MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

Abstract

Autophagy is essential for appressorium-mediated plant infection by Magnaporthe oryzae, the causal agent of rice blast disease and a major threat to global food security. The regulatory mechanism of pathogenicity-associated autophagy, however, remains largely unknown. Here, we report the identification and functional characterization of a plausible ortholog of yeast SNT2 in M. oryzae, which we term MoSNT2. Deletion mutants of MoSNT2 are compromised in autophagy homeostasis and display severe defects in autophagy-dependent fungal cell death and pathogenicity. These mutants are also impaired in infection structure development, conidiation, oxidative stress tolerance and cell wall integrity. MoSnt2 recognizes histone H3 acetylation through its PHD1 domain and thereby recruits the histone deacetylase complex, resulting in deacetylation of H3. MoSnt2 binds to promoters of autophagy genes MoATG6, 15, 16, and 22 to regulate their expression. In addition, MoTor controls MoSNT2 expression to regulate MoTor signaling which leads to autophagy and rice infection. Our study provides evidence of a direct link between MoSnt2 and MoTor signaling and defines a novel epigenetic mechanism by which MoSNT2 regulates infection-associated autophagy and plant infection by the rice blast fungus.

Abbreviations: M. oryzae: Magnaporthe oryzae; S. cerevisiae: Saccharomyces cerevisiae; F. oxysporum: Fusarium oxysporum; U. maydis: Ustilago maydis; Compl.: complemented strains of ΔMosnt2 expressing MoSNT2-GFP; ATG: autophagy-related; HDAC: histone deacetylase complex; Tor: target of rapamycin kinase; MTOR: mechanistic target of rapamycin kinase in mammals; MoSnt2: DNA binding SaNT domain protein in M. oryzae; MoTor: target of rapamycin kinase in M. oryzae; MoAtg8: autophagy-related protein 8 in M. oryzae; MoHos2: hda one similar protein in M. oryzae; MoeIf4G: eukaryotic translation initiation factor 4 G in M. oryzae; MoRs2: ribosomal protein S2 in M. oryzae; MoRs3: ribosomal protein S3 in M. oryzae; MoIcl1: isocitrate lyase in M. oryzae; MoSet1: histone H3K4 methyltransferase in M. oryzae; Asd4: ascus development 4; Abl1: AMP-activated protein kinase β subunit-like protein; Tig1: TBL1-like gene required for invasive growth; Rpd3: reduced potassium dependency; KAT8: lysine (K) acetyltransferase 8; PHD: plant homeodomain; ELM2: Egl-27 and MTA1 homology 2; GFP: green fluorescent protein; YFP: yellow fluorescent protein; YFP^CTF: C-terminal fragment of YFP; YFP^NTF: N-terminal fragment of YFP; GST: glutathione S-transferase; bp: base pairs; DEGs: differentially expressed genes; CM: complete medium; MM-N: minimum medium minus nitrogen; CFW: calcofluor white; CR: congo red; DAPI: 4', 6-diamidino-2-phenylindole; BiFC: bimolecular fluorescence complementation; RT: reverse transcription; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction; RNAi: RNA interference; ChIP: chromatin immunoprecipitation.

Keywords: Autophagy; Magnaporthe oryzae; MoSnt2; MoTor signaling; pathogenicity.

Figure 1.

MoSNT2 is critical for growth and reproduction of M. oryzae. (A) Hyphal growth of plate colonies on CM agar medium. (B) Quantified diameters of colonies. Error bars represent standard deviations. Asterisk indicates significant difference (** P < 0.01). (C) Microscopy observation of conidiphore development. Scale bar: 50 μm. (D) Quantified conidial production. (E) Microscopic observation of conidial morphology. Scale bar: 20 μm. (F) Fertility assay. The standard testing strain TH3 was crossed with each indicated strain on oatmeal medium in an inductive condition. Scale bar: 200 μm.

Figure 2.

MoSNT2 plays critical roles in autophagy of M. oryzae. (A) Epifluorescence micrographs of autophagosomes. Transformants expressing the GFP-MoATG8 fusion gene were grown in CM liquid medium for 48 h, then transferred into MM-N for the indicated time. Mycelium was stained with 10 μg/ml CFW before photographing. Scale bar: 20 μm. (B) Autophagosome number within hyphae. The mean autophagosome number was calculated from at least 25 hyphal segments, each of which was defined as a hyphal region separated by 2 neighboring CFW-stained septa. (C) Fluorescence intensity of GFP-MoAtg8. The mean value of GFP fluorescence intensity was calculated from at least 25 hyphal segments with a length of 50 μm. (D) Immunoblot analysis of GFP-MoAtg8 proteolysis. (E) Quantified intensity of GFP:GFP-MoAtg8 ratios. The GFP-MoAtg8 band in the Guy11 strain was defined as reference with an intensity of 1.0.

Figure 3.

