Cell fusion-related proteins <i>AoHam4, AoHam8 and AoPP2A</i> regulate hyphal fusion, conidiation, trap morphogenesis, and secondary metabolism in <i>Arthrobotrys oligospora</i>

The diverse mycelial networks of fungi are generated through polar growth, cell division, and cell fusion. Most of the genes are well characterized as crucial for cellular communication and fusion processes in filamentous fungi, but their functions and molecular mechanisms remain poorly understood. Here, we functionally characterized the hyphal anastamosis protein 4 (AoHam4), hyphal anastamosis-8 protein (AoHam8) and serine/threonine protein phosphatase 2A (AoPP2A) in the model nematode-trapping fungus Arthrobotrys oligospora. Our results indicate that Aoham4, Aoham8 and Aopp2a genes are essential for hyphal fusion and trap morphogenesis, and modulate mycelial growth, conidial production, and pathogenicity in A. oligospora. Staining, RT-qPCR and transmission electron microscopy (TEM) results indicated that all three genes are involved in regulating reactive oxygen species (ROS) accumulation, lipid metabolism and autophagy processes. Moreover, RNA-Seq and liquid chromatography-mass spectrometry (LC-MS) experiments further confirmed that deletion of Aoham4, Aoham8 and Aopp2a genes affects transcription and metabolic levels. Yeast-two-hybrid (Y2H) analysis showed that AoPP2A can interact with AoSO (Soft, a fungus-specific scaffolding protein, is involved in signaling and secretion with the MAK-2 cascade). Since the ΔAoham8 mutant strain was more sensitive to cell wall-disrupting reagents, speculating that Aoham8 may regulate the mitogen-activated protein (MAP) kinase cascade response by activating the cell wall integrity pathway. Collectively, our studies illuminate the crucial roles of the fungal cell-fusion genes Aoham4, Aoham8 and Aopp2a in A. oligospora, as well as laying the groundwork for clarifying the mechanisms of mycelial development and trap morphogenesis of nematode-trapping fungi.