cellulase improved the degradation efficiency of phosphoric acid‐swollen cellulose by 141%. This work provides a novel insight into finding a stable electron donor for LPMOs and a new direction for finding more efficient thermostable CDHs in newly identified fungal species. © 2023 Society of Industrial Chemistry and John Wiley & Sons Ltd.