MoSec13 combined with MoGcn5b modulates MoAtg8 acetylation and regulates autophagy in Magnaporthe oryzae

Abstract

Macroautophagy/autophagy is an evolutionarily conserved cellular degradation process that is crucial for cellular homeostasis in Magnaporthe oryzae. However, the precise regulatory mechanisms governing autophagy in this organism remain unclear. In this study, we found a multiregional localization of MoSec13 to the vesicle membrane, endoplasmic reticulum, nucleus, and perinucleus. MoSec13 negatively regulated autophagy through specific amino acid residues in its own WD40 structural domain by interacting with MoAtg7 and MoAtg8. We also found that the histone acetyltransferase MoGcn5b mediated the acetylation of MoAtg8 and regulated autophagy activity. Subsequently, we further determined that MoSec13 regulated the acetylation status of MoAtg8 by controlling the interaction between MoGcn5b and MoAtg8 in the nucleus. In addition, MoSec13 maintained lipid homeostasis by controlling TORC2 activity. This multilayered integration establishes MoSec13 as an essential node within the autophagic regulatory network. Our findings fill a critical gap in understanding the role of Sec13 in autophagy of filamentous fungi and provide a molecular foundation for developing new therapeutic strategies against rice blast fungus.ABBREVIATIONS BFA: brefeldin A; BiFC: bimolecular fluorescence complementation; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; Co-IP: co-immunoprecipitation; COPII: coat complex II; GFP: green fluorescent protein; HPH: hygromycin phosphotransferase; MM-N: nitrogen-starvation conditions; NPC: nuclear pore complex; PAS: phagophore assembly site; PE: phosphatidylethanolamine; UPR: unfolded protein response.

Keywords: Autophagy; Gcn5b; Magnaporthe oryzae; Sec13; TORC2.

Figure 1.

MoSec13 interacts with MoAtg7 and MoAtg8 to negatively regulate autophagy. (A) Observation of the subcellular localization of MoSec13-GFP during the conidial stage using confocal microscopy (Fv3000, 60×oil). FM 4–64 was utilized to label vesicular membranes; scale bar: 5 μm. ImageJ software was employed for the analysis of colocalization between MoSec13-GFP and FM 4–64 staining, and GraphPad prism software was utilized for generating linear peak plots. (B) Colocalization analysis of MoSec13-GFP and MoLhs1-DsRed during the conidial stage was conducted using confocal microscopy (zeiss LSM780, 63×oil). MoLhs1-DsRed served as a marker for the ER; scale bar: 5 μm. The colocalization analysis of MoSec13-GFP and MoLhs1-DsRed was performed using ImageJ software, and GraphPad prism software was utilized for generating linear peak plots. (C) Colocalization analysis of MoSec13-GFP and MoH₂B-mCherry during the conidial stage was conducted using confocal microscopy (Fv3000, 60×oil). MoH₂B-mCherry served as a marker for the nucleus; scale bar: 5 μm. The colocalization analysis of MoSec13-GFP and MoH₂B-mCherry was performed using ImageJ software, and GraphPad prism software was utilized for generating linear peak plots. (D) Co-IP assays were conducted to identify interactions between MoSec13 and MoAtg7, as well as MoSec13 and MoAtg8. Protein supernatants derived from co-expressing strains, including GFP and Flag-MoAtg7, GFP and Flag-MoAtg8, MoSec13-GFP and Flag-MoAtg7, and MoSec13-GFP and Flag-MoAtg8, were individually incubated with GFP agarose beads for a duration of 4 h. Subsequently, the eluates were subjected to immunoprecipitation using anti-GFP and anti-flag antibodies for detailed analysis. Note that in panel D the paired analyses were run on different gels. Thus, blots 1 and 3 were run on the same gel, whereas blots 2 and 4 were run together on a single different gel; accordingly, the positions of the molecular mass markers differ between gels. (E) Affinity-isolation assays were conducted to identify interactions between MoSec13 and MoAtg7, as well as MoSec13 and MoAtg8. Purified GST, GST-MoAtg7, and GST-MoAtg8 proteins were mixed with purified His-MoSec13 and incubated with GST beads for 4 h. The eluates were then examined through immunoprecipitation using anti-GST and anti-His antibodies for further analysis. As explained for panel D, two different sets of gels were used for the paired analyses; hence, the molecular mass markers, in particular for 27 kDa, are at different positions between the two sets of gels. (F) Fluorescence localization maps of GFP-MoAtg8 were generated for wild-type Guy11 and the ∆Mosec13 mutant hyphae in both liquid CM and MM-N medium. Vacuole staining was achieved using 7-amino-4-chloromethylcoumarin (CMAC); scale bar: 10 μm. (G) The degradation of the fusion protein GFP-MoAtg8 was observed in both wild-type Guy11 and the ∆Mosec13 mutant mycelium under conditions of liquid CM and MM-N medium. Immunoprecipitation analysis was conducted using anti-GFP and anti-ACTB antibodies. (H) The level of macroautophagy was quantified using the formula GFP:(GFP+GFP-MoAtg8). Data from three replicate experiments were analyzed using a t-test analysis, P*** < 0.001, P** < 0.01. (I) The lipidation levels of MoAtg8 were assessed in both wild-type Guy11 and the ∆Mosec13 mutant under conditions of liquid CM and MM-N medium. (J) The lipidation assessment of MoAtg8 was determined by the ratio of MoAtg8–PE:ACTB. Data from three replicate experiments were analyzed using a t-test analysis, P** < 0.01.

