Naotaka Furuichi

PiPE, a Phytophthora-associated PAMPS from P. infestans, Binds to aCa2+-Dependent Protein Kinase (CDPK) in Potato for the Induction ofHypersensitive Reaction

We report that PiPE, a Phytophthora-associated PAMPS (Pathogen Associated Molecular Patterns), induced generation of active oxygen species and hypersensitive cell death (HR) by treatment of potato tuber tissues, and that the PiPE gene from a species of Oomycete, Phytophthora infestans Mont (de Bary), was cloned. Interaction of a His-tagged PiPE from P. infestans with a His-tagged Ca2+-Dependent Protein Kinase (RiCDPK2) from potato cv. Rishiri (R1-gene) was investigated by using enzyme-linked immunosorbent assay with mouse monoclonal anti-PiPE Antibodies (Abs). We found that the PiPE and a Mycelical Homogenate (MH) from P. infestans can interact with His- RiCDPK2 in vitro. PiPE showed binding interaction with the His-fusion proteins from three other domains of RiCDPK2, indicating the existence of binding sites for PiPE of P. infestans on RiCDPK2. We suggest that after binding with the PiPE, RiCDPK2 may trigger signals that lead to the occurrence of HR in potato.

Host selective toxins and suppressor effector from plant pathogens regulate Ca2+ dependent protein kinase in the plant cell

Seven members of the family of host selective toxins were reported from Alternaria alternate and Alternaria solani by S. Nishimura lab. The suppressor effector for hypersensitive response of host cells was reported firstly by using Phytophthora infestans by K. Tomiyama lab. The host selective toxin (HST) effect on the host plant and induced the infection of the pathogen into the tissue, which has no resistant genes against HST. The suppressor effector induced the inhibition of hypersensitive cell death, accumulation of phytoalexins and the symptom of HR, hypersensitive response, in host tissues. Recently, the Ca2+ -dependent protein kinase in the plasma membrane of host cells was stimulated after the treatment of Alternaric acid, a HST, from A. solani and the suppressor effector from P. infestans in vitro, in the assay as reported. This means that the kinase, CPKs, could recognize the HST and suppressor in host plasma membrane and regulate the occurrence of HR in host cells. So far, the receptor sites for the PAMPS were reported. However, there are few reports with regard to the receptor for HST in plant cells. The CPKs in the host plasma membrane are a candidate against receptor of HST in potato and other plants. The suppressor of P. infestans has a -1,3-linkages of glucose, and also contains glucosamine. Recently, cyclic nucleotide gated protein channel (CNGC) activity was induced by the treatment of At pep peptides effector in host cells, resulting the HR response in the cells. The CNGC channel activity was reported by the Fluorescent protein method. For the activation of CPKs, the influx of calcium into the cytoplasm is important physiological phenomena in host cells. In plant, CNGC played a role for the occurrence of Ca2+ influx in host cell, as reported. The CPKs signaling cascades regulated the occurrence of HR response in host cells, and the HST effect on the inhibition of HR in potato and tomato.

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

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.