Transcriptional Regulation of Autophagy-Related Genes by Sin3 Negatively Modulates Autophagy in Magnaporthe oryzae

Abstract

Autophagy is a conserved degradation and recycling pathway in eukaryotes and is important for their normal growth and development. An appropriate status of autophagy is crucial for organisms which is tightly regulated both temporally and continuously. Transcriptional regulation of autophagy-related genes (ATGs) is an important layer in autophagy regulation. However, the transcriptional regulators and their mechanisms are still unclear, especially in fungal pathogens. Here, we identified Sin3, a component of the histone deacetylase complex, as a transcriptional repressor of ATGs and negative regulator of autophagy induction in the rice fungal pathogen Magnaporthe oryzae. A loss of SIN3 resulted in upregulated expression of ATGs and promoted autophagy with an increased number of autophagosomes under normal growth conditions. Furthermore, we found that Sin3 negatively regulated the transcription of ATG1, ATG13, and ATG17 through direct occupancy and changed levels of histone acetylation. Under nutrient-deficient conditions, the transcription of SIN3 was downregulated, and the reduced occupancy of Sin3 from those ATGs resulted in histone hyperacetylation and activated their transcription and in turn promoted autophagy. Thus, our study uncovers a new mechanism of Sin3 in modulating autophagy through transcriptional regulation. IMPORTANCE Autophagy is an evolutionarily conserved metabolic process and is required for the growth and pathogenicity of phytopathogenic fungi. The transcriptional regulators and precise mechanisms of regulating autophagy, as well as whether the induction or repression of ATGs is associated with autophagy level, are still poorly understood in M. oryzae. In this study, we revealed that Sin3 acts as a transcriptional repressor of ATGs to negatively regulate autophagy level in M. oryzae. Under the nutrient-rich conditions, Sin3 inhibits autophagy with a basal level through directly repressing the transcription of ATG1-ATG13-ATG17. Upon nutrient-deficient treatment, the transcriptional level of SIN3 would decrease and dissociation of Sin3 from those ATGs associates with histone hyperacetylation and activates their transcriptional expression and in turn contributes to autophagy induction. Our findings are important as we uncover a new mechanism of Sin3 for the first time to negatively modulate autophagy at the transcriptional level in M. oryzae.

Keywords: ATG8; fungal pathogen; histone deacetylation; rice blast; transcriptional regulation.

FIG 1

Deletion of SIN3 increases the transcriptional expression of autophagy-related genes (ATGs) in Magnaporthe oryzae. (A) Relative expression of ATGs in the Δsin3 strain compared with those of the WT strain when cultured in the liquid nutrient-rich medium (CM). Values are the average from three biological replicates, and the asterisks indicate the significant difference between the WT and Δsin3 strains (*, P < 0.05; **, P < 0.01). (B) Relative expression of ATG1, ATG13, ATG17, and ATG8 in the WT and Δsin3 strains cultured under the CM and nutrient-deficient medium (SD-N) conditions. Strains were cultured in the liquid CM for 2 days and then transferred to SD-N for 4 h. Values are the means ± SD from three biological replicates. Different letters (a, b, and c) indicate the significant differences as determined by one-way ANOVA (P < 0.05).

FIG 2

Loss of SIN3 promotes autophagy in Magnaporthe oryzae. (A) The GFP-Atg8 localization in the GFP-ATG8 and Δsin3/GFP-ATG8 strains under liquid nutrient-rich medium (CM) and nutrient-deficient medium (SD-N) conditions. Strains were cultured in the CM for 2 days and then transferred to SD-N for 4 h. Mycelia were stained with CMAC, and images were captured by fluorescence microscopy. Bars, 20 μm. (B) The autophagosome numbers of the GFP-ATG8 and Δsin3/GFP-ATG8 strains in the CM and SD-N medium. At least 25 hyphal segments were used to calculate the autophagosomes. Values are means ± SD from three replicates. The asterisks indicate the significant difference between the WT and Δsin3 strains (**, P < 0.01). (C) Immunoblot analysis of the GFP-ATG8 and Δsin3/GFP-ATG8 strains under CM and SD-N conditions. The immunoblot of GAPDH was presented as the internal loading control. The degradation rates were calculated with the following formula: GFP/(GFP + GFP-Atg8). Two biological replicates were performed with similar results.