Figure 2.

MoSec13 interacts with MoAtg7 and MoAtg8 through specific amino acid residues within the WD40 domain. (A) The AlphaFold2-model-predicted interaction sites between MoSec13 and MoAtg7, where MoSec13 is represented in green, MoAtg7 in blue, and the interaction sites between the two proteins in red. (B) The AlphaFold2-model-predicted interaction sites between MoSec13 and MoAtg8, with MoSec13 in green, MoAtg8 in blue, and the interaction sites between the two proteins in red. (C) The specific interaction sites predicted by AlphaFold2 for MoSec13 binding to MoAtg7 are located at Met1, Ser19, Asp198, and Arg222 of MoSec13. Asp198 and Arg222 are located at a WD structural domain of MoSec13. (D) The specific interaction sites predicted by AlphaFold2 for MoSec13 binding to MoAtg8 are located at Ser111, Gly134, Asn138, and Gln220 of MoSec13; all of these residues are located at a WD40 structural domain of MoSec13. (E) Affinity-isolation assays were conducted to identify interactions between MoSec13[4A–1] and MoAtg7. MoSec13[4A–1] was derived from the mutation of the four sites Met1, Ser19, Asp198, and Arg222 to alanine. (F) Co-IP assays were conducted to identify interactions between MoSec13[4A–1] and MoAtg7. (G) Affinity-isolation assays were conducted to identify interactions between MoSec13[4A–2] and MoAtg8. MoSec13[4A–2] was derived from the mutation of the four sites Ser111, Gly134, Asn138, and Gln220 to alanine. (H) Co-IP assays were conducted to identify interactions between MoSec13[4A–2] and MoAtg8.

Figure 3.

