Deciphering the role of ThHSF in the differential expression regulation of laccase isozymes in the white-rot fungus Trametes hirsuta

White-rot fungi exhibit responses to Cu2+, leading to a substantial increase in the production of certain laccase isozymes for industry utilization. Currently, studies on the differential expression mechanism of the laccase isozymes in white-rot fungi are notably insufficient. Four laccase isozymes, including LacA, LacB, as well as a newly identified isozyme, LacF, were dramatically induced to overexpress in Trametes hirsuta AH28-2 when exposed to Cu2+, accompanied by slight enhancement of LacC. Here, based on transcriptomics and proteomics, a nuclear-localized heat shock transcription factor, ThHSF, was mined and exhibited as a copper-responsive protein involved in the differential regulation of laccase isozyme expression. A decreased mycelial thickness and curved hyphal morphology were observed in three Thhsf silenced T. hirsuta AH28-2 transformants in response to Cu2+ stress. Thhsf silencing resulted in down-regulated transcriptional levels and activities of LacA, LacB, and LacF, but not LacC. EMSA assays further demonstrated the binding of ThHSF only to the promoter regions of lacA, lacB, and lacF containing HSE elements like CTTGAA. A previously reported Hsp70 homolog, ThhspA1, could interact with ThHSF and synergistically regulate the expression of three laccase isozymes. ThHSF and ThHspA1 co-overexpression led to approximately 1.5-fold increased laccase activities, providing an efficient strategy to enhance laccase production.