TbNACα negatively regulates Trichoderma breve T069 synthesis of ethyl caffeate and enhances antagonism of Sclerotium rolfsii

The nascent polypeptide-associated complex (NAC) plays crucial roles in various biological functions in eukaryotes and has been extensively studied in animals and plants; however, its role in the biocontrol mechanisms of microorganisms requires further investigation. This study examined the function of TbNACα, a NAC subunit, in the biocontrol activity of Trichoderma breve T069 against Sclerotium rolfsii. Following deletion of the TbNACα gene from T. breve T069, the ΔTbNACα mutant exhibited significantly reduced mycelial growth, spore production, and spore germination. While volatile substances from ΔTbNACα showed no significant effect on S. rolfsii, non-volatile substances demonstrated significant inhibition of S. rolfsii growth. Transcriptome sequencing analysis revealed 3,398 differentially expressed genes in the ΔTbNACα mutant compared to wild-type T069, primarily regulating genes associated with secondary metabolite biosynthetic enzymes, hydrolases, and membrane transport proteins. Untargeted metabolomics identified 50 upregulated metabolites (27 in positive ion mode and 23 in negative ion mode) in crude extracts from ΔTbNACα mutant metabolite broth. Among these metabolic substances, ethyl caffeate demonstrated the strongest activity against S. rolfsii, with an EC₅₀ of 107.15 μg mL^–1. Quantitative real-time PCR (qPCR) analysis indicated significant upregulation of genes involved in the ethyl caffeate synthesis pathway in ΔTbNACα strains. This research establishes the negative regulation of ethyl caffeate synthesis and elucidates the antagonistic inhibition mechanism of TbNACα in T. breve T069.