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Malate dehydrogenase (MDH) is a key enzyme in the tricarboxylic acid (TCA) cycle and is essential for energy balance, growth, and tolerance to cold and salt stresses in plants. However, the role of MDH in filamentous fungi is still largely unknown. In this study, we characterized an ortholog of MDH (AoMae1) in a representative nematode-trapping (NT) fungus Arthrobotrys oligospora via gene disruption, phenotypic analysis, and nontargeted metabolomics. We found that the loss of Aomae1 led to a weakening of MDH activity and ATP content, a remarkable decrease in conidia yield, and a considerable increase in the number of traps and mycelial loops. In addition, the absence of Aomae1 also caused an obvious reduction in the number of septa and nuclei. In particular, AoMae1 regulates hyphal fusion under low nutrient conditions but not in nutrient-rich conditions, and the volumes and sizes of the lipid droplets dynamically changed during trap formation and nematode predation. AoMae1 is also involved in the regulation of secondary metabolites such as arthrobotrisins. These results suggest that Aomae1 has an important role in hyphal fusion, sporulation, energy production, trap formation, and pathogenicity in A. oligospora. Our results enhance the understanding of the crucial role that enzymes involved in the TCA cycle play in the growth, development, and pathogenicity of NT fungi. [ABSTRACT FROM AUTHOR]

Ssk1, a response regulator of the two-component signaling system, plays an important role in the cellular response to hyperosmotic stress in fungi. Herein, an ortholog of ssk1 (Aossk1) was characterized in the nematode-trapping fungus Arthrobotrys oligospora using gene disruption and multi-phenotypic comparison. The deletion of Aossk1 resulted in defective growth, deformed and swollen hyphal cells, an increased hyphal septum, and a shrunken nucleus. Compared to the wild-type (WT) strain, the number of autophagosomes and lipid droplets in the hyphal cells of the ΔAossk1 mutant decreased, whereas their volumes considerably increased. Aossk1 disruption caused a 95% reduction in conidial yield and remarkable defects in tolerance to osmotic and oxidative stress. Meanwhile, the transcript levels of several sporulation-related genes were significantly decreased in the ΔAossk1 mutant compared to the WT strain, including abaA, brlA, flbC, fluG, and rodA. Moreover, the loss of Aossk1 resulted in a remarkable increase in trap formation and predation efficiency. In addition, many metabolites were markedly downregulated in the ΔAossk1 mutant compared to the WT strain. Our results highlight that AoSsk1 is a crucial regulator of asexual development, stress responses, the secondary metabolism, and pathogenicity, and can be useful in probing the regulatory mechanism underlying the trap formation and lifestyle switching of nematode-trapping fungi. [ABSTRACT FROM AUTHOR]

Aims: Phospholipase C (PLC) is a hydrolase involved in signal transduction in eukaryotic cells. This study aimed to understand the function of PLC in the nematode‐trapping fungus Arthrobotrys oligospora. Methods and Results: Orthologous PLC (AoPLC2) of A. oligospora was functionally analysed using gene disruption and multi‐phenotypic analysis. Disrupting Aoplc2 caused a deformation of partial hyphal cells (about 10%) and conidia (about 50%), decreased the number of nuclei in both conidia and hyphal cells, and increased the accumulation of lipid droplets. Meanwhile, the sporulation‐related genes fluG and abaA were downregulated in ΔAoplc2 mutants than in the wild‐type strain. Moreover, ΔAoplc2 mutants were more sensitive to osmotic stressors. Importantly, the number of traps, electron‐dense bodies in traps, and nematicidal activity of ΔAoplc2 mutants were reduced, and the shape of the traps was deformed. In addition, AoPLC2 was involved in the biosynthesis of secondary metabolites in A. oligospora. Conclusions: AoPLC2 plays an important role in the development of hyphae, spores, and cell nuclei, responses to stress, formation of traps, and predation of nematodes in A. oligospora. Significance and Impact of study: This study reveals the various functions of phospholipase C and elucidates the regulation of trap morphogenesis in nematode‐trapping fungi. [ABSTRACT FROM AUTHOR]

Inducer of meiosis 2 (Ime2), a protein kinase that has been identified in diverse fungal species, functions in the regulation of various cellular processes, such as ascospore formation, pseudohyphal growth, and sexual reproduction. In this study, AoIme2, an ortholog of Saccharomyces cerevisiae Ime2, was characterized in the nematode-trapping fungus Arthrobotrys oligospora. Disruption of the gene Aoime2 caused defective growth, with slower mycelial growth in Δ Aoime2 mutants than the wild type (WT) strain, and in the mutants, the number of hyphal septa in mycelia was higher and the number of cell nuclei in mycelia and conidia was considerably lower than in the WT strain. The conidial yields of the Δ Aoime2 mutants were decreased by ∼33% relative to the WT strain, and the transcription of several sporulation-related genes, including abaA , fluG , rodA , aspB , velB , and vosA , was markedly downregulated during the conidiation stage. The Δ Aoime2 mutants were highly sensitive to the osmotic stressors NaCl and sorbitol, and the cell wall of partial hyphae in the mutants was deformed. Further examination revealed that the cell wall of the traps produced by Δ Aoime2 mutants became loose, and that the electron-dense bodies in trap cells were also few than in the WT strain. Moreover, Aoime2 disruption caused a reduction in trap formation and serine-protease production, and most hyphal traps produced by Δ Aoime2 mutants did not form an intact hyphal loop; consequently, substantially fewer nematodes were captured by the mutants than by the WT strain. In summary, an Ime2-MAPK is identified here for the first time from a nematode-trapping fungus, and the kinase is shown to be involved in the regulation of mycelial growth and development, conidiation, osmolarity, and pathogenicity in A. oligospora. [ABSTRACT FROM AUTHOR]

• AoGlo3 is the ortholog of yeast Glo3 and a pleiotropic regulator in A. oligospora. • AoGlo3 is indispensable for conidiation, trap formation, and nematode trapping. • AoGlo3 contributes to mycelial growth and environmental adaption. • AoGlo3 is required for endocytosis and autophagy. Small GTPases of the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development and pathogenicity in yeast and filamentous fungi; however, little is known about their roles in nematode-trapping (NT) fungi. In this study, an ortholog of Arf-GAP Glo3 (AoGlo3) in Saccharomyces cerevisiae was characterized in the NT fungus Arthrobotrys oligospora. Deletion of the Aoglo3 gene resulted in growth defects and an increase in hyphal septum. Meanwhile, the sporulation capacity of the Δ Aoglo3 mutant was decreased by 98%, and 67.1–71.2% spores became gourd or claviform in shape (from obovoid), which was accompanied by a significant decrease in the spore germination rate. This reduced sporulation capacity correlated with the transcriptional repression of several sporulation-related genes including fluG , rodA , abaA , medA , and lreA. The Δ Aoglo3 mutant was also sensitive to several chemical stressors such as Congo red, NaCl, and sorbitol. Additionally, AoGlo3 was found to be involved in endocytosis, and more myelin figures were observed in the Δ Aoglo3 mutant than in the wild-type strain, which was consistent with the presence of more autophagosomes observed in the mutant. Importantly, AoGlo3 affected the production of mycelial traps and serine proteases for nematode predation. In summary, AoGlo3 is involved in the regulation of multiple cellular processes such as mycelial growth, conidiation, environmental adaption, endocytosis, and pathogenicity in A. oligospora. [ABSTRACT FROM AUTHOR]