title: Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells.
abstract: Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) 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 Ca(2+), mainly due to a Ca(2+) 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 Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) 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 Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Haruyasu
authors.initials: H
authors.lastname: Hamada
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan; Research Institute for Science and Technology (RIST), Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Kurusu
authors.affiliation: Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Okuma
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nokajima
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kiyoduka
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Koyano
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Sugiyama
authors.affiliation: Biotechnology Research Center, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Okada
authors.affiliation: Food Science Research Laboratories, Meiji Co., Ltd., 5-3-1, Chiyoda, Sakado, Saitama 350-0289, Japan.
authors.forename: Jinichiro
authors.initials: J
authors.lastname: Koga
authors.affiliation: Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, 305-8506 Japan, and.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Saji
authors.affiliation: Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Miyao
authors.affiliation: Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Hirochika
authors.affiliation: Biotechnology Research Center, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Yamane
authors.affiliation: Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Murata
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan; Research Institute for Science and Technology (RIST), Tokyo University of Science, Noda, Chiba 278-8510, Japan,. Electronic address: kuchitsu@rs.noda.tus.ac.jp.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kuchitsu
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000494:pharmacology; D005656:Fungal Proteins / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D012275:Oryza / Q000166:cytology / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D059828:Plant Cells; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism*; D000094162:Phytoalexins
chemical_list: D015220:Calcium Channels; D005656:Fungal Proteins; D010940:Plant Proteins; D012717:Sesquiterpenes; D000331:Aequorin; D043364:Endo-1,4-beta Xylanases; D002118:Calcium; D000094162:Phytoalexins