Rice phospholipase C4 enhances Ca2+ and ROS bursts during effector-triggered immunity to induce ferroptotic cell death

Phospholipases are major regulators of lipid-dependent signaling and play crucial roles in plant immunity. Rice (Oryza sativa) phospholipase C4 (OsPLC4) is a major functional enzyme in the rice phospholipase family that regulates intracellular Ca²⁺ levels. Here we show that OsPLC4 translocates primarily to the plasma membrane in a Ca²⁺-dependent manner, with its C2 domain functioning as a membrane trafficker. Transient expression of OsPLC4 and its truncated variants triggers cell death and immune responses in plants. During effector-triggered immunity (ETI) in rice, OsPLC4 expression and Ca²⁺ influx are specifically and strongly induced in response to avirulent Magnaporthe oryzae. Upon infection, the rice Osplc4 knockout mutant (ΔOsplc4) exhibits substantially reduced Ca²⁺, reactive oxygen species (ROS), and Fe³⁺ accumulation, as well as diminished lipid peroxidation and hypersensitive response (HR) cell death. Complementation of ΔOsplc4 can fully restore Ca²⁺-mediated ferroptotic cell death. OsPLC4 expression also activates HR cell death and the expression of defense-related genes such as OsRbohB, OsMEK2, OsMPK1, and OsPAL1 during avirulent M. oryzae infection. The Ca²⁺ chelator ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) substantially inhibits Ca²⁺, ROS, and Fe³⁺ accumulation and HR-related cell death in rice, whereas the Ca²⁺ influx enhancers trifluoperazine hydrochloride (TFP) and acibenzolar-S-methyl (ASM) strongly induce Ca²⁺-mediated ferroptotic cell death. Additionally, OsPLC4 overexpression triggers cell death and immune responses in Arabidopsis. Collectively, our findings demonstrate that OsPLC4 acts as a downstream target of resistosome activation and sustains the co-elevation of Ca²⁺ and ROS levels during rice ETI, thereby promoting Ca²⁺-mediated, lipid ROS-dependent ferroptotic cell death. These results establish OsPLC4 as a key regulator of Ca²⁺-dependent plant immunity.