Hypersensitive response (HR) cell death is the most effective plant immune response restricting fungal pathogen invasion. Here, we report that incompatible rice (Oryza sativa) Magnaporthe oryzae interactions induce iron-and reactive oxygen species (ROS)-dependent ferroptotic cell death in rice cells. Ferric ions and ROS (i.e., H 2 O 2 ) accumulated in tissues undergoing HR cell death of rice leaf sheath tissues during avirulent M. oryzae infection. By contrast, iron did not accumulate in rice cells during virulent M. oryzae infection or treatment with the fungal elicitor chitin. Avirulent M. oryzae infection in DOsnadp-me2-3 mutant rice did not trigger iron and ROS accumulation and suppressed HR cell death, suggesting that NADPmalic enzyme2 is required for ferroptotic cell death in rice. The small-molecule ferroptosis inhibitors deferoxamine, ferrostatin-1, and cytochalasin E and the NADPH oxidase inhibitor diphenyleneiodonium suppressed iron-dependent ROS accumulation and lipid peroxidation to completely attenuate HR cell death in rice sheaths during avirulent M. oryzae infection. By contrast, the small-molecule inducer erastin triggered iron-dependent ROS accumulation and glutathione depletion, which ultimately led to HR cell death in rice in response to virulent M. oryzae. These combined results demonstrate that iron-and ROS-dependent signaling cascades are involved in the ferroptotic cell death pathway in rice to disrupt M. oryzae infection.

Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant im-mune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVRPii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

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.

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.