Epsilon Archive for Student Projects

Abstract

This study explored the role of CFEM-domain-containing proteins in Clonostachys rosea strain IK726, a well-characterized biocontrol fungus with known mycoparasitic activity. Genome analysis of C. rosea IK726 identified 22 genes encoding CFEM proteins, including 11 proteins with a transmembrane domain, eight GPI-anchored proteins, and three proteins with predicted signal peptides. The proteins with signal peptides were predicted to be secreted. One of these secreted CFEM-domain proteins, CRV2T0013563 (named CFEM12), which also harbours an antimicrobial peptide domain, was selected for functional characterization by generating gene deletion strains. Four individual deletion mutants of cfem12 were generated through Agrobacterium tumefaciens mediated transformation and validated through molecular techniques.

A comprehensive phenotypic assessment was conducted under both standard and stress conditions. Mycoparasitic and antagonistic assays were performed using dual culture plate confrontation and culture filtrate secretion tests against the plant pathogens Botrytis cinerea, Fusarium graminearum, and Rhizoctonia solani. A climate chamber experiment evaluating biocontrol efficacy against F. graminearum; an in-planta experiment evaluating the biocontrol efficacy against B. cinerea; root colonization on wheat; subcellular localization studies; transient gene expression assays to assess differences between the wild type (WT) and mutants.

The phenotypic analysis showed no significant difference related to growth or stress tolerance (NaCl, caffeine, SDS, sorbitol) between the C. rosea WT and cfem12 deletion strains. However, conidial production in cfem12 deletion strains was significantly affected (p < 0.001). While mutants did not differ from the WT in their ability to overgrow pathogens in dual culture assays, they exhibited a significantly reduced capacity to inhibit B. cinerea in secretion assays (p < 0.001) compared to the WT. No differences were observed in self-interaction between C. rosea mutant and WT lines. Furthermore, the mutants showed no significant differences from the WT in their ability to colonize wheat roots, control F. graminearum under controlled climate chamber conditions and to control B. cinerea in an in planta experiment.

Lastly, the transient expression of cfem12 gene did not supress hyper sensitive response (HR) induced by the gene avr4 in Nicotiana benthamiana and the subcellular localization-confocal analysis did not provide any information about the localization of the gene.

In summary, although cfem12 is not essential for growth, stress tolerance, root colonization, or mycoparasitic overgrowth, it plays a significant role in conidiation and the secretion of antifungal compounds in C. rosea.