The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea

Abstract

Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8, a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (ΔBcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the ΔBcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinereaIMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea.

Keywords: BcATG8; Botrytis cinerea; autophagy; vegetative differentiation; virulence.

FIG 1

Evolutionary analysis of BcATG8. (A) Complementation of yeast ATG8 null mutant with BcATG8. In contrast to the wild-type (WT) yeast, the ATG8 mutant (ΔAtg8) and the mutant transformed with the empty pYES2 vector (pYES2) died after nitrogen starvation, while the mutants transformed with pYES2-BcATG8 (Y-1, Y-4, and Y-7) survived similarly to the WT. (B) Yeast two-hybrid analyses for interaction between BcAtg8 and BcAtg4. Serial dilutions of yeast cells transformed with bait and prey constructs indicated in the figure were examined on SD-Leu-Trp-His plates. The plasmid pair pGADT7 and pGBKT7-53 served as a positive control, and pGADT7 and pGBKT7-Lam served as a negative control.

FIG 2

Subcellular localization of GFP-BcAtg8. The GFP-BcAtg8 fused construct was introduced into the wild-type strain to examine the localization of BcAtg8 in fungal cells in various developmental stages. (A) GFP-BcAtg8 presented as punctate structures in conidia. (B) GFP-BcAtg8 was diffused in the cytoplasm and concentrated in the nuclei of germlings. The nuclei were stained by DAPI (4′,6-diamidino-2-phenylindole) and examined by fluorescence microscopy. DIC, differential interference contrast; CM, complete medium; MM-N, minimal medium without (NH₄)₂SO₄. Scale bars, 10 μm.

FIG 3

Analysis of autophagy process using GFP-BcAtg8 marker in B. cinerea. (A) GFP-BcAtg8 localized in the cytoplasm as preautophagosomal structures (PAS) under nutrient-rich conditions; with induction by starvation or rapamycin, the autophagy process was activated and GFP-BcAtg8 transferred to vacuoles. The vacuoles were stained with CMAC (7-amino-4-chloromethylcoumarin) and examined by fluorescence microscopy. DIC, differential interference contrast; CM, complete medium; MM-N, minimal medium without (NH₄)₂SO₄. Scale bars, 10 μm. (B) GFP-BcAtg8 proteolysis assays of B05.10 and the ΔBcAtg1 mutant. Mycelia were cultured at 25°C for 48 h in CM liquid medium, and autophagy was induced after 4 or 6 h of nitrogen starvation. Mycelia were collected at the indicated times, and mycelial extracts were analyzed by anti-GFP antibody Western blotting. GAPDH was used as an internal reference.

FIG 4

BcATG8 is required for autophagy. (A) Transmission electron microscopy observation of mycelia of the wild-type strain B05.10, the BcATG8 deletion mutant (ΔBcAtg8), and the complemented strain ΔBcAtg8-C cultured in nitrogen-limiting medium (MM-N) with 2 mM PMSF for 6 h. Arrows indicate autophagic bodies. V, vacuole. Scale bar, 0.5 μm. (B) The starved mycelia of each strain were stained with the fluorescent dye MDC (monodansylcadaverine) and observed under a fluorescence microscope. There was no fluorescence observed for all strains grown in CM (complete medium) or for the ΔBcAtg8 strain grown in MM-N. Scale bar, 10 μm.

FIG 5

Impacts of BcATG8 deletion on mycelial growth. (A) Colonies of the wild-type strain B05.10, the BcATG8 deletion mutant (ΔBcAtg8), and the complemented strain ΔBcAtg8-C after growth on various media at 25°C for 4 days. PDA, potato dextrose agar; CM, complete medium; MM, minimal medium; MM-N, MM without (NH₄)₂SO₄. (B) Statistical analysis of the colony diameters of the indicated strains. Error bars denote standard errors of the results from three repeated experiments, and asterisks indicate statistically significant differences (P < 0.01).

FIG 6

Involvement of BcATG8 in modulating conidiation, germination, and sclerotial formation. (A) The morphology of conidiation among the wild-type strain B05.10, the BcATG8 deletion mutant (ΔBcAtg8), and the complemented strain ΔBcAtg8-C grown on sterilized potato fragments at 25°C with a 12-h photophase for 10 days. (B) The conidia produced by B05.10, the ΔBcAtg8 strain, and ΔBcAtg8-C were washed from each fragment and counted under the microscope. (C) Conidial germination rates of B05.10, the ΔBcAtg8 strain, and ΔBcAtg8-C on a hydrophobic surface in CM or MM-N after 20 h of incubation at 25°C. CM, complete medium; MM-N, minimal medium without (NH₄)₂SO₄. Error bars denote standard errors of the results from three repeated experiments, and asterisks indicate statistically significant differences (P < 0.01); the error bars and asterisks apply to both panels B and C. (D) Comparison of sclerotial formation among B05.10, the ΔBcAtg8 strain, and ΔBcAtg8-C after 4 weeks of incubation on PDA at 4°C in the dark. PDA, potato dextrose agar. The top-right inset view in each panel is the back of the petri dish.

FIG 7

Infection tests on different host plant tissues. The plant tissue samples were incubated with the wild-type strain B05.10, the BcATG8 deletion mutant (ΔBcAtg8), and the complemented strain ΔBcAtg8-C. Agar plugs without fungal mycelia were used as negative controls (CK). (A to D) Disease symptoms on wounded cucumber leaves 60 h postinoculation (h.p.i.) (A), wounded tomato leaves 60 h.p.i. (B), wounded apple fruits 72 h.p.i. (C), and wounded grape fruits 72 h.p.i (D). (E) Diameters of disease lesions caused by each strain on different plant tissues. Error bars denote standard errors of the results from five repeated experiments, and asterisks indicate statistically significant differences (P < 0.01).

FIG 8

Involvement of BcATG8 in the regulation of lipid metabolism. (A) Lipid droplets in conidia were stained with Nile red and examined under a microscope with episcopic fluorescence. Scale bars, 20 μm. (B) Relative transcription levels of lipid droplet biosynthesis-related genes in the wild-type strain B05.10 and the BcATG8 deletion mutant (ΔBcAtg8). Genes tested are as follows. Lipid droplet biosynthesis-related genes: A, BC1G_13009; B, BC1G_11851; and C, BC1G_10262. Degradation-related genes: D, BC1G_07580; E, BC1G_03357; F, BC1G_12236; G, BC1G_09602; and H, BC1G_07986. Error bars denote standard errors from three repeated experiments, and asterisks indicate statistically significant differences (P < 0.01).