ThLaeA functions as a conditional repressor to maintain metabolic homeostasis in Trichoderma hypoxylon

ABSTRACT The global regulator LaeA is widely recognized as a master activator of secondary metabolism and development in filamentous fungi. Yet its role under genetically buffered or metabolically stable conditions—where canonical phenotypes are masked—remains poorly understood. This study aimed to characterize the LaeA ortholog ( ThlaeA ) in Trichoderma hypoxylon and to elucidate its regulatory role in a Δ Thtri5 background, where trichodiene synthase function is disrupted. We constructed single and double knockout mutants (Δ ThlaeA , Δ Thtri5 , and Δ ThlaeA Δ Thtri5 ) and performed integrated metabolomic and transcriptomic analyses to assess global regulatory effects. Additional assays evaluated oxidative stress responses and biocontrol activity. The deletion of ThlaeA alone had negligible effects on secondary metabolite production, whereas disruption of ThlaeA in the Δ Thtri5 background restored biosynthesis of major terpenoid compounds abolished in Δ Thtri5 . Metabolomics revealed that ThlaeA regulates 48.4% of metabolites in the wild-type but 31.3% in Δ Thtri5 , while transcriptomics showed restoration of 16.7% of gene expression dysregulated in Δ Thtri5 . Phenotypically, ThlaeA deletion reinstated oxidative stress sensitivity and partially attenuated biocontrol efficacy. ThlaeA acts as a conditional repressor that counterbalances Thtri5-dependent perturbations to maintain metabolic homeostasis. This work redefines the LaeA paradigm and provides a framework for understanding the context-dependent regulation of secondary metabolism in fungi. IMPORTANCE LaeA plays compelling roles in secondary metabolism and development in filamentous fungi. However, related research also found that genetic operations of LaeA have no obvious effect on the metabolic spectrum in some fungal species. Many attempts have been made to decipher the phenomenon and to explain how about the function of LaeA in these cases. Here, we identified a ThlaeA in Trichoderma hypoxylon . The deletion of ThlaeA alone did not alter secondary metabolism but restored metabolite production in a Δ Thtri5 background. Integrated metabolomic and transcriptomic analyses revealed that ThlaeA modulates metabolic homeostasis by compensating for Thtri5-related perturbations. ThlaeA-Thtri5 interaction regulates oxidative stress responses and membrane transport pathways, coupling secondary metabolism with physiological adaptation. This regulatory model broadens the understanding of the LaeA protein family. Fine-tuning this pathway can enhance the environmental adaptability and agricultural biocontrol potential of Trichoderma strains, while boosting bioactive secondary metabolite production and optimizing fungal cell factories. This study advances fundamental insights into fungal metabolic regulation and provides a rational basis for strain improvement and biotechnological applications in agriculture and industry.