Deciphering the role of <i>Th</i> HSF1 in the differential expression regulation of laccase isozymes in the white-rot fungus <i>Trametes hirsuta</i>

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 <i>Trametes hirsuta</i> 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, <i>Th</i>HSF1, 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 <i>Thhsf1</i>-silenced <i>T. hirsuta</i> AH28-2 transformants in response to Cu²⁺ stress. <i>Thhsf1</i> silencing resulted in downregulated transcriptional levels and activities of LacA, LacB, and LacF, but not LacC. EMSA assays further demonstrated the binding of <i>Th</i>HSF1 only to the promoter regions of <i>lacA</i>, <i>lacB,</i> and <i>lacF</i> containing HSE elements like CTTGAA. A previously reported Hsp70 homolog, <i>Th</i>HspA1, could interact with <i>Th</i>HSF1 and synergistically regulate the expressions of three laccase isozymes. <i>Th</i>HSF1 and <i>Th</i>HspA1 co-overexpression led to approximately 1.5-fold increased laccase activities, providing an efficient strategy to enhance laccase production.IMPORTANCEWhite-rot fungi, especially <i>Trametes</i> 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 <i>Th</i>HSF1 collaborates with <i>Th</i>HspA1 to regulate the expression of only three Cu²⁺-responsive laccase isozymes in <i>T. hirsuta</i> AH28-2. Co-overexpression of <i>Th</i>HSF1/<i>Th</i>HspA1 efficiently promotes laccase production. These findings also deepen our understanding of how white-rot fungi adapt to environmental Cu²⁺.