Characterization of the Ca(2+) -responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30.
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.
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Ling
L
Chen
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Gen
G
Zou
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Jingzhi
J
Wang
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Jin
J
Wang
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Rui
R
Liu
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Yanping
Y
Jiang
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Guoping
G
Zhao
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Zhihua
Z
Zhou
D000595:Amino Acid Sequence; D019703:Calcineurin / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002480:Cellulase / Q000096:biosynthesis*; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D015966:Gene Expression Regulation, Fungal; D011401:Promoter Regions, Genetic / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics; D014242:Trichoderma / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D015854:Up-Regulation; D016335:Zinc Fingers / Q000235:genetics
D014157:Transcription Factors; D002482:Cellulose; D019703:Calcineurin; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D002118:Calcium