Received date: July 04, 2015; Accepted date: August 05, 2015; Published date: August 10, 2015
Citation: Furuichi N, Yokokawa K, Ohnishi K, Ohta M (2015) 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. J Plant Pathol Microbiol S4:001. doi:10.4172/2157-7471.S4-001
Copyright: © 2015 Furuichi N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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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.
NADPH oxidase; CDPK; potato; Phytophthora infestans;, signal transduction of HR
Plants are exposed to pathogen attach in their environment and have developed mechanisms to respond with biotic elicitor and the suppressor or host specific toxin from the pathogens. Among the earliest cellular responses to such attach and stimuli are the production of superoxide anion generation [1-4], the hypersensitive response [5-8] and phytoalexin production [7-9] in host cells, and a specific calcium signature is often reported [7,10-12]. Suppressor, a glucan, for hypersensitive response and phytoalexin production was well documented [6,9,11,13,14]. Suppressor glucan inhibited the active oxygen generation at an early period of infection with Pi [9,15,16]. Yet, the receptor site for the inhibition of HR in plant cells has not been reported. The suppressor glucan from Pi well control the production of phytoalexin and host cell death in compatible interaction between Pi and the potato cultivar [9,11,15,17,18]. Calcium dependent protein kinases (CDPKs) may function as a potential sensor that decodes and translates the elevation of calcium concentration into enhanced CDPK activity and subsequent downstream signaling events [13,19-24]. CDPKs are calcium-binding Ser/Thr protein kinases .
Upon elicitor stimulation of host cells, the NADPH oxidase produce active oxygen species (AOS) in the defense response [16,26-29]. The generation of superoxide anion (O2-) was reported in incompatible interactions between potato and the oomycete pathogen, Pi , and has been considered that production of AOS in the cell was the earliest events in the plant defense response and a signal for induction of hypersensitive cell death (HR) [1,29,30]. So far, CDPKs have not been identified in yeast and animal cells. Calmodulin-dependent protein kinases are well characterized as major mammalian calcium dependent signaling molecules . CDPKs comprise a large gene family (34 members in Arabidopsis sp.) [21,25]. This suggests that individual isoforms have different functions and participate in multiple distinct signaling pathways. For measuring such random diffusional motion of an individual CDPKs, fluorescence correlation laser microscopy (FCCS) is, at present, the only practical method. [31-33]. FCCS detects fluctuation of the fluorescence intensity in a confocally defined volume with a sharply focused laser, 0.25 fl. This method has been developed as a unique technique to measure rotational diffusion coefficients of molecules in a solution and in living cells [31,33-35]. Application of this technique to biological systems has brought to light novel aspects of various molecular dynamics, such as the status of the pathogen suppressor, toxin and the cell receptor in plants [15,36,37]. Recently, we reported the isolation of PiPE elicitor [38,39] and the suppressor in HR from P. infestans. The PiPE elicitor binds to the CDPK in the plasma membrane of host potato cells .
Here we report whether downstream CDPK-regulated processes control the AOS production in HR response. The challenge that currently faces the CDPK field is not only to defined biological functions to specific CDPK isoforms, but also to integrate CDPK signals into the AOS generation [15,24], HR response  and phytoalexin accumulation. The localization of effector suppressor from P. infestans by using Alexa-antibodies immune staining methods were well discussed in the present report. A part of this report was published in the annual Meeting of Plant and Cell Physiology, Japan (Abstructs, 2005), and in the Annual Meeting of Plant Pathology, Japan (Abstracts, 2005).
Potato tuber cvs. Eniwa (R), Rishiri (R), Konafubuki (R1,3), Mayqueen (r) and Irish Cobbler (r) were stored at 4°C until use. Potato and A. thaliana ecotype Colombia were grown in soil to extract DNA or RNA in a green house at 25°C. Pi (Mont.) de Bary isolate DN101 (Race 0) and Pi831 (Race 1) were maintained on rye agar medium supplemented with 2% sucrose and 0.2% bacto yeast extract at 18°C in the dark. For liquid culture of the fungus, the mycelia were grown in the dark at 18°C for 2-3 weeks on the synthetic medium as described . The mycelial mats were harvested by gentle filtration, washed, and frozen at -20°C. Elicitor of Pi was prepared from frozen mycelia of race 0 grown on rye meal liquid medium at 15°C for 4 weeks. Mycelial mats were thawed in 0.05 M acetate buffer, pH 4.5 and homogenized with polytron homogenizer for 30 min as reported . The homogenate was autoclaved at 120°C for 10 min. The autoclaved suspension was cooled in an ice bath and centrifuged at 12000 rpm for 10 min at 4. The supernatant was collected and the pellet was resuspended in 100 mM borate buffer (1:2 v/w). It was autoclaved at 120°C for 10 min and cooled on ice and centrifuged at 12000 rpm for 10 min at 4. The supernatant was collected and mixed with previously collected.