MoSNT2 is essential for plant infection by M. oryzae. (A) Rice leaf segments infected with fungal mycelium. (B) Measurement of fungal biomass in infected leaves based on qPCR analysis of the MoPOT2 repetitive element. CK, rice leaf segments inoculated with agar plugs without fungal mycelium. (C) Rice root infection assay. Arrows indicate typical necrotic lesions. (D) qPCR analysis of fungal biomass in inoculated rice roots. (E) Appressorium development on hydrophobic coverslip. Scale bar: 10 μm.

Figure 4.

MoSNT2 is crucial for infection structure development and autophagic cell death. (A) Infection structure development on onion epidermis at 36 hpi. The black arrowhead and arrow indicates conidium and appressorium respectively, while the white arrowhead indicates invasive hypha. (B) Percentage of appressorium and penetration peg formation on onion epidermis (n > 50, triple replications, ** P < 0.01). (C) Percentage of spores containing 3 totally-collapsed conidial cells on onion epidermis (n > 50, triple replications). (D) Infection structure development on rice leaf sheath. Scale bar: 10 μm.

Figure 5.

MoSNT2 regulates cell wall integrity and oxidative stress response. (A) Growth of M. oryzae on CM agar medium containing 200 μg/ml CFW or 200 μg/ml CR for 5 days. (B) CFW staining and epifluorescence microscopy of cell wall chitin of mycelium grown in liquid CM. Scale bar: 20 μm. (C) Increased hyphal melanization as a consequence of MoSNT2 deletion. (D) qRT-PCR analysis on the expression levels of melanin biosynthesis genes in mycelium grown in liquid CM. (E) Mycelial growth on CM agar medium in the presence of different concentrations of H₂O₂. (F) Statistical analysis of the inhibition rate under H₂O₂-induced oxidative stress on mycelial growth. Asterisks represent significant differences (** P < 0.01).

Figure 6.

MoSnt2 mediates H3 deacetylation and regulates expression of autophagy genes. (A) Visualization of the interaction between proteins as shown in the BiFC assay. Vegetative hyphae were stained with DAPI and then analyzed by epifluorescence microscopy. Scale bar: 10 μm. (B) GST-PHD1 coimmunoprecipitates H3 histones. E. coli-expressed fusion proteins were used for affinity isolation of histones of calf thymus and immunoblot analysis conducted with the antibodies indicated. The star indicates GST-PHD1 and GST-PHD2, while arrowhead indicates GST. (C) Histone deacetylase activity in affinity isolation complexes. (D) Immunoblot analysis of histone proteins in M. oryzae with the indicated primary antibodies. (E) qRT-PCR analysis on the expression levels of autophagy genes. (F) In vitro affinity isolation of autophagy gene DNA by MoSnt2. GST-MoSnt2-F1, GST-MoSnt2-F2 or GST alone were incubated with sheared chromatin, affinity isolated, washed and subjected to qPCR for autophagy genes. Similar results were obtained from 3 independent biological experiments.

Figure 7.

MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR. (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

Figure 8.

Effects of rapamycin on vegetative growth, autophagic cell death and pathogenicity of M. oryzae. (A) Plate colonies of the Guy11 strain in CM agar medium. The Guy11 strain was grown on CM medium supplemented with rapamycin (rapa.) at the indicated concentration. Solvent DMSO was seperately added into medium as a control. (B) Diameters of plate colonies recorded every 2 days. (C) Autophagic conidial cell death of the Guy11 strain at 24 hpi of appressorium development on hydrophobic coverslip in the presence of rapamycin. Scale bar: 10 μm. (D) Percentage of Guy11 strain spores containing 3 totally-collapsed conidial cells at 24 hpi (n > 100, triple replications, ** P < 0.01). (E) Glycogen distribution during appressorium development on hydrophobic surface. Glycogen was stained by iodine solution and microscopically visualized as yellowish-brown deposits. (F) Percentage of spores containing glycogen content in conidial cells (n > 100, triple replications, * P < 0.05, ** P < 0.01). (G) Lipid body translocation and degradation during appressorium morphogenesis as revealed by Bodipy staining. (H) Blast lesions on rice leaf segments infected with Guy11 strain. (I) Mean width and length of lesions calculated from at least 15 independent blast lesions. (J) qPCR analysis of fungal biomass in the infected rice leaf segments.

Figure 9.

Model for MoSnt2-mediated epigenetic control of pathogenicity in M. oryzae. MoTor promotes the expression of MoSNT2 through unidentified effector(s) or transcription factor(s). Nucleus-localized MoSnt2 recognizes acetylated histone H3 and recruits the histone HDAC deacetylase complex to targeted chromatin regions. The MoSnt2-recruited HDAC then deacetylates H3 and alters expression of genes. MoSnt2-regulated gene expression functions to repress autophagy to promote hyphal proliferation during vegetative growth in nutrient rich conditions, while promoting autophagic conidial cell death and assisting pathogenic growth on host rice.