AoMae1 Regulates Hyphal Fusion, Lipid Droplet Accumulation, Conidiation, and Trap Formation in Arthrobotrys oligospora

Abstract

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.

Keywords: hyphal fusion; lipid droplet; malate dehydrogenase; pathogenicity; trap formation.

Figure 1

Comparison of mycelial growth and hyphal fusion between the WT and ∆Aomae1 mutant strains. (A) Colony morphology of fungal strains incubated on different media for seven days at 28 °C. (B) Comparison of mycelial growth rate. (C) Observation of hyphal fusion in WA (water agar) plates. The red arrow indicates the hyphal fusion site, scale bar = 10 μm. (D) Comparison of the number of hyphal fusion sites under different medium conditions. The WT and mutant strains were observed using CFW staining for hyphal fusion after five days of incubation in PDA, WA, and MM media, and 30 random photographs were used to count the number of hyphal fusion sites. An asterisk indicates a significant difference between the ∆Aomae1 mutant and the WT strain (Tukey’s HSD, * p < 0.05).

Figure 2

Comparison of the cell length and nuclei between WT and mutant strains. (A) Observation of hyphal septa of the WT strain and ∆Aomae1 mutant cultured on PDA, WA, and WA (after nematode induction). White arrows indicate hyphal septa; scale bar = 5 μm. The mutant isolate ∆Aomae1-90 was used as a representative strain. (B) Comparison of cell length of WT and ∆Aomae1 mutant strains. The WT and mutant strains were incubated on PDA, WA, and WA-N media for five days, and 50 random photographs were used to determine the hyphal cell length. (C) Observation of cell nuclei of WT and ∆Aomae1 mutant, scale bar = 5 μm. White arrows indicate hyphal septa and red arrows indicate nuclei. (D) Comparison of the number of nuclei. The WT and mutant strains were incubated on a PDA medium for five days, and 50 random photographs were used to determine the number of nuclei. An asterisk (B,D) indicates a significant difference between the ∆Aomae1 mutant and the WT strain (Tukey’s HSD, * p < 0.05).

Figure 3

Observation of LD accumulation in the mycelia and conidia of the WT and ∆Aomae1 mutant strains. The mutant isolate ∆Aomae1-90 was used as a representative strain. (A) BODIPY staining under different culture conditions, scale bar = 5 μm. The WT and mutant strains were stained with BODIPY for observation of the LD morphology after five days of incubation in PDA, WA, and WA-N media. (B) LD staining for conidia, scale bar = 5 μm. The arrows in (A,B) indicate the LDs. (C) Observation of LDs via TEM; the WT and mutant strains were incubated on a PDA medium. LD, lipid droplet; V, vacuole.

Figure 4

Comparison of conidiation, MDH activity, and ATP content. (A) Conidial morphology observation using SEM. (B) Comparison of conidia yield. (C) Comparison of spore germination rate. (D) MDH activity assay. (E) ATP content assay. An asterisk (B–E) indicates a significant difference between the ∆Aomae1 mutant and the WT strain (Tukey’s HSD, * p < 0.05).

Figure 5

Comparison of trap formation and pathogenicity. (A) Representative images of trap formation and nematode predation at 24 and 36 h, scale bar = 100 μm. (B) Comparison of the trap morphology of each strain at 48 h, scale bar = 20 μm. (C) Comparison of the number of traps. (D) Comparison of mycelial loops. A total of 30 random traps were used to determine the mycelial loops. (E) Comparison of nematode mortality. An asterisk (C–E) indicates a significant difference between the ∆Aomae1 mutant and the WT strain (Tukey’s HSD, * p < 0.05). CK indicates the natural death rate of nematodes incubated on the WA medium.

Figure 6

Comparison of the metabolic profiling between WT and ∆Aomae1 mutant strains. (A) Comparison of the high-performance liquid chromatography profiles of the WT and ∆Aomae1 mutant strains. (B) Comparison of the chromatograms of arthrobotrisin anion peaks. The histogram shows that the arthrobotrisin content was comparable to the peak area of the WT and ∆Aomae1 mutant strains. (C) Volcano plot of differentially expressed metabolites between WT and ∆Aomae1 mutants. (D) The enriched KEGG pathways of differentially expressed metabolites.