All step were performed at 4°C using the ice bath or cold room. 3 weeks old hyphal mat dried by dessication and frozen in liquid nitrogen was ground to powder with dry-autoclaved pestle and morter as reported . The powder was homogenized with 0.05 M sodium acetate buffer, pH 4.5 (2 mg/g fresh weight of mycelia) added with 100 mM/ml of PMSF (1μl/4 ml of acetate buffer) and homogenized till it turned into viscose paste. The homogenate was sonicated twice for 1 and 1.5 min on ice and centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was collected and the pellet was kept for elicitor extraction. The supernatant was mixed with an equal volume of 80% phenol, incubated on ice for 3 hrs and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected and mixed with chloroform: methanol (1:1 v/v) mixture (1:1 v/v). Mixed by inverting the tube 50 times, incubated on ice for 30 min and the supernatant was collected. Added the equal volume of chloroform and mixed by inverting the tube 50 times. Incubated on ice for 30 min and the supernatant were collected. The supernatant was evaporated in evaporator by adding water till the smell of phenol disappeared. The diluted sample was stored at -20°C.
Genomic DNA isolation and southern blot analysis
Genomic DNA was prepared from frozen potato and A. thaliana leaves (100 mg fresh weight) by DNeasy Plant Mini Kit (QIAGEN) according to the reported methods . 10 μg of genomic DNA was digested with restriction enzyme, and electrophoresed on ethidium bromide stained 1.2% agarose gel. Alkaline DNA was transferred to the nylon membrane (Hybond N+, Amersham) and hybridized at 55°C with partial of labeled AtrbohF cDNA probe. Probe DNA was labeled by AlkPhos Gene images DIRECT+LABBELING DETECTION kit (Amersham Bioscience) according to the manufactures instructions. Membrane was washed at 65°C for 10 min in primary wash buffer (2 M urea, 0.1% SDS, 50 mM Na phosphate; pH 7.0, 150 mM NaCl, 1 mM MgCl2 and 0.2% Blocking reagent), and washed at 55°C for 10 min in secondary wash buffer (50 mM Tris base, 100 mM NaCl and 2 mM MgCl2). Positive cDNA clones were detected according to the manufactures instructions.
The CaMV35S promoter which was fused to a synthetic green fluorescent protein gene, was used to construct the chemeric gene with SdCDPK1 or SdCDPK2, and Enrboh1 and Mqrboh1 to create the GFPCDPK and GFP-RBOH reporter construct. A CDPK1 and others were inserted in C-end of the CaMV 35S minimal promoter [41-43].
RNA isolation and RT-PCR
Total RNA was prepared from frozen healthy potato leaves (100 mg fresh weight), using the guanidine hydrochloride RNA extraction method as reported [40,44]. Extracted total RNA was treated with DNase to remove traces of contaminating DNA and then separated by electrophoresis on a 1.2% agarose-formaldehyde gel. 2μg of total RNA was reverse transcribed into single strand cDNA by the Ready-To- GoTM RT-PCR beads (GE Healthcare) with 1 pM oligo d(T)16~18 primer according to the manufactures instructions. The incubation conditions were as follows: 30 min, 42°C; 5 min, 95°C. 0.3 μg of the cDNA reaction was used in a 50μl PCR with the sets of AtrbohF specific primers. The PCR reaction mixture contained 0.3 μg of template cDNA, 2 units of Ex Taq polymerase (TaKaRa), 4 μl of dNTP mixture, 5 μl of 10×Ex Taq buffer, and 50 pM of each of the primer 5’ and 3’ for EnRBOH1 as reported for AtRBOHF , fw 5’-ATG AAA CCG TTC TCA AAG-3’, rv 5’-ATG ATC ATG ATC GGA TTA-3’, for SdCDPK1 (Primers; fw 5’-ATGGGGAACACTTGTGTAGGA C-3’, rv 5’-TAG TTT TAC TGC TTC TCT GAA TCC-3’) and - 2 (fw 5’-ATG GAA CCA AAA CCA GCA ACT G-3’, rv 5’-TAA GAT TTC TTC ACT CTG TAC GAG-3’) and PCR reaction was carried out in Eppendorf PCR thermal cycler. The incubation conditions were as follows: an initial step of 5 min at 95°C followed by 35 cycles of 1 min at 95°C, 1 min at 55~65°C, 1~3 min at 72°C, and a final step of 10 min at 72°C.
