The CcMAK2 gene is required for proper asexual fruiting body formation in Cordyceps cicadae

Cordyceps cicadae is a traditional precious medicinal and edible entomogenous fungus. Its asexual fruiting bodies are highly valued in natural health food. Currently, the intrinsic mechanisms regulating fruiting body development in C. cicadae remain largely elusive. The Fus3/MAK2 homolog within the MAPK signaling pathway plays a crucial role in sexual reproduction and pathogenesis in filamentous fungi, yet its function in the fruiting body development of C. cicadae has not been reported. Therefore, this study focused on CcMAK2 from C. cicadae, constructing gene overexpression and knockout strains to analyze its impact on fungal growth and development. The results demonstrated that the knockout strain (ΔCcMAK2) failed to penetrate the insect cuticle from inside, and completely lost the ability to form fruiting bodies. In contrast, the wild-type (WT) and overexpression strain (CcMAK2^OE) developed normally, indicating that CcMAK2 is essential for fruiting body formation in C. cicadae. Functional analysis revealed that the ΔCcMAK2 mutant exhibited significantly reduced levels of H₂O₂ and O₂ ^.-during the fruiting stage, accompanied by increased activities of CAT and GR enzymes, as well as elevated chitinase activity. These findings suggest that the deletion of CcMAK2 is associated with alterations in ROS homeostasis and cell wall-related enzyme. Gene expression analysis further showed that the deletion of CcMAK2 led to altered transcript levels of the downstream transcription factor Ste12 and the cell wall integrity pathway-related gene CcSO. These results suggest that CcMAK2 may be involved in regulating these genes, potentially contributing to its role in fruiting body development. This study provides foundational insights into the role of CcMAK2 in fruiting body development in C. cicadae and lays groundwork for further mechanistic studies in Cordyceps and other fungi. Graphical abstract.