MoSec13 regulates growth, pathogenicity and autophagic activity through specific amino acid residues within the WD40 domain. (A,B) Growth of the wild-type strain Guy11, the ∆Mosec13 mutant, the complemented strain ∆Mosec13-C and (A) ∆Mosec13-C[4A–1] or (B) ∆Mosec13-C[4A–2] on CM medium for 4 days. (C,D) Pathogenicity evaluation of the wild-type strain Guy11, the ∆Mosec13 mutant, the complemented strain ∆Mosec13-C and (C) ∆Mosec13-C[4A–1] or (D) ∆Mosec13-C[4A–2] on barley leaves caused by mycelial plugs. (E,F) The degradation of the fusion protein GFP-MoAtg8 was observed in both wild-type Guy11, the complemented strain ∆Mosec13-C and (E) ∆Mosec13-C[4A–1] or (F) ∆Mosec13-C[4A–2] mycelium under conditions of liquid CM and MM-N medium. Immunoprecipitation analysis was conducted using anti-GFP and anti-ACTB antibodies. The level of macroautophagy was quantified using the formula GFP:(GFP+GFP-MoAtg8). Data from three replicate experiments were analyzed using a t-test analysis, P**** < 0.0001, P*** < 0.001, P** < 0.01, ns p > 0.05. (G,H) The lipidation levels of MoAtg8 were assessed in both wild-type Guy11, the complemented strain ∆Mosec13-C and (G) ∆Mosec13-C[4A–1] or (H) ∆Mosec13-C[4A–2] under conditions of liquid CM and MM-N medium. The lipidation assessment of MoAtg8 was determined by the ratio of MoAtg8–PE:ACTB. Data from three replicate experiments were analyzed using a t-test analysis, P** < 0.01, P* < 0.05, ns p > 0.05.

Figure 4.

MoGcn5b-mediated acetylation of MoAtg8 regulates autophagy. (A) Colocalization analysis of GFP-MoGcn5b and MoH₂B-mCherry during the conidial stage was conducted using confocal microscopy (Fv3000, 60×oil). MoH₂B-mCherry served as a marker for the nucleus; scale bar: 5 μm. The colocalization analysis of GFP-MoGcn5b and MoH₂B-mCherry was performed using ImageJ software, and GraphPad prism software was utilized for generating linear peak plots. (B) MoAtg8 was acetylated by MoGcn5b in vitro. The GFP-MoGcn5b protein, purified from M. oryzae, was separately incubated with GST-MoAtg7 and GST-MoAtg8 proteins, both purified from E. coli. Following the incubations, the reaction solutions were subjected to analysis through immunoprecipitation using anti-AcK, anti-GFP, and anti-GST antibodies. (C) A yeast two-hybrid assay was conducted to identify the interaction between MoGcn5b and MoAtg8. Plasmids MoGcn5b-AD and MoAtg8-BD, MoGcn5b-AD and pGBKT7, and MoAtg8-BD and pGADT7 were assessed for growth in SD-Leu-Trp and SD-Leu-Trp-Ade-His media. Plasmids pGBKT7–53 and pGADT7-T were used as positive controls. (D) A co-IP assay was conducted to examine the interaction between MoGcn5b and MoAtg8. Protein supernatants derived from co-expressed strains, including GFP and mCherry-MoAtg8, and GFP-MoGcn5b and mCherry-MoAtg8, were individually incubated with GFP agarose beads for a duration of 4 h. Subsequently, the eluates were subjected to immunoprecipitation using anti-GFP and anti-mCherry antibodies for detailed analysis. (E) The acetylation level of MoAtg8 in wild-type Guy11 and ∆Mogcn5b mutant was examined in CM liquid medium and MM-N liquid medium. The acetylation level was assessed by the formula AcK:GFP-MoAtg8. (F) Data from three replicate experiments were analyzed using a t-test analysis, P*<0.05. (G) Colocalization of GFP-MoAtg8 and MoH₂B-mCherry under nutrient-rich and nitrogen-deficient conditions using confocal microscopy (Fv3000, 60×oil). Scale bar: 5 μm. (H) The degradation of the fusion protein GFP-MoAtg8 was observed in both wild-type Guy11 and the ∆Mogcn5b mutant mycelium under conditions of liquid CM and MM-N medium. Immunoprecipitation analysis was conducted using anti-GFP and anti-ACTB antibodies. (I) The level of autophagy was quantified using the formula GFP:(GFP+GFP-MoAtg8). Data from three replicate experiments were analyzed using a t-test analysis, P***<0.001, P*<0.05, ns p > 0.05. (J) The lipidation levels of MoAtg8 were assessed in both wild-type Guy11 and the ∆Mogcn5b mutant under conditions of liquid CM and MM-N medium. (K) The lipidation assessment of MoAtg8 was determined by the ratio of MoAtg8–PE:ACTB. Data from three replicate experiments were analyzed using a t-test analysis, P**<0.01, P*<0.05.

