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

Phospholipases are major regulators of lipid-dependent signaling and play a crucial role in plant immunity. Rice (Oryza sativa) phospholipase C4 (OsPLC4) is a major functional enzyme in the rice phospholipase family that regulates intracellular Ca²⁺ levels. OsPLC4 translocates primarily to the plasma membrane in a Ca²⁺-dependent manner, with its C2 domain acting as a membrane trafficker. Transient expression of OsPLC4 and its truncated variants triggered cell death and immune responses in plants. OsPLC4 expression and Ca²⁺ influx were specifically and significantly induced during effector-triggered immunity (ETI) in rice in response to avirulent Magnaporthe oryzae. The Osplc4 knockout mutant (ΔOsplc4) rice exhibited significantly decreased levels of Ca²⁺, reactive oxygen species (ROS), and Fe³⁺ accumulation, lipid peroxidation, and hypersensitive response (HR) cell death upon infection. However, OsPLC4 complementation of the ΔOsplc4 mutant completely restored Ca²⁺-mediated ferroptotic cell death response. OsPLC4 expression triggered HR cell death and 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) significantly inhibited Ca²⁺, ROS, and Fe³⁺ accumulation and HR-related cell death in rice, whereas the Ca²⁺ influx enhancers trifluoperazine hydrochloride (TFP) and asibenzolar-S-methyl (ASM) strongly induced Ca²⁺-mediated ferroptotic cell death. Additionally, OsPLC4 overexpression triggered cell death and immune responses in Arabidopsis plants. Collectively, our findings provide evidence that OsPLC4, as a downstream target of resistosome activation, sustains the co-elevation of Ca²⁺ and ROS levels during rice ETI and promotes Ca²⁺-mediated, lipid ROS-dependent ferroptotic cell death, establishing OsPLC4 as a key regulator of Ca²⁺-dependent plant immunity.