Investigating the role of CFEM domain proteins in mycoparasitic and beneficial-fungus plant interactions

Common Fungal Extracellular Membrane (CFEM) domains containing protein are reported only in fungi and are crucial for fungal virulence. These proteins mediate interaction with the host plant by evading and suppressing the host´s immune response mechanisms. Although the role of CFEM domain-containing proteins in pathogenic fungus-plant interactions is known, their role in beneficial fungus-plant interactions and mycoparasitic interactions remains elusive. This study aims to understand the function of CFEM domain proteins in the fungal biocontrol agents Clonostachys rosea with a focus on their role in the interactions with the fungal and plant hosts. Twenty-two CFEM domain proteins have been identified in the C. rosea genome. The weblogo analysis revealed that the consensus motif for the CFEM domain in fungal pathogens aligns with that in C. rosea, indicating shared evolutionary features and functional similarities. Among those, three CFEM domain proteins (CFEM1, protein ID CRV2T00010487; CFEM2, protein ID CRV2T00013563, CFEM3 protein ID CRV2T00016446) contain signal peptides and are predicted to be secreted. Those three putative effectors were selected for functional characterization. Gene expression analysis showed the downregulation of cfem1 and cfem2 (P = 0.001) in C. rosea during interaction with fungal host Botrytis cinerea at mycelial contact and after contact time points, compared to self-interaction control. However, no differences in gene expression were found during the interactions with wheat roots. The preliminary findings indicate a potential role of CFEM domain proteins in mycoparasitic interactions while they may not be necessary for initial interactions with plant hosts. Furthermore, gateway cloning was used to create gene deletion cassettes, and Agrobacterium tumefaciens-mediated transformation is underway to generate gene deletion strains in C. rosea. To identify potential receptors for these three CFEM proteins in plants and to test if CFEM proteins can induce or suppress the host immune response, overexpression constructs were generated. The transient expression of CFEM proteins in Nicotiana benthamiana by agroinfiltration is underway. Total RNAs will be isolated from N. benthamiana leaves filtrated with overexpression cassettes, and the expression pattern of the immune response marker gene will be compared with the empty vector control. Further study on C. rosea mutant phenotyping is required to comprehend more on the function of these genes in mycoparasitic and beneficial-fungus plant interactions.