RNA transcriptional analysis
Potato leaves cvs. Eniwa (R) and Mayqueen (r) grown in a greenhouse were sampled and washed. Both of 100 mg of leaves (1.5 cm in diameter) were prepared in the dark. HWC elicitor, PiE elicitor and suppressor were prepared from the mycelium of Pi, as described above. The volume of elicitors (200 μg/ml) and suppressor (200 μg/ml) solutions applied to the leaf surface was 100 μl, and solution applied to leaves were permeated by infiltration for 2 min. In all experiments, the treated leaves were incubated at 20°C in the dark. Total RNA after incubation was prepared from frozen leaves (100 mg fresh weight), using the guanidine hydrochloride RNA extraction method as described by Logemann et al. . Extracted total RNA was treated with DNase to remove traces of contaminating DNA and then separated by electrophoresis on a 1.2% agarose-formaldehyde gel. 2μg of total RNA was reverse transcribed into single strand cDNA by the Ready-To- GoTM RT-PCR beads (GE healthcare) with 1 pM oligo d(T)16~18 primer according to the manufactures instructions. The RT-PCR incubation conditions were as follows: 30 min, 42°C; 5 min, 95°C. 0.1 μg of the cDNA reaction was used in a 25μl PCR with the sets of β-actin as a control and specific primers. The PCR reaction mixture contained 0.1 μg of template cDNA, 1 units of Ex Taq polymerase (TaKaRa), 2 μl of dNTP mixture, 2.5 μl of 10×Ex Taq buffer, and 25 pM of each of the primer 5’ and 3’ and PCR reaction was carried out in PCR thermal cycler. To stop the PCR amplification of DNA under log increase, we analyzed kinetics of PCR amplification. In all PCR experiments were performed in 20 cycles by the result of kinetics experiment. The incubation conditions were as follows: an initial step of 5 min at 95 C followed by 20 cycles of 1 min at 95°C, 1 min at 55~65°C, 1~3 min at 72°C, and a final step of 10 min at 72°C.
Cloning and sequence analysis
For nucleotide sequence determination, PCR products were cloned to the pCR T7/CT TOPO plasmid vector (Invitrogen). The nucleotide sequencing was performed by using Thermo Sequenase fluorescent labeled primer cycle sequencing kit (Amercham Bioscience) and ALFexpressTM DNA sequencer (Amercham Bioscience) according to the manufactures instructions.
The expression of recombinant protein in High Five insect cells
Seed cells in 24-well plates that were coated poly/lysine at 2.4×105cells/well in Express Five SFM, and allow to attach for at least 1 h. Prepare DNA which is pXINSECT-DEST39 plasmid vector (Invitrogen) containing insert cDNA and CELLFECTIN Reagent mixtures as follows for each well of cells to be transfected as reported. Dilute 1μl DNA into 25μl SFM, dilute 4 to 8μl of CELLFECTIN Reagent into 25 μl of SFM and combine the diluted DNA with the diluted CELLFECTIN, mix, and incubated at room temperature for 15 min. Add the DNA/CELLFECTIN complexes (to 50 μl) directly into the growth medium of cells in each well. Mix by rocking the plate back and forth and incubate at 27°C. After 5 h replace the medium in each well with 1 ml of SFM and return cells to incubator. Assay was conducted for transient gene expression at 48 h post-transfection.
Purification of recombinant protein
For purification of fusion protein, XpressTM System Protein Purification protocol (Invitrogen) was followed. The polyhistidinetagged fusion protein was loaded onto a ProBond His-bind resin column equilibrated with lysate buffer. The column was washed with 8 ml of denaturing binding buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8). Then, the column was washed with 8 ml of denaturing buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride) pH 6.0 and pH 5.3 successively. Finally, the protein was eluted with 5 ml of denaturing elution buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 4.0). the elute was dialyzed against 10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100 overnight at 4°C to remove urea. During this time, the dialysis buffer was replaced 4 times. The purified protein concentration was determined using the BCA protein assay kit (Pierce) with bovine serum albumin (BSA) as a standard.
SDS-PAGE and electroblotting of proteins
The fusion proteins were separated with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli  by using mini PAGE system (ATTO) and detected by staining with coomassie brilliant blue G-250. For western immunoblotting, fusion proteins were transferred electrophoretically to 0.45 μm difluoride (PVDF) membrane, ImmobilonTM P (MILLIPORE). The membranes were blocked with 3% (w/v) BSA in TBS (20 mM Tris-20) and incubated overnight with anti-His (C-terminus) mouse monoclonal IgG2b antibody diluted to 1:5000 in dilution buffer (TBST +1% BSA). After two washing with TBST, the membrane was incubated for 1 h at room temperature with alkaline phosphatase-conjugated rabbit anti-mouse antibody (D0314, DAKO) diluted to 1:250. The membrane was washed with TBST and alkaline phosphatase buffer (0.1 M Tris-HCl, 0.1 M NaCl, 5 mM MgCl2, pH 9.5) successively and finally developed using AP conjugated substrate kit (BioRad) according to the manufactures instructions.
Measurement of active oxygen species (AOS) generation
Accumulated O2- was measured by using firefly luciferase inducer (CLA) as reported . Sample protein was added to a 15 ml sample tube containing 426 μl of 39 mM HEPES (pH 7.0), 5 μl of 10 mM MgCl2 or CaCl2, 5 μl of 10 mM EGTA, 1.5 μl of 10 mM guanosine 5’-triphosphate (GTP)-γ-S, 15 μl of 500 mM CLA. CLA was added to the tube at last. The tube containing sample was incubated at 37°C for 3 minutes. The count of the radiated light was taken just after adding 23 μl of 3.3 mM NADPH for 15 second interval (ATTO luminecencer) as reported .
