AoSsk1, a Response Regulator Required for Mycelial Growth and Development, Stress Responses, Trap Formation, and the Secondary Metabolism in Arthrobotrys oligospora

Abstract

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.

SNARE Protein AoSec22 Orchestrates Mycelial Growth, Vacuole Assembly, Trap Formation, Stress Response, and Secondary Metabolism in Arthrobotrys oligospora

“…The WT and mutant strains were incubated for 5-day radial growth on PDA, TYGA, and TG agar plates at 28 °C [ 41 ]. The spore yield of each strain was measured from the 15-day-old cultures grown on corn meal yeast extract (CMY) medium, as previously described [ 44 , 45 ]. Freshly harvested conidia and hyphae of WT and mutant strains were stained with 20 μg/mL of cell wall-specific calcofluor white (CFW, Sigma-Aldrich, St. Louis, MO, USA) or nucleus-specific 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA), and visualized for hyphal morphology under an inverted fluorescence microscope [ 46 , 47 ].…”

“…Three hyphal mass discs (7 mm) of each strain were incubated in PD broth for 7 days at 28 °C and 180 rpm, followed by mass collection through vacuum filtration [ 44 ]. The biomass was dried, weighed (WT, 3.14 g; Δ Aosec22 , 2.89 g), and used to prepare extracts of the same concentration.…”

“…Ethyl acetate (250 mL) was added to extract the fermentation broth and ultrasonic extraction was performed for 40 min. The fermentation broth was allowed to stand for 12 h and the crude extract was concentrated in ethyl acetate under vacuum [ 44 ]. The crude extract was dissolved in 500 µL of analytic-grade methanol and the solution was filtered through a 0.22 µm membrane filter for liquid chromatography–mass spectrometry (LC–MS) analysis.…”

The cAMP‐PKA signalling pathway regulates hyphal growth, conidiation, trap morphogenesis, stress tolerance, and autophagy in Arthrobotrys oligospora

“…In addition, the fermentation broths of the WT and mutant strains after 7 days of culture were used for LC–MS assays, as described previously (Xie et al, 2022a). The peak areas and diagnostic fragment ions at m/z 139, 393, and 429 were calculated for the arthrobotrisin content (Jiang et al, 2022).…”

The Arf-GAP Proteins AoGcs1 and AoGts1 Regulate Mycelial Development, Endocytosis, and Pathogenicity in Arthrobotrys oligospora

“…To assess the sporulation of each strain, 7 mm equal-sized fungal colonies from each strain were inoculated in the center of the CMY medium nad incubated at 28 °C for 14 days as previously described [ 45 ]. The number and morphology of conidia and conidiophores were observed by the side-shot method on the WA plate [ 46 , 47 ]. Conidial germination rates were determined as previously described [ 45 ].…”