FIG 3

Sin3 negatively regulates the transcription of autophagy-related genes in Magnaporthe oryzae. (A) Schematic representation of genomic regions of ATG1, ATG13, and ATG17 genes that were examined. (B) Transient luciferase (LUC) assays showing that the overexpression of SIN3 negatively regulates transcription of ATG1, ATG13, and ATG17. Representative images of N. benthamiana leaves are shown to indicate the relative LUC activity between different combinations at the top. Values are means ± SD from three replicates, and the asterisks indicate the significant difference (**, P < 0.01). (C) EMSA indicated the binding of Sin3 to the loci of ATG1, ATG13, and ATG17. The unlabeled probes for the competition assay were added as 20-fold excess. (D) ChIP-qPCR analysis showing the relative enrichment of Sin3-FLAG in the F1 locus of ATG1, ATG13, and ATG17 in the WT and SIN3-FLAG strains. Mycelia cultured in the liquid CM were collected for ChIP experiments with the FLAG antibody. The relative fold of enrichment in the SIN3-FLAG strain over that of the WT strain with two independent replicates is shown. Values are means ± SD from three technical replicates. The asterisks indicate the significant difference between the WT and SIN3-FLAG strains (**, P < 0.01). (E) ChIP-qPCR analysis showing the relative enrichment of H4K16ac in the F1 locus of ATG1, ATG13, and ATG17 in the WT and Δsin3 strains. Mycelia cultured in the liquid CM were collected for ChIP experiments with the H4K16ac antibody. The relative enrichment in the Δsin3 strain over that in the WT strain with two independent replicates is shown. Values are means ± SD from three technical replicates.

FIG 4

Transcriptional expression and protein accumulation of Sin3 were decreased during autophagy induction. (A) The expression level of SIN3 in the indicated treatment of the WT strain. Strains were cultured in the CM for 2 days and transferred to the SD-N medium for an additional 2 and 4 h. Values are means ± SD from three biological replicates. Different letters (a or b) indicate significant differences tested by one-way ANOVA (P < 0.01). (B and C) Relative abundance of Sin3-GFP detected by immunoblot analysis in the indicated treatment of the Δsin3-C strain. The main band (top) of Sin3-GFP is estimated to be 190 kD (molecular weight for protein). The asterisk indicates the unspecific band. Values are means ± SD from three biological replicates. Different letters (a or b) indicate significant differences tested by one-way ANOVA (P < 0.01). (D) The localization and relative abundance of Sin3-GFP in mycelia with indicated culture conditions. Images were photographed by a confocal microscope. The relative intensities of Sin3-GFP and H2B-mCherry were analyzed by ImageJ software. Bar, 5 μm. (E) ChIP-qPCR analysis showing H4K16ac enrichment on the F1 region of ATG1, ATG13, and ATG17. Mycelia cultured in the liquid CM and SD-N medium were collected for ChIP experiments with the anti-H4K16ac antibody. The relative enrichment of H4K16ac in the WT strain from the SD-N medium over that from the CM medium with two independent replicates is shown. Values are means ± SD from three biological replicates. The asterisks indicate the significant difference of the enrichment of H4K16ac between the CM and SD-N growth conditions (*, P < 0.05; **, P < 0.01).

