# Functional characterization of a CFEM domain-containing protein in the mycoparasitic fungus Clonostachys rosea reveals antimicrobial activity and a role in conidiation

## TL;DR

This study shows that a CFEM domain-containing protein in the fungus Clonostachys rosea has antimicrobial properties and affects spore production, but not plant interactions or biocontrol abilities.

## Contribution

The study identifies a CFEM protein with antimicrobial activity and a role in conidiation in a mycoparasitic fungus.

## Key findings

- CFEM10 exhibits antimicrobial activity against E. coli and S. cerevisiae.
- Deletion of CFEM10 significantly reduces conidial production in C. rosea.
- CFEM10 does not affect fungal antagonism or biocontrol capabilities in C. rosea.

## Abstract

Common fungal extracellular membrane (CFEM) domain-containing proteins are small cysteine-rich proteins exclusive to fungi. They are shown to contribute to fungal virulence by promoting appressorium development and suppressing plant immune response. This study aimed to investigate the role of CFEM-domain-containing proteins in fungal antagonism and beneficial fungus-plant interactions using the mycoparasitic fungus Clonostachys rosea IK726, a biocontrol agent against several fungal pathogens. Gene expression analysis of 21 C. rosea IK726 CFEM-encoding genes during in vitro interactions with fungal hosts Botrytis cinerea and Rhizoctonia solani showed that their expression patterns depend on the host and interaction stage. CFEM10, predicted to have antimicrobial activity, was expressed in Escherichia coli and purified. An in vitro assay using purified CFEM10 protein revealed its antimicrobial activity against E. coli and Saccharomyces cerevisiae. Functional analysis of CFEM10 using gene deletion strains showed a significant difference (P = 0.01) in conidial production between the WT and Δcfem10 strains. However, no significant difference was found in fungal antagonisms against B. cinerea, Fusarium graminearum or R. solani, root colonization ability and biocontrol of fusarium foot and root rot between the WT and Δcfem10 strains. Similarly, transient expression of cfem10 in tobacco leaves failed to suppress hypersensitive response (HR) induced by Avr4/Cf4 complex. In summary, our results demonstrated the antimicrobial activity of CFEM10 and its involvement in fungal conidiation. Functional analysis of several CFEM-domain-containing proteins is needed to comprehensively evaluate their roles in fungal antagonism and beneficial interactions with plant hosts.

The online version contains supplementary material available at 10.1007/s00438-026-02390-7.