Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata

Abstract

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy-mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP-AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H₂ O₂ ) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H₂ O₂ triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down-regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium-like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild-type toxin levels in axenic culture, the mutant induced a lower level of H₂ O₂ and smaller necrotic lesions on citrus leaves. In addition to H₂ O₂ , nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy-mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.

Keywords: Atg8; ROS detoxification; autophagy; peroxisome; pexophagy; stress tolerance; virulence.

FIGURE 1

Induction of autophagy by nitrogen starvation in Alternaria alternata. (a) Ttransmission electron microscopy images of wild‐type (WT) hyphae grown in potato dextrose broth (PDB), minimal medium (MM), or nitrogen‐depleted MM (MM−N) for 4 h, showing lipid bodies (L), peroxisomes (P), autophagic vacuoles (AV), and autophagosomes (AP). (b) WT expressing a GFP‐AaAtg8 fusion protein or GFP alone was grown in PDB, MM, or MM−N for 4 h and treated with 2 mM phenylmethylsulfonyl fluoride (PMSF). The vacuoles were stained with 7‐amino‐4‐chloromethylcoumarin (CMAC) and examined microscopically. Phagophore assembly sites (indicated by arrows) were observed in the hyphae grown in PDB. Scale bars, 10 μm. (c) Western blot analysis of proteins prepared from WT expressing GFP‐AaAtg8 with an anti‐GFP antibody. The percentage of free GFP band intensity in relation to total intensity is also shown. An SDS‐PAGE gel after Coomassie blue staining is shown to ensure equal loading of the samples.

FIGURE 2

AaAtg8 is required for autophagy and cell wall integrity. (a) Hyphae of the wild type (WT) and ∆AaAtg8 were cultured in nitrogen‐depleted minimal medium (MM−N) for 4 h in the presence of 2 mM phenylmethylsulfonyl fluoride (PMSF), stained with monodansylcadaverine (MDC, for detecting autophagosomes) or MM 4‐64 (for staining vacuoles), and examined microscopically. Scale bars, 25 μm. (b) Transmission electron microscopy images of WT and ∆AaAtg8 hyphae grown in MM−N and spores grown in potato dextrose broth, showing autophagic vacuoles (AV), vacuoles (V), and peroxisomes (indicated by arrows). (c) Quantitative analysis of autophagic vacuoles of hyphae and spores (n = 30). (d) Cell wall width (μm) of the hyphae and spores (n = 15). The significance of tests was analysed by one‐way analysis of variance and Tukey's HSD post hoc test (**p < 0.01; *p < 0.05).

FIGURE 3

AaAtg8 is associated with fungal growth and development. (a) Conidia (Cn) produced by two ∆AaAtg8 mutants (D1 and D2) were much smaller and slender than those produced by the wild type (WT) and the Cp17 strain. Conidia germinated to produce a germ tube (Gt), which formed a nonmelanized enlargement resembling an appressorium‐like structure at the end of the germ tube (indicated by an arrow). ∆AaAtg8 produced more hyphal fragments (Hf) than WT and Cp17. (b) Quantity of conidia produced by different strains of Alternaria alternata. (c) The size (μm²) of conidia. (d) Percentage of germinated conidia of different strains grown in potato dextrose broth (PDB) or liquid minimal medium (MM). (e) Percentage of the formation of appressorium‐like structures. The significance of the difference between means was analysed using Tukey's HSD post hoc test (p ≤ 0.01). Means (n = 30) indicated by the same case letters are not significantly different.

FIGURE 4

AaAtg8 contributes to virulence. (a) The formation of necrotic lesions on detached calamondin leaves inoculated with a piece of mycelium obtained from the wild type (WT), ∆AaAtg8 mutants (D1 and D2), or the Cp17 strain. Leaves treated with water were used as mock control. Similar results were obtained on citrus leaves inoculated with conidial suspensions (data not shown). Photographs were taken 5 to 6 days postinoculation (dpi). (b) Quantitative assays of the size of necrotic lesions. The significance of the difference between means was analysed using Tukey's HSD post hoc test (p ≤ 0.01). Means (n = 5) indicated by the same case letters are not significantly different. (c) H₂O₂ accumulation in detached calamondin leaves inoculated with conidial suspensions prepared from WT or D1, revealing the formation of dark‐brown insoluble precipitates surrounding the infection sites after reaction with 3,3′‐diaminobenzidine (DAB). (d) Microscopic lesions (brownish) were observed on the surface of citrus leaves inoculated with WT conidia at 1 dpi and coalesced to become large necrotic lesions at 2 dpi. Greyish lesions were observed on leaves inoculated with D1 conidia at 2 dpi. Scale bars, 50 μm.