Microprojectile bombardment and confocal laser scanning microscopy
Microprojectile bombardment was performed with a Biolistic PDS-1000/He particle Deliverly System (Bio-rad). Potato suspension cells in MS medium in petri dishes were bombarded with a rupturedisk pressure of 1,100 p.s.i. (~7.6 MPa) at a target distance of 6 Cm. At 2-40 h after bombardment , they were analyzed with Carl Zeiss LSM confocal laser scanning microscope. Green fluorescence of GFP and red fluorescence of rhodamin were excited with 488 nm with an argon laser and collected sequentially with a filter set (HQ530/60 and ES70LP). Images of both fluorescences were processed and merged with the Lasersharp program system (Carl Zeiss).
Phosphorylation assay of NADPH oxydase- and His-CDPK
PiPE elicitor purified as describe above, microsomal fraction containing NADPH oxidase or NADPH oxidase purified as describe above (Approximately 2 μg PiPE elicitor) were incubated with purified His-CDPK1 in phosphorylation buffer (20 mM Tris-HCl (pH 7.1), 50 μM CaCl2, 50 mM KCl, 2 mM MgCl2, 1 mM DTT, 20 μM ATP, 1% tritonX-100, 10% glycerol) containing 1 μCi [γ-32P] ATP at 30°C for 10 min. Thus treated proteins were incubated at 4°C for 10min, added 2 mM ATP, incubated at 4°C for 10 min and precipitated by the method of MeOH/CH3Cl precipitation as described . Precipitated proteins dissolved in 1% SDS, separated by 12% SDS-PAGE and visualized by CBB R-250. After drying the gel, phosphorylation was detected by using Thyphoon (GE Healthcare).
Live cell fluorescence imaging was performed using an inverted confocal laser scanning microscope LSM510 (Carl Zeiss). GFP was excited at the 488 nm laser line of a CW Ar+ laser and Alexa 633 was excited at the 543 nm laser line of a CW He-Ne laser through a water immersion objective (C-Apochromat, 40x, 1.2NA; Carl Zeiss). Emmision signal were detected at 505-550 nm for GFP and >560 nm for Alexa by sequential scanning.
FCCS measurement and data analysis
FCCS measurement were carried out mainly with a ConfoCor2 (Carl Zeiss), which consisted of a CW Ar+ Laser and He-Ne laser, a water immersion objective (C-Apochromat, 40x, 1.2NA; Carl Zeiss), and two channels of avalanche photodiodes (SPCM-200-PQ; EG&G) as reported . The confocal pinhole diameter was adjusted to 90 um. GFP was excited at the 488 nm laser line and Alexa was excited at the 543 nm laser line. The emission signals were split by a dichroic mirror (570 nm beam splitter) and detected at 505-530 nm by the green channel for GFP and at 600-650 nm by the red channel for Alexa.
The fluorescence auto-cross correlation functions of the red and green channels, Gr (τ) and Gg (τ), and the fluorescence correlation function, Gc (τ), are calculated as reported previously . For quantitative evaluation of correlation among various samples, Gc (0) is normalized by Gg (0) (relative correlation amplitude; Gc(0)/Gg (0).
Expression analysis of signal genes in resistance and susceptible hosts
We have investigated a signal transduction pathway between potato Pi, and that isolated two homologues of gp91phox, plasma membrane NADPH oxidase, from the resistant and susceptible potato cDNA library of cvs. Eniwa (R1-) and Mayqueen (r-gene). The two genes designated Enrboh1 (DDBJ accession No, AB059744) and Mqrboh1 (DDBJ accession No, AB 300445), respectively. It was showed that the genes were new Rboh family in potato. The RNA expression analysis of the Rboh1 gene showed that Enrboh1 was transiently expressed followed by expression of Ser/Thr type protein kinase genes, which was peaked at 6 h after the treatment of PiPE, a AOS inducing elicitor from Pi, and inhibited by the glucan suppressor from Pi. On the other hand, Mqrboh1 was expressed slowly and showed low expression level compared to Enrboh1 after the treatment of PiPE. It was suggested that Enrboh1 and Mqrboh1 were induced in different levels in response to PiPE and suppressor of Pi. Moreover, to analyze whether Rboh1 protein involved in AOS generation, we expressed His-Rboh1 and measured the activity in the host cells. Addition of His-Enrboh1 to potato microsomal fraction (MF) of cv. Eniwa showed drastic and transient AOS generation in MF depend on His-rboh1 concentration immediately after the treatment. It was suggested that the His-rboh1 strongly produce the AOS generation. The AOS also play an important role in the inductin of systemic resistance .
FCCS analysis of the binding GFP-CDPK1 and the suppressor of Pi.
