The Hog1-Nmd5 signaling pathway regulates asexual development, lipid metabolism, stress response, trap morphogenesis, and secondary metabolism of Arthrobotrys oligospora.

The high-osmolarity glycerol (HOG) signalling pathway, comprising Ste11/Ssk2/Ssk22 (MAPKKK), Pbs2 (MAPKK), and Hog1 (MAPK), is an important and conserved pathway in fungi. However, the functions and downstream regulatory factors of Hog1 in nematode-trapping (NT) fungi remain poorly understood. Here, three proteins (AoNmd5, AoPyp1, and AoPtp) interacting with Hog1 were screened in a representative NT fungus Arthrobotrys oligospora using yeast screening library and verified using yeast two-hybrid (Y2H) assay. The function of AoNmd5 was furtherly characterized by phenotypic comparison, staining technique, and multi-omics analyses. AoNmd5 was essential for vegetative growth, conidial development, trap morphogenesis, and nematode predation ability. In addition, AoNmd5 played crucial roles in endocytosis, lipid metabolism, reactive oxygen species, stress response, autophagy, and other metabolic processes. Furthermore, we constructed an AoNmd5 interaction network based on transcriptomic analysis and Y2H, revealing its significant role in the respiratory chain and redox processes as well as its interaction with the small GTPase Ran1, which mediates Hog1 nucleocytoplasmic shuttling. These findings suggest that the Hog1-Nmd5 signalling pathway has pleiotropic roles in A. oligospora. This study deepens our understanding of the HOG pathway and its interaction with importins in NT fungi.