AoHal4b interacts with AoAdv-1 governs hyphal fusion, nematode predation ability, and secondary metabolism in Arthrobotrys oligospora

INTRODUCTION: Serine/threonine kinases (STKs) are crucial for contribute to the growth, pathogenicity, and potassium homeostasis in filamentous fungal pathogens. However, the functions of STKs in hyphal fusion, trap morphogenesis, and lifestyle transitions of nematode-trapping (NT) fungi remain unclear. OBJECTIVES: We sought to investigate the functions and underlying regulatory mechanisms of the STK-encoding gene Aohal4b in Arthrobotrys oligospora, a model NT fungus. METHODS: Two putative STKc_Hal4-encoding genes (Aohal4a and Aohal4b) were identified in the A. oligospora genome by querying with the conserved STKc_Hal4-like domain from S. cerevisiae. AoHal4b functions were investigated using targeted gene deletion, phenotypic characterization, subcellular localization, quantitative real-time PCR, RNA-seq, and untargeted metabolomics. In addition, we employed a combination of yeast two-hybrid, GST pull-down, split-luciferase complementation, and phosphorylation analyses to dissect the underlying regulatory mechanisms. RESULTS: The ΔAohal4b mutant completely lost hyphal fusion capacity, which abolished the formation of functional three-dimensional networks and resulted in the production of aberrant spiral hyphae. The mutant also exhibited a severe deficiency in secondary metabolism, with arthrobotrisin levels reduced by 171-fold; this metabolic shift contributes to a hyper-trapping yet functionally impaired phenotype. Beyond these core functions, AoHal4b additionally governs mycelial growth, sporulation, and ion homeostasis. Furthermore, we demonstrated that AoHal4b directly interacts with the transcription factor AoAdv-1 through multiple protein-protein interaction assays, and AoHal4b regulates the nuclear localization of AoAdv-1 by phosphorylation. Moreover, comparative transcriptomic analysis revealed that AoHal4b and AoAdv-1 antagonistically co-regulate several genes associated with cell wall remodeling, including those encoding chitinases and glucanases. CONCLUSION: Our study demonstrates AoHal4b as a key pleiotropic regulator in A. oligospora. It orchestrates hyphal fusion and trap morphogenesis through direct interaction with the transcription factor AoAdv-1, and concurrently modulates secondary metabolism and ion homeostasis. These findings provide deep mechanistic insight into the STK-mediated developmental switch governing the carnivorous lifestyle of NT fungi.