For the analysis of the binding between GFP-CDPK1 as a receptor and the ligand glucan suppressor from Pi in potato cells in vivo, we have used the fluorescence cross correlation spectroscopy, FCCS, laser microscopy. GFP-CDPK1 was constructed for the assay, transformation was done into the potato cells by using particle gun, and the glucan suppressor was incubated with the potato cell under the FCCS system at time intervals. Then the potato cells were treated with the Alexa Fluoru 568-anti-glucan-monoclonal Abs [9,49]. In this study we first tested whether the diffusion or binding of GFP-CDPK1 with the glucan suppressor of Pi in situ is measurable. We then studied how the dynamics change in the localization of GFP-CDPK1 in the potato cells and the binding between them in the cells after suppressor glucan addition in the host cells at various levels of microdynamics.
To test whether we could see the GFP-CDPK1 binding with the glucan suppressor of Pi in vivo, we first examined the FCCS analysis by using the GFP-CDPK1 transformed potato suspension cells. A recombinant GFP-CDPK1 was used to analyze the protein for localization of the CDPK1 in potato cells. Images of cells expressing the GFP-CDPK1 are shown in Figure 1. The intact potato cells exhibits GFP fluorescence that is distinct from the plasma membrane and strongly suggestive of an association with cell walls (Figure 1d). Intact suspension cell show fluorescence that remains with the plasma membrane (Figure 1e). Electron microscopic analysis by using the potato leaves with anti-CDPK1-kinase domain Abs with gold particle labeling (5 nm in diameter) showed that the CDPK1 localized on potato cell wall and the border between cell wall and plasma membrane in the host cells.
Figure 1: (a) Potato suspension cell was infected with Phytophthora infestans (Pi) and the plasma membrane bound to infecting hyphae tip within 5min after the penetration of the cell wall. (b) The hypersensitive cell death was occurred in the suspension cells of cv. Rishiri (R1-gene) at 5 h after infection. The cell death was measured by loss of plasmolysis, loss of stainability by neutral red (5 mM), and granulation of cytoplasmic strands and particles in the cell. PM:Plasma membren of potato cell. Z: Zoospores of Pi. (c) Effect of hyphal cell wall elicitor and suppressor glucan on potato leaves (cv. Irish Cobbler, r-gene, and cv. Eniwa, R1-gene). The suppressor for hypersensitive response from Pi caused little effect on the leaves within 48 h after treatment, although elicitor caused severe HR responses on those ones.
Further analyses confirmed the plasma membrane location of GFP-CDPK1 western blots comparing intact plasma membranes and microsomes prepared from potato tubers clearly show the presence of CDPK1 in the plasma membrane fraction. In addition, proteomic analysis of the plasma membrane from potato suspension cells or potato leaves identified CDPK1 as the only the glucan binding protein (Furuichi et al., unpublished data), a finding that we have independently verified with potato leaves material. It is showed that CDPK1 is relatively abundant in plant plasma membrane.
AOS generating activity of the CDPK1 transformed potato cells
To identify which of the previously reported CDPK activities might be associated with the presence of CDPK1, we investigated the kinase activity veryfing in their level of CDPK1 expression. For this pursues, we created a recombinant potato suspension culture cells (cv. Rishiri), by using gene transformation from the potato cells under the control of the CaMV 35S promoter. The GFP-CDPK1-overexpressing recombinant cultured cells were reacted with elicitor more rapidly and generated high level of AOS than the non-recombinant potato suspension cells (Figure 2a). These cells were found to have a 1.4-fold increase in AOS generation levels when assessed by CLA index (Figure 2b). The presence of four Ca2+-binding EF hands in the CDPK protein, as well as its plasma membrane location , prompted us to investigate whether CDPK1 is, or forms part of , the Ca2+ sensor kinase, which has a steep  cytsolic calcium dependence. We therefore analyzed the effect of Ca2+ addition or the addition with elicitor and suppressor on the kinase activity. Ca2+ (100 uM) and Mg2+ (50 mM), bi-ionic condition with elicitor (100 ug/ml) stimulated the auto-phosphorylation of the His-CDPK1 within 10 min (Figure 2). The suppressor addition with these bi-iones also stimulated the auto-phosphorylation of CDPK1.
