Filamentous fungi have wide applications in biotechnology. The CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations of genomes in a variety of organisms. However, a genome-editing approach has not been reported in filamentous fungi. Here, we demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

For understanding solvation by ionic liquids, it is mandatory to characterize their static relative dielectric permittivities ε ("static dielectric constants"). Exploiting the definition of ε in terms of the zero-frequency limit of the frequency-dependent dielectric dispersion curve, the static dielectric constant of an electrically conducting liquid can be extrapolated from dielectric relaxation spectra in the microwave regime. On the basis of this method, we report dielectric constants of 42 ionic liquids at 25 °C.

Calcium signaling plays pivotal roles in the hyphal growth, conidiation, and osmosis sensitivity of fungi through the Ca(2+) /calmodulin-calcineurin-dependent pathway. This study found that an appropriate extracellular Ca(2+) concentration markedly stimulated the hyphal growth, cellulase production, and total protein secretion of the cellulase hyper-producing strain, Trichoderma reesei Rut-C30. Transcription analysis revealed upregulation of not only encoding genes of cellulases and the transcriptional activator XYR1 but also several genes encoding endoplasmic reticulum-chaperones after Ca(2+) addition. The function of CRZ1, T. reesei calcineurin-responsive zinc finger transcription factor 1, was further characterized by gene disruption. Electrophoretic mobility shift assays (EMSAs) in combination with chromatin immunoprecipitation (ChIP) verified that CRZ1 could bind directly to the upstream regions of xyr1 and cbh1 (cellobiohydrolase I-encoding gene) in response to Ca(2+) . A DNase I footprinting assay identified its putative binding consensus site (5'-[T/G]GGCG-3' or 5'-GGGC[G/T]-3'). EMSAs confirmed that CRZ1 competed for occupancy of the xyr1 promoter with another transcription factor, ACE1. These results revealed putative signaling pathways downstream of calcineurin in response to extracellular Ca(2+) involved in upregulation of cellulose degradation-related genes, reflecting progress in the study of Ca(2+) signaling in filamentous fungi. This study also provides insight that will facilitate further improvement of (hemi-)cellulase production by T. reesei.