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Abstract

The Binding of Ca2+-Dependent Protein Kinase to the Suppressor of Potato Late Blight Pathogen Proved by Fluorescence Correlation Spectroscopy (FCS) Inhibits the NADPH Oxidase and Active Oxygen Generation in Potato Cell

Naotaka Furuichi, Masatoshi Ohta , Hisakazu Okamura, Tuneyoshi Horigome and Kazutoshi Yokokawa

Representing the suppressor for hypersensitive cell death of plant cell, glucan from Phytophthora infestans (Pi) was reported, and that the suppressor inhibited the accumulation of phytoalexin and hypersensitive cell death. To evaluate the activation of calcium dependent protein kinase (CDPK) after the binding of suppressor from Pi, fluorescence correlation spectroscopy (FCS) was applied to single GFP-CDPK and Alexa labeled suppressor. We constructed chimeric protein, tandemly fused green FP (GFP)-CDPK and suppressor with the alexa 633 labeled-suppressor antibodies (Abs). Dual color FCS provides information about the coincidence of spectrally two fluorescent molecules at a single-molecule level. Here we report that for the inhibition of NADPH oxidase of potato, the suppressor bound to CDPK peptide (kinase domain-I and -III peptides), CDPK phosphorylated the NADPH oxidase in gel kinase assay, and that the generation of active oxygen by NADPH oxydase was inhibited by the suppressor. This means HR inhibiting suppressor from the plant pathogen control the superoxide radical formation in plant cell by signal of the phosphorylation by a CDPK.