Figure 2: (a) Effect of smGFP-CDPK overexpressing potato cells on the generation of AOS in the cells after PiP elicitor treatment at different time intervals (Control: without smGFP-CDPK1). : CLA was used as a chemiluminescent substrate for measuring the AOS generation by NADPH oxydase in potato cells by using luminometer. GFP::SdCDPK1+PiPE, Suspension cells (cv.Rishiri) +PiPE, GFP:: SdCDPK1, Suspension cells (cv. Rishiri). (b) smGFP-GFP-CDPK1 was transformed into the potato suspension cells by the particle gun. MF (5 μg) + NADPH oxydase- His (36 nM), MF (5 μg) + NADPH oxydase-His (3.6 nM), MF (5 μg) + NADPH oxydase-His (36 nM) + Tiron (10 mM), MF (5 μg), MF (5 μg) + Tiron (10 mM). (c) Effect of insect cell expressed NADPH oxydase activity on the generation of AOS in potato membrane fraction (cv. Eniwa, R1-gene). CLA index showing generation of AOS in the suspension cells. Tiron was added for a specific scavenger of the AOS. MF (5 μg) + His-rboh1 (36 nM), MF (5 μg) + His-rboh1 (36 nM) + anti-p47phox and - antibodies(Abs) 67phox Abs, MF (5 μg) + His-rboh1 (36 nM) + anti-p67phox Abs, MF (5 μg) + His-rboh1 (36 nM) + anti-p47phox Abs, MF (5 μg). (d) Effect of predicted epitopes of p67phox and p47phox (NCF1) on generation of AOS in potato microsomal fractions. Predicted amino acid sequences of p67phox and p47phox. Amino acids showed are recognized amino acid residues by p67phox (DEPKESEKADANNQ) , and p47phox (PGPQSPGSPLEEERQ) and p47phox Abs. CLA assay of generation of AOS in potato microsomal fractions. MF+His-rboh1 (132μg/ml), MF+ His-rboh1 (132μg/ml)+ anti-p47phox Abs, MF+ His-rboh1 (132μg/ml)+ anti-p67phox Abs, MF+ His-rboh1 (132μg/ml)+ anti-p47phox and p67phox Abs, MF. (e) Effect of predicted antigen epitopes of CDPK on generation of AOS in potato microsomal proteins and His-rboh1. MF+ His-rboh1 (132μg/ml), MF+ His-rboh1 (132μg/ml)+ anti-CDPK-Abs, MF+ His-rboh1 (132μg/ml)+anti-CDPK-Abs +anti-p47phox and p67phox Abs, MF only.
The CDPK is ubiquitous in plants and one of the first plant Ca2+ dependent kinase to be characterized. It is suggested that the CDPK stimulated the generation of AOS in potato membrane fractions by the predominant activation with NADPH oxidase. In contrast, we tested that by the inhibition of AOS with the CDPK-1 and -2 with Abs or glucan suppressor of Pi treatment in potato membrane fractions containing with His-Enrboh1.
FCCS analysis of the binding between GFP-CDPK1 and suppressor
Figure 3 shows FCCS analysis in a potato suspension cells of cv. Rishiri (R1-gene). The GFP-CDPK1 transformed potato cells were treated with the glucan suppressor, and then the Alexa anti-glucan suppressor-monoclonal Abs . When GFP-CDPK1 were observed with FCCS, the presence of slow movement on the plasma membrane of potato cell, suggesting the presence of the CDPK1 on the membrane of potato. CDPK1 had merythtylation site from the sequencing analysis (Figure 3). The suppressor glucan was introduced into the potato cytosol, the membrane location and the plasma membrane, after staining with the anti-suppressor-binding monoclonal Abs. In the FCS analysis, it was showed that GFP-CDPK1 was bound with the suppressor glucan, within a short time intervals, 0.01 msec. Then its binding was dispersed in the following time intervals. Gc(0) is directly proportional to the number of linked proteins Nc. The decrease of the binding between the GFP-CDPK and Alexa labeled-suppressor could be monitored as a reduction of Gc (0)/Gg (0) . We thus measured how correlation amplitude of the binding (Figure 3a). We suggested that during this time period (within 0.01 msec), the suppressor signals were transduced onto the NADPH oxidase in the host cells. Figure 4 shows that the CDPK1 phosphorylated the NADPH-oxidase from cv. Rishiri in vitro, suggesting the CDPK signals were downstreaming to the NADPH oxidase in vivo in potato single cell.
Figure 3: (a) Fluorescent cross correlation spectroscopy measurements of GFP-CDPK1 and the suppressor glucan with rhodamine-anti- suppressor antibodies (Abs) in potato suspension cells, cv. Rishiri (R1-gene). Typical auto- and cross-correlation spectroscopy of the GFP-CDPK1 and the suppressor glucan. Measurement positions are indicated by the cross-hair (+) in the green channel laser scanning microscopy (LSM) images of GFP-CDPK1 and the rhodamine suppressor in a potato cell. The potato cells were treated with suppressor and then anti-suppressor monoclonal Abs (see Figure 3c). The control treatment without suppressor and the antisuppressor Abs were also tested (data not shown). (b) FCCS analysis showed that potato cells were treated with suppressor and anti-suppressor monoclonal Abs. (c) The potato cell was treated with suppressor and then anti-suppressor Abs with rhodamine anti-mouse monoclonal Abs. (d) Schatterd plots of the binding between suppressor and His-CDPK. Suppressors were at 0,25,50,75, and 100μM. (e) The smGFP-CDPK1 overexpressing potato cells were observed by using green filter of laser scanning microscopy. 1:smGFP-CDPK1 was localized on to the cell wall and plasma membrane of potato cell. 2: Light microscope observation of potato cell. 3: The same images in a potato cell (GFP images were filed up in No.2 photo). (f) The smGFP-CDPK1 overexpressing potato cells were treated with the suppressor of Phytophthora infestans(Pi) and anti-glucan Abs. Green color showed smGFP-CDPK1 proteins. Red colors showed the rhodamine labeled anti-suppressor Abs.