Figure 5.

MoSec13 coordinates the acetylation of MoAtg8 by modulating the interaction between MoAtg8 and MoGcn5b. (A) A yeast two-hybrid assay was conducted to identify the interaction between MoSec13 and MoGcn5b. Plasmids MoGcn5b-AD and MoSec13-BD, MoGcn5b-AD and pGBKT7, and MoSec13-BD and pGADT7 were assessed for growth in SD-Leu-Trp and SD-Leu-Trp-Ade-His media. Plasmids pGBKT7–53 and pGADT7-T were used as positive controls. (B) An affinity-isolation assay was conducted to identify the interaction between MoSec13 and MoGcn5b. Purified proteins GST and GST-MoGcn5b were mixed with purified protein His-MoSec13 and incubated with GST beads for 4 h. The eluates were then examined through immunoprecipitation using anti-GST and anti-His antibodies for further analysis. (C) A co-IP assay was conducted to identify the interaction between MoSec13 and MoGcn5b. Protein supernatants derived from co-expressed strains, including GFP and Flag-MoGcn5b, and MoSec13-GFP and Flag-MoGcn5b, were individually incubated with GFP agarose beads for a duration of 4 h. Subsequently, the eluates were subjected to immunoprecipitation using anti-GFP and anti-flag antibodies for detailed analysis. (D) Fluorescence microscopy was performed to observe YFP fluorescence of MoGcn5b-YFPC and YFPN-MoSec13, MoGcn5b-YFPC and YFPN, YFPC and YFPN-MoSec13, and colocalization of MoGcn5b-MoSec13 with H₂B-mCherry during the mycelial period. Colocalization analysis was performed using ImageJ software, and GraphPad prism software was utilized for generating linear peak plots for panels D-F. Scale bar: 10 μm. (E) Confocal microscopy (Fv3000) was performed to observe YFP fluorescence of YFPC-MoAtg8 and MoSec13-YFPN, YFPC-MoAtg8 and YFPN, and YFPC and MoSec13-YFPN, and colocalization of MoAtg8-MoSec13 with H₂B-mCherry during the mycelial period. Scale bar: 5 μm. (F) Confocal microscopy (Fv3000) was performed to observe the colocalization of MoAtg8-MoSec13 with mCherry-MoApe1 and mCherry-MoAtg17 during the mycelial period. Scale bar: 5 μm. (G) The acetylation level of MoAtg8 in wild-type Guy11 and ∆Mosec13 mutant strains was examined in CM liquid medium and MM-N liquid medium. (H) The acetylation level was assessed by the formula AcK:GFP-MoAtg8. Data from three replicate experiments were analyzed using a t-test analysis, P** < 0.01, P* < 0.05. (I) Detection of the strength of MoGcn5b-MoAtg8 interactions in wild-type Guy11 and ∆Mosec13 mutant cells. Wild-type and ∆Mosec13 mutant strains with GFP-MoGcn5b and mCherry-MoAtg8 double tags were incubated in CM liquid medium for 36–48 h. The extracted total protein supernatant was incubated with GFP agarose gel beads for 4 h and the eluate obtained was analyzed by immunoprecipitation with anti-GFP antibody and anti-mCherry antibody. (J) Interaction strength was assessed with the formula mCherry-MoAtg8:GFP-MoGcn5b. Data from three replicate experiments were analyzed using a t-test analysis, P** < 0.01.

Figure 6.

