Phosphatidylethanolamines link ferroptosis and autophagy during appressorium formation of rice blast fungus.

A cell death pathway, ferroptosis, occurs in conidial cells and is critical for formation and function of the infection structure, the appressorium, in the rice blast fungus Magnaporthe oryzae. In this study, we identified an orthologous lysophosphatidic acid acyltransferase (Lpaat) acting at upstream of phosphatidylethanolamines (PEs) biosynthesis and which is required for such fungal ferroptosis and pathogenicity. Two PE species, DOPE and SLPE, that depend on Lpaat function for production were sufficient for induction of lipid peroxidation and the consequent ferroptosis, thus positively regulating fungal pathogenicity. On the other hand, both DOPE and SLPE positively regulated autophagy. Loss of the LPAAT gene led to a decrease in the lipidated form of the autophagy protein Atg8, which is probably responsible for the autophagy defect of the lpaatΔ mutant. GFP-Lpaat was mostly localized on the membrane of lipid droplets (LDs) that were stained by the fluorescent dye monodansylpentane (MDH), suggesting that LDs serve as a source of lipids for membrane PE biosynthesis and probably as a membrane source of autophagosome. Overall, our results reveal novel intracellular membrane-bound organelle dynamics based on Lpaat-mediated lipid metabolism, providing a temporal and spatial link of ferroptosis and autophagy.

The Histone Deacetylases MoRpd3 and MoHst4 Regulate Growth, Conidiation, and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae.

As the causal agent of the blast disease, Magnaporthe oryzae is one of the most destructive fungal pathogens of rice. Histone acetylation/deacetylation is important for remodeling of chromatin superstructure and thus altering gene expression. In this study, two genes encoding histone deacetylases, namely, MoRPD3 and MoHST4, were identified and functionally characterized in M. oryzae. MoHst4 was required for proper mycelial growth and pathogenicity, whereas overproduction of MoRpd3 led to loss of pathogenicity, likely due to a block in conidial cell death and restricted invasive growth within the host plants. Green fluorescent protein (GFP)-MoRpd3 localized to the nucleus and cytoplasm in vegetative hyphae and developing conidia. By comparative transcriptomics analysis, we identified potential target genes epigenetically regulated by histone deacetylases (HDACs) containing MoRpd3 or MoHst4, which may contribute to conidia formation and/or conidial cell death, which is a prerequisite for successful appressorium-mediated host invasion. Taken together, our results suggest that histone deacetylases MoRpd3 and MoHst4 differentially regulate mycelial growth, asexual development, and pathogenesis in M. oryzae. IMPORTANCE HDACs (histone deacetylases) regulate various aspects of growth, development, and pathogenesis in plant-pathogenic fungi. Most members of HDAC classes I to III have been functionally characterized, except for orthologous Rpd3 and Hst4, in the rice blast fungus Magnaporthe oryzae. In this study, we assessed the function of MoRpd3 and MoHst4 by reverse genetics and found that they differentially regulate M. oryzae vegetative growth, asexual development, and infection. Particularly, MoRpd3 negatively regulates M. oryzae pathogenicity, likely through suppression of conidial cell death, which we recently reported as being critical for appressorium maturation and functioning. Overall, this study broadens our understanding of fungal pathobiology and its critical regulation by histone modification(s) during cell death and in planta differentiation.

Karyopherin MoKap119-mediated nuclear import of cyclin-dependent kinase regulator MoCks1 is essential for Magnaporthe oryzae pathogenicity.

Nuclear import of proteins relies on nuclear import receptors called importins/karyopherins (Kaps), whose functions were reported in yeasts, fungi, plants, and animal cells, including cell cycle control, morphogenesis, stress sensing/response, and also fungal pathogenecity. However, limited is known about the physiological function and regulatory mechanism of protein import in the rice-blast fungus Magnaporthe oryzae. Here, we identified an ortholog of ß-importin in M. oryzae encoded by an ortholog of KAP119 gene. Functional characterisation of this gene via reverse genetics revealed that it is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The mokap119Δ mutant was also defective in formation of appressorium-like structure from hyphal tips. By affinity assay and liquid chromatography-tandem mass spectrometry, we identified potential MoKap119-interacting proteins and further verified that MoKap119 interacts with the cyclin-dependent kinase subunit MoCks1 and mediates its nuclear import. Transcriptional profiling indicated that MoKap119 may regulate transcription of infection-related genes via MoCks1 regulation of MoSom1. Overall, our findings provide a novel insight into the regulatory mechanism of M. oryzae pathogenesis likely by MoKap119-mediated nuclear import of the cyclin-dependent kinase subunit MoCks1.

Label-Free Quantitative Proteomics of Lysine Acetylome Identifies Substrates of Gcn5 in Magnaporthe oryzae Autophagy and Epigenetic Regulation.

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.

Phototrophy and starvation-based induction of autophagy upon removal of Gcn5-catalyzed acetylation of Atg7 in Magnaporthe oryzae.

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.