bZIP63.5 Regulates a Zinc-Finger Mediated Detoxification Network in Trichoderma Harzianum for Enhanced Biocontrol of Alternaria alternata.

Trichoderma species are widely recognised as biocontrol agents, yet the transcriptional mechanisms underlying their antagonism against foliar pathogens remain largely unexplored. In this study, we identified 32 bZIP genes in Trichoderma harzianum Tha739 and demonstrated that bZIP63.5 exhibited the strongest upregulation under Alternaria alternata metabolite stress. Overexpression and knockdown strains of bZIP63.5 were constructed, and phenotypic assays revealed that bZIP63.5 significantly enhanced the antifungal activity of T. harzianum through direct confrontation, volatile organic compound production, and secreted metabolites. Using yeast one-hybrid and electrophoretic mobility shift assays, we identified that bZIP63.5 binds to known cis-acting elements (TGTCACA, TGTCA, GTGA, TGAC) as well as a novel motif (TACGGAC). Yeast two-hybrid screening further revealed that bZIP63.5 interacts with multiple proteins, including histone H4 and heat shock protein HSP70, suggesting its involvement in chromatin regulation and stress response. Transcriptome analysis of the bZIP63.5 overexpression strain showed that it directly upregulates a set of zinc-finger transcription factors, which in turn activate downstream detoxification genes (ABC transporters, cytochrome P450s) and defence-related enzymes (glycosyl hydrolases, peroxidases). ChIP-PCR confirmed the direct binding of bZIP63.5 to the promoters of these zinc-finger TFs. Functional validation through overexpression and knockout of three core zinc-finger TFs (Zn2CyS6ZF56.5, C2H2ZF36.8, C2H2ZF40.8) demonstrated their essential roles in enhancing the biocontrol efficacy of T. harzianum against A. alternata. This study elucidates a hierarchical transcriptional network centered on bZIP63.5 that coordinates detoxification and defence responses in T. harzianum, providing novel insights into the molecular mechanisms of fungal biocontrol and potential targets for improving disease management in forestry systems.