Figure 4: Predicted amino acid sequence alignment of cv. Eniwa rboh1 (Enrboh 1), Mayqueen rboh1 (Mqrboh 1), StrbohA and StrbohB. Amino acids different from each group are in black boxes. Six potential transmembrane-spanning domains (TMD1-6) are indicated by a bar. Dots under the sequences indicate amino acid residues involved in N-glycosylation. Dashes indicate gaps in sequence to allow for maximal alignment. The alignment was conducted with program DNASIS.
Kinase assay of CDPK1 with NADPH oxidase
Microsomal fraction containing NADPH oxidase and recombinant NADPH oxidase isolated as His-fusion describe above were purified and incubated with purified His-SdCDPK1 in phosphorylation buffer (20 mM Tris-HCl,pH 7.1, 50 μM CaCl2, and Ca2+ and Mg2+) containing 1 μCi [γ-32P]ATP at 30 C for 10 min. Thus treated proteins were incubated at 4 C for 10min, added 2 mM ATP, incubated at 4 C for 10 min and precipitated by the method of MeOH/CH3Cl precipitation as described . Figure 4b shows that this purified SdCDPK1 was auto-phosphorylated, and this phosphorylation activity was inhibited by EGTA and PiPE (an elicitor from Pi.). The result suggest that the auto-phosphorylation activity of CDPK2 regulated by PiPE and Ca2+ ion. We performed the phosphorylation assay using microsomal fraction containing the CDPK2 with the purified His-NADPH oxidase phosphrylated the His-NADPH oxidase, ca. 97 kD, and the CDPK1 per se. The result show that purified NADPH oxidase (arrow heads) was phosphorylated by CDPK1 in vitro, but NADPH oxidase in microsome fraction, was not. It was suggested that CDPK1 was inhibited by unknown effector in microsome fraction. We observed that the 84 KD arrowhead may contain the decomposed peptides from the NADPH oxidase, which was bound with and confirmed by using p67- or p47- Abs (Furuichi et al., unpublished data). It was suggested from the present data that CDPK1 signal tranceduced to the downstreams of the gp91phox in potato cell. These findings suggest that the suppressor passed through the host cell wall and the plasma membrane and bound to CDPK1 receptor sites, which was localized on the cell wall and the plasma membrane of potato cells as shown in Figure 3f.
CDPK1 phosphorylated the NADPH oxidase in plasma membrane of potato cells to inhibit the hypersensitive response and the cell death of the host cell (Figure 5).
Figure 5: SdCDPK2 autophosphorylation and RT-PCR gene expression analysis. (a) SdCDPK2 is autophosphorylated by the PiPE and elicitor treatment. Purified His-CDPK2 in phosphorylation buffer (lane 1), phosphorylation buffer containing 2 mM EGTA (lane 2) or phosphorylation buffer containing about 2 μg PiPE (lane 3) was incubated at 30°C for 10 min under the existence of 1 μCi [γ-32P] ATP. Thus treated His-CDPK2 was precipitated, subjected to 12% SDS-PAGE and detected by autoradiography as described in” Materials and Methods”. (b) NADPH oxydase is phosphorylated by His-CDPK2. Microsomal fraction containing NADPH oxydase (lane 1) and purified NADPH oxydase (lane 2) was incubated with purified His-CDPK2 and detected by autoradiography as shown in (a). Arrows indicate His- NADPH oxydase and arrowhead indicates His-CDPK2, respectively. Lines shown on the left side indicate molecular markers, 97, 66, 43 and 29 kDa (up to bottom), respectively. (c) Quantitative RT-PCR of defence signal genes in cv. Eniwa (R1-gene) and Mayqueen (r-gene). After the treatment of elicitor, PiPE, and suppressor of Pi, total RNA was extracted and PCR using the primers (See Methods). All PCR reactions were done at 20 cycles from the result. HWC elicitor and PiPE treatment, and suppressor treatment were observed, and also Pto homologs were observed.
Suppressor controls hypersensitive cell death
The data suggest that SdCDPK1 phosphorylated the NADPH oxidase in plasma membrane of potato cells to downstream the suppressor signal for the inhibition of hypersensitive response and of cell death in host cell. We have reported in a previous paper that the suppressor of the compatible race of Pi. inhibit the hypersensitive cell death and phytoalexin, rishitin, accumulation in host cells . The suppressor also inhibited the AOS generation in the compatible interaction of the host-pathogen interaction . These findings strongly suggest that the AOS signal is an upstream signal than the caspase homolog (VPE) cascade in host cells for the hypersensitivity in planta, and that the AOS generation trigger the hypersensitive cell death per se. This is now underway by using FCCS in vivo, which contains the direct detection of a single photon from plant cell membrane .
