Mitogen-activated protein kinase (MAPK) signaling is required for plant cell death responses to invading microbial pathogens. Iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death occurs in rice (Oryza sativa) during an incompatible rice–Magnaporthe oryzae interaction. Here, we show that rice MAP kinase (OsMEK2 and OsMPK1) signaling cascades are involved in iron- and ROS-dependent ferroptotic cell death responses of rice to M. oryzae infection using OsMEK2 knock-out mutant and OsMEK2 and OsMPK1 overexpression rice plants. The OsMPK1:GFP and OsWRKY90:GFP transcription factor were localized to the nuclei, suggesting that OsMPK1 in the cytoplasm moves into the nuclei to interact with the WRKY90. M. oryzae infection in ΔOsmek2 knock-out plants did not trigger iron and ROS accumulation and lipid peroxidation, and also downregulated OsMPK1, OsWRKY90, OsRbohB, and OsPR-1b expression. However, 35S:OsMEK2 overexpression induced ROS- and iron-dependent cell death in rice. The downstream MAP kinase (OsMPK1) overexpression induced ROS- and iron-dependent ferroptotic cell death response to virulent M. oryzae infection. The small-molecule ferroptosis inhibitor ferrostatin-1 suppressed iron- and ROS-dependent ferroptotic cell death in 35S:OsMPK1 overexpression plants. However, the small-molecule inducer erastin triggered iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death during M. oryzae infection. Disease (susceptibility)-related cell death was lipid ROS-dependent, but iron-independent in the ΔOsmek2 knock-out mutant during the late M. oryzae infection stage. These combined results suggest that OsMEK2 and OsMPK1 expression positively regulates iron- and ROS-dependent ferroptotic cell death, and blast disease (susceptibility)-related cell death was ROS-dependent but iron-independent in rice–M. oryzae interactions.

Ferritin is a ubiquitous iron storage protein that regulates iron homeostasis and oxidative stress in plants. Iron plays an important role in ferroptotic cell death response of rice (Oryza sativa) to Magnaporthe oryzae infection. Here, we report that rice ferritin 2, OsFER2, is required for iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death and defense response against the avirulent M. oryzae INA168. The full-length ferritin OsFER2 and its transit peptide were localized to the chloroplast, the most Fe-rich organelle for photosynthesis. This suggests that the transit peptide acts as a signal peptide for the rice ferritin OsFER2 to move into chloroplasts. OsFER2 expression is involved in rice resistance to M. oryzae infection. OsFER2 knock-out in wild-type rice HY did not induce ROS and ferric ion (Fe3+) accumulation, lipid peroxidation and hypersensitive response (HR) cell death, and also downregulated the defense-related genes OsPAL1, OsPR1-b, OsRbohB, OsNADP-ME2-3, OsMEK2 and OsMPK1, and vacuolar membrane transporter OsVIT2 expression. OsFER2 complementation in ΔOsfer2 knock-out mutants restored ROS and iron accumulation and HR cell death phenotypes during infection. The iron chelator deferoxamine, the lipid-ROS scavenger ferrostatin-1, the actin microfilament polymerization inhibitor cytochalasin E and the redox inhibitor diphenyleneiodonium suppressed ROS and iron accumulation and HR cell death in rice leaf sheaths. However, the small-molecule inducer erastin did not trigger iron-dependent ROS accumulation and HR cell death induction in ΔOsfer2 mutants. These combined results suggest that OsFER2 expression positively regulates iron- and ROS-dependent ferroptotic cell death and defense response in rice–M. oryzae interactions.

Nodule inception (NIN)-like proteins (NLPs) have a central role in nitrate signaling to mediate plant growth and development. Here, we report that OsNLP2 negatively regulates ferroptotic cell death and immune responses in rice during Magnaporthe oryzae infection. OsNLP2 was localized to the plant cell nucleus, suggesting that it acts as a transcription factor. OsNLP2 expression was involved in susceptible disease development. ΔOsnlp2 knockout mutants exhibited reactive oxygen species (ROS) and iron-dependent ferroptotic hypersensitive response (HR) cell death in response to M. oryzae. Treatments with the iron chelator deferoxamine, lipid-ROS scavenger ferrostatin-1, actin polymerization inhibitor cytochalasin A, and NADPH oxidase inhibitor diphenyleneiodonium suppressed the accumulation of ROS and ferric ions, lipid peroxidation, and HR cell death, which ultimately led to successful M. oryzae colonization in ΔOsnlp2 mutants. The loss-of-function of OsNLP2 triggered the expression of defense-related genes including OsPBZ1, OsPIP-3A, OsWRKY104, and OsRbohB in ΔOsnlp2 mutants. ΔOsnlp2 mutants exhibited broad-spectrum, nonspecific resistance to diverse M. oryzae strains. These combined results suggest that OsNLP2 acts as a negative regulator of ferroptotic HR cell death and defense responses in rice, and may be a valuable gene source for molecular breeding of rice with broad-spectrum resistance to blast disease.

Mitogen-activated protein kinase (MAPK) signaling is required for plant cell death responses to invading microbial pathogens. Ferric ions and reactive oxygen species (ROS) accumulate in rice (Oryza sativa) tissues undergoing cell death during Magnaporthe oryzae infection. Here, we report that rice MAP kinase (OsMEK2 and OsMPK1) signaling cascades are involved in iron- and ROS-dependent ferroptotic cell death responses of rice to M. oryzae infection. OsMEK2 interacted with OsMPK1 in the cytoplasm, and OsMPK1 moved from the cytoplasm into the nucleus to bind to the OsWRKY90 transcription factor. OsMEK2 expression may trigger OsMPK1-OsWRKY90 signaling pathways in the nucleus. Avirulent M. oryzae infection in ΔOsmek2 mutant rice did not trigger iron and ROS accumulation and lipid peroxidation, and also downregulated OsMPK1, OsWRKY90, OsRbohB, and OsPR-1b expression. However, OsMEK2 overexpression induced ROS-and iron-dependent cell death in rice during M. oryzae infection. The downstream MAP kinase (OsMPK1) overexpression induced ROS- and iron-dependent ferroptotic cell death in the compatible rice-M. oryzae interaction. These data suggest that the OsMEK2-OsMPK1-OsWRKY90 signaling cascade is involved in the ferroptotic cell death in rice. The small-molecule inducer erastin triggered iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death in ΔOsmek2 mutant plants during M. oryzae infection. Disease-related cell death was lipid ROS-dependent and iron-independent in the ΔOsmek2 mutant plants. These combined results suggest that OsMEK2 and OsMPK1 expression positively regulates iron- and ROS-dependent ferroptotic cell death via OsMEK2-OsMPK1-OsWRKY90 signaling pathways, and blast disease (susceptibility)-related cell death was ROS-dependent but iron-independent in rice-M. oryzae interactions.