Label-Free Quantitative Proteomics of Lysine Acetylome Identifies Substrates of Gcn5 in <named-content content-type="genus-species">Magnaporthe oryzae</named-content> Autophagy and Epigenetic Regulation

Abstract

ABSTRACT The rice blast fungus Magnaporthe oryzae poses a great threat to global food security. During its conidiation (asexual spore formation) and appressorium (infecting structure) formation, autophagy is induced, serving glycogen breakdown or programmed cell death function, both essential for M. oryzae pathogenicity. Recently, we identified an M. oryzae histone acetyltransferase (HAT) Gcn5 as a key regulator in phototropic induction of autophagy and asexual spore formation while serving a cellular function other than autophagy induction during M. oryzae infection. To further understand the regulatory mechanism of Gcn5 on M. oryzae pathogenicity, we set out to identify more Gcn5 substrates by comparative acetylome between the wild-type (WT) and GCN5 overexpression (OX) mutant and between OX mutant and GCN5 deletion (knockout [KO]) mutant. Our results showed that Gcn5 regulates autophagy induction and other important aspects of fungal pathogenicity, including energy metabolism, stress response, cell toxicity and death, likely via both epigenetic regulation (histone acetylation) and posttranslational modification (nonhistone protein acetylation). IMPORTANCE Gcn5 is a histone acetyltransferase that was previously shown to regulate phototropic and starvation-induced autophagy in the rice blast fungus Magnaporthe oryzae, likely via modification on autophagy protein Atg7. In this study, we identified more potential substrates of Gcn5-mediated acetylation by quantitative and comparative acetylome analyses. By epifluorescence microscopy and biochemistry experiments, we verified that Gcn5 may regulate autophagy induction at both the epigenetic and posttranslational levels and regulate autophagic degradation of a critical metabolic enzyme pyruvate kinase (Pk) likely via acetylation. Overall, our findings reveal comprehensive posttranslational modification executed by Gcn5, in response to various external stimuli, to synergistically promote cellular differentiation in a fungal pathogen.

Keywords

• comparative acetylome
• Gcn5
• histone acetyltransferase (HAT)
• Magnaporthe oryzae