Functional diversification of epidithiodiketopiperazine methylation and oxidation towards pathogenic fungi

This study is led by Dr. Wen-Bing Yin and Dr. Jie Fan from the Institute of Microbiology, Chinese Academy of Sciences. The genus Trichoderma is widely recognized for its critical role in sustainable agriculture, particularly in promoting plant growth and protecting crops from pathogens. This is largely attributed to its production of various secondary metabolites (SMs), including epidithiodiketopiperazines (ETPs), which possess strong antifungal properties. These compounds are essential to Trichoderma's biocontrol mechanisms, enabling it to effectively outcompete harmful plant pathogens. Despite their recognized importance, the biological activities of ETPs, especially their structural diversity, remain underexplored in the context of ecological and agricultural applications.

In a previous study, the research team elucidated the biosynthesis of a, b'-disulfide bridged ETPs and uncovered the hidden diversity driven by the flexible substrate selection of Tda enzymes. For example, the deletion of tdaH and tdaG eliminated C6'-O-methylation and C4, C5-epoxidation of ETPs, respectively, thereby leading to divergent pathways to give diverse ETP derivatives. In this study, the team aims to investigate the antagonistic effects of ETP diversity on various pathogenic fungi by comparing Trichoderma hypoxylon wild type (WT) to gene deletion mutants.

To explore the mechanisms, the researchers adopted a comprehensive approach. By fermentation and analyzing their secondary metabolites through LC-MS, the team summarized the biosynthetic network of a, b'-disulfide bridged ETPs, particularly that in the single and double gene deletion mutants of tdaH and tdaG. Single or double gene deletion not only led to the accumulation of the corresponding precursor but also triggered the divergent pathways to afford diverse ETP derivatives.

Dual confrontation assays were then conducted between the T. hypoxylon strains and eleven pathogenic fungi, including Fusarium, Aspergillus, and Botrytis species, to assess the antagonistic effects of the different ETP modifications. Elimination of C6'-O-methylation and C4, C5-epoxidation reduced the antagonistic effects of T. hypoxylon against various pathogenic fungi. The deletion mutants exhibited varying antagonistic effects against different fungi, highlighting the importance of ETP structural diversity in T. hypoxylon's ecological adaptation. For instance, the DtdaH mutant showed a reduced ability to inhibit the growth of Aspergillus fumigatus and Botrytis cinerea, while the DtdaG mutant showed a significant decrease in growth inhibition against Fusarium nivale. The double deletion of tdaH and tdaG led to distinct antagonistic effects compared to the single deletions, further emphasizing the role of both modifications in mediating T. hypoxylon's interactions with pathogenic fungi.

“This work bridges the gap between fungal chemical ecology and practical agricultural applications,” said Dr. Yin. “By understanding how these modifications function, we can design more effective biofungicides that could replace harmful synthetic chemicals.” This study provides a roadmap for developing targeted biocontrol agents, aligning with global efforts to promote sustainable agriculture development.

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Functional diversification of epidithiodiketopiperazine methylation and oxidation towards pathogenic fungi