| Summary: | Small GTPases from the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development, endocytosis, and virulence in fungi. Here, we identified two orthologous Arf-GAP proteins, AoGcs1 and AoGts1, in a typical nematode-trapping fungus <i>Arthrobotrys oligospora</i>. The transcription of <i>Aogcs1</i> and <i>Aogts1</i> was highly expressed in the sporulation stage. The deletion of <i>Aogcs1</i> and <i>Aogts1</i> caused defects in DNA damage, endocytosis, scavenging of reactive oxygen species, lipid droplet storage, mitochondrial activity, autophagy, serine protease activity, and the response to endoplasmic reticulum stress. The combined effects resulted in slow growth, decreased sporulation capacity, increased susceptibility to chemical stressors and heat shock, and decreased pathogenicity of the mutants compared with the wild-type (WT) strain. Although deletion of <i>Aogcs1</i> and <i>Aogts1</i> produced similar phenotfypic traits, their roles varied in conidiation and proteolytic activity. The Δ<i>Aogts1</i> mutant showed a remarkable reduction in conidial yield compared with the WT strain but not in proteolytic activity; in contrast, the Δ<i>Aogcs1</i> mutant showed an increase in proteolytic activity but not in sporulation. In addition, the growth of Δ<i>Aogcs1</i> and Δ<i>Aogts1</i> mutants was promoted by rapamycin, and the Δ<i>Aogts1</i> mutant was sensitive to H-89. Collectively, the Δ<i>Aogts1</i> mutant showed a more remarkable difference compared with the WT strain than the Δ<i>Aog</i><i>cs1</i> mutant. Our study further illustrates the importance of Arf-GAPs in the growth, development, and pathogenicity of nematode-trapping fungi.
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