We have reported that Ca2+ addition with elicitor onto the potato tuber disks induce enhancement of the hypersensitive cell death and phytoalexin accumulation. Plasma membrane from potato tuber cells have contained the binding receptor for the elicitor, suppressor and the fungal cell wall components from Pi [24,37-39,52]. In contrast, the potato lectin isolated from the membrane fraction of potato [37,52] had the non-specific binding activity for the elicitor . These results suggested that the binding between the germ tube of Pi and the host plasma membrane was a key role in the induction of hypersensitive cell death in potato. Bidwai and Takemoto  reported that syringomycin stimulated the protein kinase mediated phosphorylation of membrane protein and the ATPase in red beet plasma membrane for the toxin signal. This stimulation was controlled by Ca2+ ion, suggesting the role of CDPK in the cell membrane. Recently, Furuichi et al. that a Ca2+ channel protein, TPC1, existed on the plasma membrane [55-57], and that the channel control the signals for stomatal opening and germination . We also tested that by the treatment of elicitor of Pi with bi-ionic (Ca2+ and Mg2+ )conditions, the CDPK-1 and CDPK-2 autophosphorylation activity was increased more strongly with the elicitor and ions than ions only, and that the suppressor of Pi shows stimulation on the kinase with ions than ions only  (data not shown). From these and the present data in Figure 2 indicates that both compatible and incompatible interaction for the hypersensitivity in potato cell, the CDPK-1 is a switch kinase to induce or to inhibit the hypersensitive reaction of host cell.
Recently, it was reported [59-61], and Yang et al.  identified a tobacco and Arabidopsis MAPKK, NtMEK2. Expression of a constitutively active mutant of NtMEK2 in tobacco leaves led to the induction of hypersensitive cell death and the expression of defence genes in the absence of pathogens. The results suggest that a MAPK cascade containing NtMEK2, WIPK, and SIPK is involved in the expression of fungal pathogen hypersensitivity in tobacco [59,62].
The data in Figure 4 suggest that the potato-suppressor CDPK cascade may be involved in the inhibition of fungal defence responses without eliciting hypersensitive cell death and the accumulation of phytoalexins in potato as in the case of the host selective toxins. This, in turn, suggests that signaling events initiated by diverse pathogens, e.g. Alternaria and Helminthosporium, converge into a conserved CDPK cascade (shown in Figure 6). However, additional quantitative analyses in both transiently and stably transformed plants are possible to confirm the suggested role of suppressor CDPK defense inhibiting pathway .
Figure 6: Localization of CDPK in potato cells by means of fluorescent immunochemistry by using anti-CDPK-antibodies(Abs). (a) Potato suspension cells, which was taken by laser microscopy (bar means 10 μm). (b) The fluorescent microscopy shows localization of CDPK in potato cell. Note that potato cell was stain with the CDPK kinase domain I-Abs, in plasma membrane and on the cell wall. Some intracellular space, specially cytozol and on the surface of the nucleus. c, Model of CDPK signaling in potato activated by the suppressor of Phytophthora infestans (Pi). A membrane bound CDPK1 could control gp91phox activity because their phosphorylation by CDPK1 induced AOS generation in potato cells. The signaling cascade of the suppressor binding against the receptor CDPK in planta shows that the toxin from the pathogen induced CDPK signals to gp91phox, and then inhibit the hypersensitive response in compatible interaction. The suppressor inhibiting HR response are summarized on the basis of recent reviews [54,63] from the pathogen. CDPK, calcium dependent protein kinase; N, nucleus.
As shown in Figure 2, our data also reveal the previously unexpected existence of the induction of the receptor of suppressor from Pi. Analysis of this induction in compatible and incompatible interaction between potato and the pathogen shows the delay of the CDPK1 receptor in compatible combinations. The data suggests that the timing after the receptor recognition to signal transduction is important for the rapid occurrence of hypersensitive cell death in a resistance cultivar. Characterization of this induction for the signal proteins, i.e. gp91phox in potato, which appears to be as important as the p67, p47 and p22 proteins and MAPK cascade for the induction of ROS, will probably uncover new recognition mechanisms than animal cells. Future work may also allow the specific switch cascade (SSC) for the induction of hypersensitive response and inhibition of the cell death in host cells. The present data indicate that SdCDPK1 and SdCDPK2 is important candidate to discriminate these SSC phenomena in the cell .
These results strongly suggest that we can modify the receptor sites of the suppressor in plant cell for control to the late blight disease by inhibiting the pathogen toxin primary target site in SSC (Specific Switch Cascade in HR). The study of this is now in preparation to elucidate the mechanisms of hypersensitive cell death in planta.
The authors thank to all the members of Center for Transdisciplinary Research of potato project, Niigata University. We also thank to MESC, Japan, for grant support.
We thank to the grant supports of Intelligent Cosmos Science Foundation to N.F. We also thank to J. Suzuki, M. Hirano, T. Horigome (Niigata University) for phosphorylation assays, K. Igarashi and Tutomu Nakada (Brain Function Center, Niigata University) for discussion of receptor expression analysis. We thank to A. Shirata, M. Kinjo, K. Kato and K. Tomiyama for their encouragement during this work. We also thank to J. Harper (University of Nevada, Reno, USA) and A.J. Anderson (Utah State U, USA), for their suggestions during the experiments.