FIG 5

Constitutive expression of ATG1, ATG13, and ATG17 promotes autophagy in Magnaporthe oryzae. (A) The GFP-Atg8 localization in the GFP-ATG8, ATG1-OE/GFP-ATG8, ATG13-OE/GFP-ATG8, and ATG17-OE/GFP-ATG8 strains in CM and SD-N medium. Strains were cultured in CM for 2 days and then transferred to SD-N medium for 4 h. Mycelia were stained with CMAC and then photographed with a confocal microscope. BF, bright field. Bars, 5 μm. (B) The autophagosome numbers in the GFP-ATG8, ATG1-OE/GFP-ATG8, ATG13-OE/GFP-ATG8, and ATG17-OE/GFP-ATG8 strains under CM and SD-N conditions. At least 25 hyphal segments were used to calculate the number of autophagosomes. Values are means ± SD from three technical replicates. The asterisks indicate the significant difference between the WT and ATG-OE strains (*, P < 0.05). (C) Immunoblot analysis of GFP in the GFP-ATG8, ATG1-OE/GFP-ATG8, ATG13-OE/GFP-ATG8, and ATG17-OE/GFP-ATG8 strains under the CM and SD-N conditions. The immunoblot of GAPDH was presented as the internal loading control. The degradation rates were calculated with the following formula: GFP/(GFP + GFP-Atg8). Three biological replicates were performed with similar results.

FIG 6

Sin3 is required for the fungal growth, conidiation, and appressoria formation in Magnaporthe oryzae. (A and B) Radical growth and statistical analysis of the indicated strains grown in the CM for 7 days. Both the top and bottom of colonies were imaged. Values are the means ± SD from three biological replicates. Different letters (a or b) indicate significant differences tested by one-way ANOVA (P < 0.05). (C) The conidiophore morphology of the indicated strains. Strains were cultured in CM for 7 days to observe the conidiophores. Bar, 200 μm. (D) Statistical analysis of conidiation of the indicated strains in the CM. Values are means ± SD from three biological replicates. Different letters (a or b) indicate the significant differences as determined by one-way ANOVA (P < 0.05). (E) The appressoria formation of the indicated strains. Strains were cultured in CM for 7 days, and then the conidia were collected to induce appressoria on the hydrophobic surface for 16 h. Bar, 20 μm. (F) The appressoria formation rates of the indicated strains. Values are means ± SD from three biological replicates. Different letters (a or b) indicate the significant differences determined by one-way ANOVA (P < 0.05).

FIG 7

Glycogen degradation and lipid droplet translocation were delayed in the Δsin3 strain during appressoria formation. (A and B) Observation and quantification of conidia containing glycogen at 0, 4, 8, and 24 hpi. Conidial suspensions were inoculated on the hydrophobic surfaces and stained with I₂/KI solution. Bars, 10 μm. (C and D) Observation and quantification of conidia containing lipid droplets at 0, 4, 8, and 24 hpi. Conidial suspensions were inoculated on the hydrophobic surfaces and stained with boron-dipyrromethene (BODIPY) solution. Bars, 10 μm.

FIG 8

Sin3 is required for the virulence of Magnaporthe oryzae. (A) BLAST infection assays on the barley leaves inoculated with mycelial plugs from the indicated strains for 4 days. Representative inoculated leaves were shown. (B) Rice seedling assays were used to evaluate pathogenicity. The conidial suspensions (5 × 10⁴ conidia/mL) from the indicated strains and 21-day-old rice seedlings were used for spraying inoculation. Representative inoculated leaves were shown. (C) Observation and statistical analysis of invasive hyphae growth in rice sheath cells at 40 hours postinoculation (hpi). Four types of invasive hyphae (illustrated at the right with the corresponding column) were quantified as no penetration, penetration with primary hyphae, penetration with secondary invasive hyphae in the first invaded cell, and invasive hyphae spreading into neighboring cells. Data represent the means ± SD from three independent repeats, with over 100 appressoria per analysis. Bars, 10 μm.

FIG 9

A model of Sin3 in the transcriptional regulation of ATG genes under the nutrient-rich and -deficient conditions. Under the nutrient-rich conditions, Sin3 inhibits autophagy with a basal level through directly repressing the transcription of ATG1-ATG13-ATG17 genes. Upon nutrient-deficient treatment, the transcriptional level of SIN3 decreases and the dissociation of Sin3 from those ATG genes associates with histone hyperacetylation and activates their transcriptional expression and in turn contributes to autophagy induction.