FIGURE 5

Autophagy is involved in the resistance to reactive oxygen species. (a) Deletion of AaAtg8 increased sensitivity to 20 mM hydrogen peroxide (H₂O₂), 3.75 mM tert‐butyl‐hydroperoxide (tBHP), 3.4 mM cumyl hydroperoxide (cumyl‐H₂O₂, dissolved in ethanol), and 7 mM diethyl malonate (DEM, dissolved in methanol). Potato dextrose agar amended with ethanol (EtOH) or methanol (MeOH) was used as mock controls. The percentage of growth inhibition of ∆AaAtg8 in relation to the wild type (WT) is also shown. The significance of tests was analysed by one‐way analysis of variance and Tukey's HSD post hoc test (**p < 0.01). (b) Reverse transcription‐quantitative PCR analyses revealed that deletion of AaAtg8 decreased the gene expression of NoxA, encoding an NADPH oxidase, and Yap1, encoding a redox‐responsive transcription factor.

FIGURE 6

H₂O₂ induces the formation of autophagy, and autophagy is required for the detoxification of reactive oxygen species (ROS). (a) Fluorescence microscopy images of wild‐type (WT) hyphae expressing a green fluorescent protein (GFP)‐AaAtg8 fusion protein. Fungal hyphae cultured in potato dextrose broth (PDB) amended with or without 15 mM H₂O₂ were treated with 2 mM phenylmethylsulfonyl fluoride (PMSF) for 8 h. The vacuoles were stained with 7‐amino‐4‐chloromethylcoumarin (CMAC) and examined microscopically. H₂O₂ treatment induced the formation of condensed green spots colocalized with vacuoles (indicated by arrows). (b) Western blot analysis of proteins prepared from WT expressing GFP‐AaAtg8 treated with various concentrations of H₂O₂ for 8 h with an anti‐GFP antibody. The free GFP band intensity in relation to total intensity is also shown. A Ponceau S stain image is shown to ensure equal loading of the samples. (c) Accumulation of ROS in WT and ∆AaAtg8 hyphae after H₂O₂ treatment. Fungal hyphae grown in PDB with or without H₂O₂ for 8 h were stained with 2′‐7′‐dichlorofluorescein diacetate (DCFHDA) and examined microscopically, displaying green fluorescence (indicating ROS accumulation).

FIGURE 7

AaAtg8 is required for the turnover of peroxisomes under oxidative stress. (a) Fluorescence microscopy images of wild‐type (WT) and ∆AaAtg8 hyphae expressing an mCherry fluorescent protein tagging with conserved serine‐lysine‐leucine (SKL) tripeptides at the C‐terminus. Fungal hyphae were cultured in potato dextrose broth amended with or without 15 mM H₂O₂ for 8 h. Red fluorescence, indicative of peroxisomes, was colocalized in the vacuoles (indicated by arrows) in WT but not ∆AaAtg8 hyphae after H₂O₂ treatment. (b) Colocalization of peroxisomes in the vacuoles was also observed in WT hyphae after 24 h regardless of H₂O₂ treatment.

FIGURE 8

AaAtg8 is required for the turnover of peroxisomes under starvation conditions. (a) Identification of peroxisomes in ∆AaAtg8 spores expressing an mCherry fluorescent protein tagged with SKL tripeptides. No red fluorescence was observed in the wild‐type (WT) spores. Spores were collected from fungal strains grown in potato dextrose broth and examined microscopically. Colocalization of peroxisomes in the vacuoles (indicated by arrows) in WT, but not ∆AaAtg8, hyphae grown in (b) minimal medium (MM) or (c) nitrogen‐depleted MM (MM‐N) for 24 h.