Plant innate immunity requires immune receptors that sense the presence of microbes and activate defense reactions. Phosphatidylinositol-phospholipase C (PI-PLC) activity was previously shown to be important for several types of plant defenses although its signaling mechanism is not fully understood. It is also not clear why plants possess several PI-PLC isoforms and how triggering immune receptors activates them. PI-PLC activation induces a transient release of free cytosolic Ca2+ and the turnover of specific low abundant signaling phospholipids in the plasma membrane. Both events are important signals in animals and plants. Here, the first genetic evidence linking PI-PLC signaling to plant defense against pathogens is provided. The structure of the tomato PI-PLC family was determined, the corresponding genes were cloned and the function of individual isoforms during defense was studied. We found that PLC4 and PLC6 encode active enzymes and that they have distinct roles in defense. Optimum activity requirements and substrate preferences were determined for three PI-PLC enzymes and their enzyme activity was found to be important for immune receptor-activated responses. The available information was used to draw a model explaining the role of PI-PLC signaling in immune receptor-mediated defense and resistance in plants.