Involvement of AoMdr1 in the Regulation of the Fluconazole Resistance, Mycelial Fusion, Conidiation, and Trap Formation of Arthrobotrys oligospora

Abstract

Multidrug resistance (Mdr) proteins are critical proteins for maintenance of drug resistance in fungi. Mdr1 has been extensively studied in Candida albicans; its role in other fungi is largely unknown. In this study, we identified a homologous protein of Mdr (AoMdr1) in the nematode-trapping (NT) fungus Arthrobotrys oligospora. It was found that the deletion of Aomdr1 resulted in a significant reduction in the number of hyphal septa and nuclei as well as increased sensitivity to fluconazole and resistance to hyperosmotic stress and SDS. The deletion of Aomdr1 also led to a remarkable increase in the numbers of traps and mycelial loops in the traps. Notably, AoMdr1 was able to regulate mycelial fusion under low-nutrient conditions, but not under nutrient-rich conditions. AoMdr1 was also involved in secondary metabolism, and its deletion caused an increase in arthrobotrisins (specific compounds produced by NT fungi). These results suggest that AoMdr1 plays a crucial role in the fluconazole resistance, mycelial fusion, conidiation, trap formation, and secondary metabolism of A. oligospora. Our study contributes to the understanding of the critical role of Mdr proteins in mycelial growth and the development of NT fungi.

Keywords: Arthrobotrys oligospora; conidiation; fluconazole resistance; multidrug resistance protein; trap formation.

Figure 1

Comparisons of mycelial growth, septa, and nuclei of the WT and ∆Aomdr1 mutants. (A) Colony morphologies of fungal strains cultured at 28 °C for 5 days. (B) Comparisons of mycelial growth rates. (C) Observations of mycelial septa on PDA plates. White arrows indicate mycelial septa; scale bar: 2 μm. (D) Comparison of mycelial cell lengths. (E) Mycelial cell nuclei on PDA plates; scale bar: 2 μm. White arrows indicate mycelial septa and red arrows indicate nuclei. (F) Comparison of the number of nuclei. Asterisks indicate significant differences between the ∆Aomdr1 mutant and WT strains (Tukey HSD, * p < 0.05).

Figure 2

Comparisons of LD accumulation and mycelial fusion in WT and ∆Aomdr1 mutant strains. (A) LD accumulation in mycelial cells was observed with BODIPY staining (upper pane) and TEM images (lower pane). L indicates LDs; scale bar: 5 µm. (B) BODIPY staining of LDs in conidia; scale bar: 10 µm. (C) Observation of hyphal fusion in WA plates. The red arrows indicate the hyphal fusion sites; scale bar: 5 µm. (D) Comparison of the number of hyphal fusion sites under different media. The WT and mutant strains were observed using CFW staining for hyphal fusion after 5 days of incubation in PDA, WA, MM, and WA + N media, and 30 random photographs were used to count the number of hyphal fusion sites. The representative images in (A–C) were chosen from ∆Aomdr1-1 and ∆Aomdr1-2 mutants. Asterisks indicate significant differences between the ∆Aomdr1 mutant and WT strains (Tukey HSD; *** p < 0.001).

Figure 3

Comparisons of stress responses for fluconazole resistance, osmotic pressure, and SDS treatment. (A) Growth of WT and mutant strains under different concentrations of fluconazole. (B) RGI values of WT and mutant strains under fluconazole treatment. (C) Comparisons of mycelial growth after treatment with 0.2 M of NaCl, 0.5 M of sorbitol, and 0.03% SDS. (D) RGI values of WT and mutant strains under chemical stresses. Asterisks indicate significant differences between the ∆Aomdr1 mutant and WT strains (Tukey HSD; * p < 0.05).

Figure 4

Comparisons of sporulation and spore morphology. (A) Observation of conidiophores of ∆Aomdr1 mutant and WT strains; scale bar: 50 µm. (B) CFW staining of spores of the ∆Aomdr1 mutant and WT strains; scale bar: 10 µm. The black arrows indicate the conidia. (C) SEM observations of spore morphologies of ∆Aomdr1 mutant and WT strains. The representative images in (B,C) were chosen from ∆Aomdr1-1 and ∆Aomdr1-2 mutants. (D) Comparison of the spore yield between the mutant and WT strains. (E) Percentages of normal and abnormal spores. A total of 100 random spores were used to calculate the ratio of normal and abnormal spores. Asterisks indicate significant differences between the ∆Aomdr1 mutant and WT strains (Tukey HSD; * p < 0.05).

Figure 5

Comparisons of trap formation, morphology, and nematode predation efficiency. (A) Trap formation at different time points; scale bar: 50 μm. (B) Numbers of traps at different times of nematode induction. (C) Nematode mortality at different times. Asterisks indicate significant differences between the ∆Aomdr1 mutant and WT strains (Tukey HSD; * p < 0.05).

Figure 6

Comparisons of metabolic profiles of the WT and ∆Aomdr1 mutant strains. (A) Comparison of high-performance liquid chromatography profiles of the WT and ∆Aomdr1 mutants. (B) Analysis of the number of differentially up- and downregulated compounds. (C) KEGG enrichment analysis of differential compounds. (D) Comparison of the content of arthrobotrisins. Asterisks indicate significant differences between the ∆Aomdr1 mutant and WT strains (Tukey HSD; * p < 0.0 5).