Deciphering the role of ThHSF1 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 limited. Three laccase isozymes, LacA, LacB, and LacF, were significantly induced in Trametes hirsuta AH28-2 when exposed to Cu2+, accompanied by a slight enhancement in the isozyme LacC level. Here, based on transcriptomics and proteomics, a nuclear-localized heat shock transcription factor, ThHSF1, was mined and exhibited as a copper-responsive protein involved in the differential regulation of laccase isozyme expression. A changed colony morphology and curved hyphal morphology were observed in three Thhsf1-silenced T. hirsuta AH28-2 transformants in response to Cu2+ stress. Thhsf1 silencing resulted in downregulated transcriptional levels and activities of LacA, LacB, and LacF, but not LacC. EMSA assays further demonstrated the binding of ThHSF1 only to the promoter regions of lacA, lacB, and lacF containing HSE elements like CTTGAA. A previously reported Hsp70 homolog, ThHspA1, could interact with ThHSF1 and synergistically regulate the expressions of three laccase isozymes. ThHSF1 and ThHspA1 co-overexpression led to approximately 1.5-fold increased laccase activities, providing an efficient strategy to enhance laccase production.IMPORTANCEWhite-rot fungi, especially Trametes species, are important producers of laccase. They typically express multiple laccase isozymes with distinct physicochemical properties in response to Cu2+. Elucidating the molecular mechanisms underlying differential laccase expression induced by Cu2+ is critical for enhancing laccase production through strain modification. This study demonstrates that ThHSF1 collaborates with ThHspA1 to regulate the expression of only three Cu2+-responsive laccase isozymes in T. hirsuta AH28-2. Co-overexpression of ThHSF1/ThHspA1 efficiently promotes laccase production. These findings also deepen our understanding of how white-rot fungi adapt to environmental Cu2+.