Metarhizium anisopliae is an entomopathogenic fungus which may enhance plant growth and resistance when acting as an endophyte in host plants. However, little is known about the protein interactions nor their activating mechanisms. Common in fungal extracellular membrane (CFEM) proteins have been identified as plant immune regulators that suppress or activate plant resistance responses. Here, we identified a CFEM domain-containing protein, MaCFEM85, which was mainly localized in the plasma membrane. Yeast two-hybrid (Y2H), glutathione-S-transferase (GST) pull-down, and bimolecular fluorescence complementation assays demonstrated that MaCFEM85 interacted with the extracellular domain of a Medicago sativa (alfalfa) membrane protein, MsWAK16. Gene expression analyses showed that MaCFEM85 and MsWAK16 were significantly upregulated in M. anisopliae and M. sativa, respectively, from 12 to 60 h after co-inoculation. Additional yeast two-hybrid assays and amino acid site-specific mutation indicated that the CFEM domain and 52th cysteine specifically were required for the interaction of MaCFEM85 with MsWAK16. Defense function assays showed that JA was up-regulated, but Botrytis cinerea lesion size and Myzus persicae reproduction were suppressed by transient expression of MaCFEM85 and MsWAK16 in the model host plant Nicotiana benthamiana. Collectively, these results provide novel insights into the molecular mechanisms underlying interactions of M. anisopliae with host plants.

The entomopathogen Metarhizium anisopliae is a facultative rhizosphere or endophytic fungus available for managing pests and improving plant growth. The CFEM (common in fungal extracellular membrane) proteins form a unique group in fungi but are rarely reported in entomopathogens. In this study, we cloned and identified 13 CFEM genes from M. anisopliae (MaCFEMs). Sequence alignment and WebLogo analysis showed that eight cysteines were the most conserved amino acids in their CFEM domain. Phylogenic analysis suggested that these 13 proteins could be divided into 4 clades based on the presence of the transmembrane region and the position of CFEM domain in the whole sequence. Six MaCFEM proteins with a signal peptide and without a transmembrane domain were considered candidate effector proteins. According to Phyre2 analysis, the MaCFEM88 and MaCFEM85 have the most homologous to Csa2 in Candida albicans. Subcellular localization analysis revealed that five effectors were located in the plasma membrane, while MaCFEM88 may locate in both plasma membrane and nucleus in the treated Nicotiana benthamiana. Expression pattern analysis showed that MaCFEM81, 85, 88, and 89 expression level was significantly higher in the sporulation stage compared to other growth stages. Furthermore, the yeast secretion assay showed that six candidate effectors were able to secrete out of the cell. All of the MaCFEMs couldn’t affect INF1-induced programmed cell death (PCD), but MaCFEM85 and 88 could trigger a slight hypersensitive response both when applied separately or in combination with INF1 in N. benthamiana leaves. These findings showed that six MaCFEM potential effectors with various structures and subcellular localizations in host cells might be used to illustrate the roles of MaCFEM proteins during M. anisopliae-plant interactions.

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