Glycogen synthase AfGsy2 regulates mycelial growth, stress response, predatory activity, and chlamydospore development in the nematode-trapping fungus Arthrobotrys flagrans

Abstract

Glycogen is a universal carbon and energy reserve that plays crucial roles in fungal stress adaptation, cellular homeostasis, and predatory activity. However, the role of glycogen metabolism in nematode-trapping fungi has largely remained unexplored. Here, we performed a systematic functional characterization of AfGsy2 , the glycogen synthase-encoding gene in the biocontrol fungus Arthrobotrys flagrans . Using a CRISPR-Cas9-mediated deletion mutant, together with overexpression and complementation strains, we investigated the involvement of AfGsy2 in mycelial growth, stress tolerance, trap formation, nematicidal activity against Caenorhabditis elegans , chlamydospore production and germination, and glycogen accumulation. Subcellular localization was examined using an AfGsy2-GFP fusion protein. Deletion of AfGsy2 reduced hyphal growth on both nutrient-rich and nutrient-poor media but enhanced tolerance to osmotic, salt, and oxidative stresses, suggesting that blocked glycogen synthesis may trigger metabolic rerouting toward alternative stress-protective compounds. Notably, the ΔAfGsy2 mutants exhibited reduced trap production and nematicidal activity against C. elegans , whereas overexpression of AfGsy2 accelerated these processes, suggesting a link between glycogen metabolism and predatory activity. Moreover, AfGsy2 positively regulated chlamydospore production, glycogen accumulation, and chlamydospore germination. AfGsy2-GFP predominantly localized to the vacuoles of chlamydospores, and complementation restored the mutant phenotypes to wild-type levels. Collectively, these findings uncover a link between glycogen metabolism and growth, stress resilience, sporulation, and predatory activity in A. flagrans , and establish a molecular foundation for the rational improvement of nematode-trapping fungi. Together, these results identify AfGsy2 as a pleiotropic regulator linking glycogen metabolism to biocontrol-related traits.