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.

Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca²⁺ acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca²⁺ influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca²⁺ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). During Magnaporthe oryzae infection, 14 different Ca²⁺ influx regulators altered Ca²⁺, ROS and Fe²⁺ accumulation, glutathione reductase (GR) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca²⁺ levels inhibited the reduction of glutathione isulphide (GSSG) to GSH in vitro. Ca²⁺ chelation by ethylene glycol-bis (2-aminoethylether)-N, N, N’, N’-tetra-acetic acid (EGTA) suppressed apoplastic Ca²⁺ influx in rice leaf sheaths during infection. Blocking apoplastic Ca²⁺ influx into the cytoplasm by Ca²⁺ chelation effectively suppressed Ca²⁺-mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca²⁺ influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca²⁺ influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reduced GR expression levels in rice immune responses.

The reactive oxygen species (ROS) burst is the most common plant immunity mechanism to prevent pathogen infection, although the exact role of ROS in plant immunity has not been fully elucidated. We investigated the expression and translocation of Oryza sativa respiratory burst oxidase homologue B (OsRBOHB) during compatible and incompatible interactions between rice epidermal cells and the pathogenic fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We characterized the functional role of ROS focal accumulation around invading hyphae during P. oryzae infection process using the OsRBOHB inhibitor diphenyleneiodonium (DPI) and the actin filament polymerization inhibitor cytochalasin (Cyt) A. OsRBOHB was strongly induced during incompatible rice–P. oryzae interactions, and newly synthesized OsRBOHB was focally distributed at infection sites. High concentrations of ROS focally accumulated at the infection sites and suppressed effector biotrophy-associated secreted (BAS) proteins BAS4 expression and invasive hyphal growth. DPI and Cyt A abolished ROS focal accumulation and restored P. oryzae effector BAS4 expression. These results suggest that ROS focal accumulation is able to function as an effective immune mechanism that blocks some effectors including BAS4-expression during P. oryzae infection. Disruption of ROS focal accumulation around invading hyphae enables successful P. oryzae colonization of rice cells and disease development.

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.

The oriental fruit moth, Grapholita molesta, is one of the most destructive orchard pests worldwide. Due to its behavior of boring into twigs and fruits, controlling the pest at the larval stage using insecticides is challenging. We studied the enemy species at the larval stage of G. molesta from seven fruit-growing areas by sampling peach shoots and fruits bored by its larvae. The occurrence and biological characteristics of the dominant enemy were verified using field investigation and laboratory experiments. The results showed that the dominant enemy species of larval G. molesta was identified as Therophilus festivus. The larval, cocoon, and pupal stages of T. festivus could not survive at 35 °C. The developmental durations of these three stages at 20 °C were significantly longer than those at 25 °C and 28 °C. Consecutive years of investigation in Xinxiang, China, indicated that T. festivus can parasitize G. molesta larvae collected at each generation. The parasitism rates in June (45.16%), July (63.50%), and August (48.37%) were significantly higher than those in April (15.27%). Most field research in China has focused on the release of parasitoids to control G. molesta eggs. Our study establishes a foundation for the control of G. molesta larvae using T. festivus.