AoPEX1 and AoPEX6 Are Required for Mycelial Growth, Conidiation, Stress Response, Fatty Acid Utilization, and Trap Formation in Arthrobotrys oligospora

Abstract

Arthrobotrys oligospora (A. oligospora) is a typical nematode-trapping (NT) fungus that can capture nematodes by producing adhesive networks. Peroxisomes are single membrane-bound organelles that perform multiple physiological functions in filamentous fungi. Peroxisome biogenesis proteins are encoded by PEX genes, and the functions of PEX genes in A. oligospora and other NT fungi remain largely unknown. Here, our results demonstrated that two PEX genes (AoPEX1 and AoPEX6) are essential for mycelial growth, conidiation, fatty acid utilization, stress tolerance, and pathogenicity in A. oligospora. AoPEX1 and AoPEX6 knockout resulted in a failure to produce traps, conidia, peroxisomes, and Woronin bodies and damaged cell walls, reduced autophagosome levels, and increased lipid droplet size. Transcriptome data analysis showed that AoPEX1 and AoPEX6 deletion resulted in the upregulation of the proteasome, membranes, ribosomes, DNA replication, and cell cycle functions, and the downregulation of MAPK signaling and nitrogen metabolism. In summary, our results provide novel insights into the functions of PEX genes in the growth, development, and pathogenicity of A. oligospora and contribute to the elucidation of the regulatory mechanism of peroxisomes in trap formation and lifestyle switching in NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are important resources for the biological control of plant-parasitic nematodes. They are widely distributed in various ecological environments and capture nematodes by producing unique predatory organs (traps). However, the molecular mechanisms of trap formation and lifestyle switching in NT fungi are still unclear. Here, we provided experimental evidence that the AoPEX1 and AoPEX6 genes could regulate mycelial growth and development, trap formation, and nematode predation of A. oligospora. We further analyzed the global transcription level changes of wild-type and mutant strains using RNA-seq. This study highlights the important role of peroxisome biogenesis genes in vegetative growth, conidiation, trap formation, and pathogenicity, which contribute to probing the mechanism of organelle development and trap formation of NT fungi and lays a foundation for developing high-efficiency nematode biocontrol agents.

Keywords: Arthrobotrys oligospora; conidiation; fatty acid utilization; peroxisome biogenesis-genes; transcriptome analysis; trap formation.

FIG 1

Comparison of hyphal growth of wild-type (WT) and mutant strains in A. oligospora. (A) Colonial shapes of fungal strains cultured on PDA, TG, and TYGA plates at 28°C. The strains were photographed after 6 days of incubation. (B) The quantification data for (A). (C) Aerial hyphae of the WT and mutant strains cultured on TYGA medium for 5 d. (D) Hyphae of the WT and each mutant strain were stained with CFW and 4′,6-diamidino-2-phenylindole (DAPI) after the fungal strains were grown for 7 days on CMY medium. One hundred hyphal cells were randomly selected for counting cell nuclei. Bar: 3 μm. Measurements represent the average of three independent experiments. The asterisk indicates a significant difference between the mutant and the WT strains (***P < 0.001).

FIG 2

Comparison of sporulation, trap formation, and nematocidal activity in the wild-type (WT) and mutant strains. (A) Conidiophore differentiation in the WT and mutant lines. (B) Spore yields of the WT and mutant lines. (C) Trap formation in the WT and mutant lines induced by nematodes at different time points. Bar: 100 μm. (D) The percentage of nematode mortality by the WT and mutant lines at different time points. CK is the negative control using WA plate to assess the viability of the C. elegans in the absence of fungal strains. The asterisk (B and D) indicates a significant difference between the mutant and the WT strains (***P < 0.001).

FIG 3

Comparison of stress tolerance to oxidative agents between wild-type (WT) and mutant strains of A. oligospora. (A) Colony morphologies of the WT and mutant strains incubated on TG medium supplemented with menadione or H₂O₂. (B) Relative growth inhibition (RGI) values of the WT and mutants after incubation on TG medium supplemented with 0.02–0.04 mM menadione for 6 days. (C) RGI values for the WT and mutant strains after incubation on TG medium supplemented with 2.5–10 mM H₂O₂ for 6 days. The asterisk (B and C) indicates a significant difference between the mutant and the WT strains (*P < 0.05, **P < 0.01, ***P < 0.001).

FIG 4

Comparison of fatty acid utilization and hyphae morphology between wild-type (WT) and mutant strains of A. oligospora. (A) The LDs of WT, ΔAopex1, and ΔAopex6 strains were stained with BODIPY. Bar: 3 μm. (B) RGI values of fungal strains under different fatty acids as the only carbon source. The asterisk indicates a significant difference between the mutant and the WT strains (**P < 0.01, ***P < 0.001). (C) The CFW staining of WT and mutant strains. Bar: 3 μm.

FIG 5

Observation of autophagosomes and ultrastructure of peroxisomes and Woronin bodies in the wild-type (WT) and mutant strains. (A) The autophagosomes of WT strain, ΔAopex1, and ΔAopex6 mutant strains were stained with MDC. Bar: 3 μm. (B) The WT and mutant strains were observed by transmission electron microscopy. WB: Woronin body; P: peroxisome; S: septum.

FIG 6

Transcription analysis in wild-type (WT), ΔAopex1, and ΔAopex6 mutant strains. (A) DEGs on the 3rd and 5th days. Red, upregulated DEGs; green, downregulated DEGs. (B) Venn analysis of WT, ΔAopex1, and ΔAopex6 mutant strains at two time points. (C) KEGG enrichment analysis of DEGs in WT and ΔAopex1 strains. (D) KEGG enrichment analysis of DEGs in WT and ΔAopex6 strains. DEGs, differentially expressed genes.

FIG 7

Differentially expressed genes (DEGs) associated with peroxisome and lipid metabolism. (A) Heat map showing the DEGs involved in peroxisomes. (B) Heat map showing the DEGs involved in lipid metabolism. Gene expression patterns are in log₁₀-scale. Red boxes, upregulated clusters; blue boxes, downregulated clusters.

FIG 8

Schematic illustration of the regulation of AoPEX1 and AoPEX6 in A. oligospora. In A. oligospora, AoPEX1 and AoPEX6 play critical roles in fatty acid metabolism, nutrient metabolism, nucleus formation, autophagy, and oxidative stress processes, thereby regulating hyphal growth, conidiation, and trap formation. CW, cell wall.