Abstract
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 Ca(2+) levels. Here we show that OsPLC4 translocates primarily to the plasma membrane in a Ca(2+)-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 Ca(2+) influx are specifically and strongly induced in response to avirulent Magnaporthe oryzae. Upon infection, the rice Osplc4 knockout mutant (ΔOsplc4) exhibits substantially reduced Ca(2+), reactive oxygen species (ROS), and Fe(3+) accumulation, as well as diminished lipid peroxidation and hypersensitive response (HR) cell death. Complementation of ΔOsplc4 can fully restore Ca(2+)-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 Ca(2+) chelator ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) substantially inhibits Ca(2+), ROS, and Fe(3+) accumulation and HR-related cell death in rice, whereas the Ca(2+) influx enhancers trifluoperazine hydrochloride (TFP) and acibenzolar-S-methyl (ASM) strongly induce Ca(2+)-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 Ca(2+) and ROS levels during rice ETI, thereby promoting Ca(2+)-mediated, lipid ROS-dependent ferroptotic cell death. These results establish OsPLC4 as a key regulator of Ca(2+)-dependent plant immunity.