MoSec13 regulates reticulophagy and mitophagy. (A) Affinity-isolation assay to detect MoSec13 and MoSec31 protein interaction. Purified GST and GST-MoSec31 proteins were mixed with purified His-MoSec13 and incubated with GST beads for 4 h. The eluates were then examined through immunoprecipitation using anti-GST and anti-His antibodies for further analysis. (B) A co-IP assay was used to detect MoSec13 and MoSec31 protein interaction. Protein supernatants derived from co-expressed strains, including GFP and MoSec31-flag, and MoSec13-GFP and MoSec31-flag, were individually incubated with GFP agarose beads for a duration of 4 h. Subsequently, the eluates were subjected to immunoprecipitation using anti-GFP and anti-flag antibodies for a detailed analysis. (C) PCR was employed to identify the splicing state of the UPR transcription factor MoHac1 in both wild-type Guy11 and the ∆Mosec13 mutant strains. ACTB was utilized as an internal reference gene. The unspliced state of MoHac1 is denoted as uMoHAC1, while the spliced state is represented as sMoHAC1. (D) The expression levels of UPR pathway-related target genes in the ∆Mosec13 mutant were assessed using qRT-PCR. Data analysis was conducted using a t-test, P**** < 0.0001, P*** < 0.001, P** < 0.01. (E) The expression levels of target genes associated with the UPR pathway were examined via qRT-PCR in the wild-type strain upon the addition of BFA. Subsequent data analysis was conducted using the t-test, P**** < 0.0001, P*** < 0.001. (F) The degradation of the fusion protein GFP-MoSec62 was assessed in both wild-type Guy11 and the ∆Mosec13 mutant strains under conditions of liquid CM and added DTT. (G) Reticulophagy levels were determined using the formula GFP:(GFP+GFP-MoSec62). Data from three replicate experiments were analyzed using a t-test analysis, P*** < 0.001, P** < 0.01. (H) The degradation of the Porin protein was examined in both wild-type Guy11 and the ∆Mosec13 mutant strains under conditions of liquid CM and MM-N medium. (I) Mitophagy levels were quantified by determining the ratio of Porin to the internal reference ACTB. Data from three replicate experiments were analyzed using a t-test analysis, P** < 0.01, P* < 0.05.

Figure 7.

TORC2 activity is compromised in the ∆Mosec13 strain. (A) The phosphorylation level of MoYpk1 was assessed in the wild-type strain Guy11 and the ∆Mosec13 mutant under both CM and myriocin-treatment conditions. (B) Data from three replicate experiments were analyzed using a t-test analysis, P*** < 0.001, P* < 0.05. (C) The colony morphology of the wild-type strain Guy11, the ∆Mosec13 mutant, and the complemented strain ∆Mosec13-C after 8 days of cultivation in CM medium supplemented with 0.5 M NaCl, 0.5 M KCl, and 1 M sorbitol. (D) Growth diameter analysis of the wild-type strain Guy11, the ∆Mosec13 mutant, and the complemented strain ∆Mosec13-C in medium supplemented with NaCl, KCl, and sorbitol. The data underwent a t-test analysis using GraphPad Prism software, P**** < 0.0001. (E-H) The phosphorylation levels of (E) Hog1 (detected with anti-MAPK/p38) and (G) MoYpk1 were assessed in the wild-type strain Guy11 and the ∆Mosec13 mutant under both CM and 0.5 M NaCl-treatment conditions. Data from three replicate experiments were analyzed using a t-test analysis, P**** < 0.0001, P*** < 0.001, P** < 0.01, P* < 0.05, ns p > 0.05.

Figure 8.

A model of MoSec13 regulation in M. oryzae. MoSec13, as a component shared by COPII and NPC complexes, not only negatively regulates autophagy by modulating the interactions of specific amino acid residues in its own WD40 structural domain with MoAtg7 and MoAtg8, but also regulates MoAtg8 acetylation status in the nucleus by binding to MoGcn5b. Moreover, MoSec13 can control TORC2 activity to regulate lipid homeostasis.