Regulation of a Proteinaceous Elicitor-induced Ca2+ Influx and Production of Phytoalexins by a Putative Voltage-gated Cation Channel, OsTPC1, in Cultured Rice Cells

Pathogen/microbe-or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca2+ followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca2+, mainly due to a Ca2+ influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon- insertional Ostpc1 knock-out cell lines harboring a Ca2+-sensitive photoprotein, aequorin, TvX-induced Ca2+ elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca2+-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca2+ influx as a plasma membrane Ca2+ permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.