TbNACα negatively regulates Trichoderma breve T069 synthesis of ethyl caffeate and enhances antagonism of Sclerotium rolfsii1

The nascent polypeptide-associated complex (NAC) is involved in various biological functions in eukaryotes which have been extensively studied in animals and plants, but its role in the biocontrol action of microorganisms needs to be better understood. In this study, we investigated the function of TbNACα, one of the subunits of NAC, in the biocontrol activity of Trichoderma breve T069 against Sclerotium rolfsii. The TbNACα gene was deleted from T. breve T069, and the ΔTbNACα mutant showed significantly reduced mycelial growth, spore production, and spore germination. Additionally, volatile substances from ΔTbNACα had no significant effect on S. rolfsii, while non-volatile substances significantly inhibited the growth of S. rolfsii. Transcriptome sequencing results showed that compared to wild-type T069, the ΔTbNACα mutant had 3,398 differentially expressed genes, mainly regulating the expression of genes related to 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. Moreover, the metabolic substances of ethyl caffeate had the strongest activity against S. rolfsii, with an EC₅₀ of 107.15 μg·mL^-1. Quantitative Real-time PCR (qPCR) analysis revealed that genes involved in the ethyl caffeate synthesis pathway were significantly upregulated in ΔTbNACα strains. This study provides a basis for the negative regulation of ethyl caffeate synthesis and elucidates the antagonistic inhibition mechanism of TbNACα in T. breve T069.