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 (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca²⁺. High Ca²⁺ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca²⁺ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca²⁺, ROS, and Fe²⁺ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca²⁺ influx, and H₂O₂ accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca²⁺ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca²⁺ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection.

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.

The rice-Magnaporthe oryzae pathosystem is presented as a host-pathogen ferroptosis model. Multi-omics insights connect ferroptosis to several cellular processes, such as iron homeostasis, reactive oxygen species (ROS)/redox imbalance, and polyunsaturated fatty acid-containing phospholipid (PUFA-PL) peroxidation, under multiple regulators in rice and M. oryzae, respectively. Interestingly, ferroptosis drives two opposite biological outcomes: fungal infection-related development and rice immunity.