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Resistance Gene-Dependent Activation of a Calcium-Dependent Protein Kinase in the Plant Defense ResponseTina Romeisa, Pedro Piedrasa, and Jonathan D. G. Jonesaa Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, Norfolk, United Kingdom Correspondence to: Tina Romeis, tina.romeis{at}bbsrc.ac.uk (E-mail), 44-1603-450011 (fax)
In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9–dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca2+ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9–dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.
A plant's successful defense against invading microorganisms depends on early perception of the pathogen and initiation of the appropriate signaling processes to recruit the multicomponent defense response. In gene-for-gene interactions, recognition of pathogens requires a plant resistance (R) gene that confers resistance to specific pathogen races that carry the corresponding avirulence (Avr) gene (
Many R genes that confer resistance to different pathogens have been cloned from various plant species (
The plant defense is triggered by a signaling network of parallel pathways that may be interlinked at single components. The increase in the cytosolic Ca2+ concentration, which occurs within seconds after elicitation, appears to be a master regulator required for many subsequent signaling steps. AOS production, MAP kinase activation, defense gene activation, and phytoalexin production, singly or in combination, were shown to be compromised in the presence of Ca2+-chelating or Ca2+ channel–inhibiting compounds (
In animal systems, protein kinase C isotypes and calmodulin-dependent protein kinases have been characterized in detail as Ca2+ modulators. For example, protein kinase C activity is required for induction of the defense signal–related oxidative burst in macrophages (
CDPKs are a class of serine/threonine protein kinases that are unique to plants and some protists. Conserved in structure, they consist of an N-terminal variable domain, a kinase catalytic domain, and a highly conserved junction domain that functions as autoinhibitor; at the C terminus is a calmodulin-like domain that in most cases contains conserved Ca2+ motifs (
We have established transgenic tobacco plants that express the Cf-9 disease resistance gene from tomato. This gene confers recognition of the corresponding Avr9 elicitor peptide from the fungus Cladosporium fulvum (
Using this system, we can dissect early signaling events in a gene-for-gene interaction. In impaled Cf9 tobacco guard cells, both a rapid Avr9-induced K+ efflux and an inhibition of K+ influx have been demonstrated ( In this study, we report the identification of a CDPK that undergoes an Avr9/Cf-9–dependent transition from a nonelicited to an elicited form. This transition was detected by an in-gel kinase assay with histone as substrate and by using protein gel blot analysis with antibodies to CDPK. In addition, the phosphorylation-dependent conversion into the elicited 70-kD form was accompanied by an increase in CDPK activity. Our system thus allows us to investigate the activation of a CDPK isoform after an in vivo stimulation. Inhibitor studies demonstrated that the identified CDPK is independent of or located upstream from a signaling pathway that is required for AOS synthesis. We discuss CDPK function and potential in vivo targets that may contribute to the Avr9/Cf-9–mediated induction of the plant defense response.
Avr9/Cf-9–Dependent Activation of a CDPK After elicitation with Avr9, a protein kinase of ~70 kD became activated within 5 min. High activity was maintained between 10 and 60 min after elicitation, whereas during the next 2 hr the activity returned to its basal value (Fig 1A). Intriguingly, an inverse change in the pattern of phosphorylation activity was evident at 68 kD. The signal intensity decreased within 5 to 10 min after elicitation with Avr9, remained low, and returned to its basal value only 2 hr later. These changes in protein phosphorylation did not occur after challenge with IF(Avr9-) (Fig 1A and Fig 1D, lane 2). Also, no changes were observed when the nontransformed parental tobacco cell line Petite Havana was treated with IF(Avr9+) (Fig 1D, lane 3) or IF(Avr9-) (lane 4). Chemically synthesized Avr9 peptide was able to induce the CDPK transition in Cf9 cells, indicating that the Avr9 peptide in the intercellular fluid is responsible for the observed changes (Fig 1D, lane 5).
Quantitative analysis (see Methods) revealed an increase of phosphorylation activity at 70 kD from 24% (of total phosphorylation activity at 68 and 70 kD) at time zero to 70% at 15 min after IF(Avr9+) elicitation, whereas in the control experiment with IF(Avr9-), phosphorylation activity remained unaltered at 28% (Fig 2A). Complementary changes were deduced from the phosphorylation signal at 68 kD (Fig 2B). Protein kinase activity decreased from 76% before elicitation to 30% at 15 min after the Avr9 treatment. These experiments were repeated five times; although the overall kinetics remained unchanged, the proportion of phosphorylation activity at the 70-kD signal could vary from 0 to 40% at time zero and from 65 to 100% at 15 min after the addition of Avr9, depending on the cell batch. Interestingly, in contrast to the MAP kinase activation pattern, in which the transient kinase signal clearly peaks at 15 min (
The 68- and 70-kD phosphorylation signals shown in Fig 1A were no longer detectable when the in-gel assay was performed in the presence of 2 mM EGTA (Fig 1B), indicating that both phosphorylation activities are Ca2+ dependent. Also, no protein kinase activity could be seen at 68 or 70 kD when histone was omitted from the gel. This demonstrates that the observed changes in Fig 1A are attributable to phosphorylation of histone used as substrate and not to a kinase autophosphorylation (Fig 1C). Also, with the in-gel kinase protocol used (see Methods), no changes in the protein phosphorylation pattern at 68 and 70 kD were observed when histone was replaced by casein (data not shown).
Ca2+ binding protein kinases, such as CDPKs, have been reported to migrate in gels at different rates in the Ca2+-bound versus the Ca2+-free state (
The Avr9/Cf-9–dependent induction of the 70-kD band paralleled by the decrease in the 68-kD signal was confirmed in a dose–response experiment (Fig 4) in which Cf9 cells were challenged with either IF(Avr9-) or various dilutions of IF(Avr9+) for 15 min. The proportion of the 70-kD kinase increased with Avr9 concentrations (Fig 4A), whereas at the same time, no signal was detectable at 68 kD. An immunoblot with affinity-purified antibodies directed against the calmodulin-like domain of soybean CDPK
The 68- and 70-kD Kinase Activities Reflect Distinct Forms of a Membrane-Anchored CDPK
To distinguish between these two interpretations, we incubated solubilized membrane samples from cells elicited for 15 min with IF(Avr9+) or IF(Avr9-) with nonspecific A cell fractionation study revealed that substantial amounts of both the elicited and nonelicited CDPK forms were localized in the membrane fraction (Fig 3D, lanes 5 and 6) and could be solubilized by treatment with detergent (lanes 7 and 8) but not with 0.5 M NaCl (lanes 9 and 10). Some histone phosphorylation activity at ~70 kD was seen in the soluble fraction as well (Fig 3D, lanes 3 and 4), possibly through the action of a CDPK isoenzyme of similar molecular mass that possesses a constitutive, Avr9-independent phosphorylation activity. Additional CDPK signals could be distinguished from the 68-/70-kD enzyme by protein gel blot analysis after separation on a high-resolution SDS gel (data not shown).
Enzymatic Activities of Elicited and Nonelicited CDPK
Solubilized membrane extracts from cells challenged with or without Avr9 were prepared, and protein kinase activity toward the peptide syntide-2 was determined (Fig 5A and Fig 5B). Syntide-2 was reported previously to be utilized by CDPKs (
Further evidence that the Ca2+-dependent increase in enzymatic activity in the elicited sample was the result of CDPK activity was obtained from studies with various antagonists of protein kinase C and calmodulin, which have been reported also to inhibit CDPKs (Fig 5B;
To further test Avr9/Cf-9–dependent CDPK activation, we separated elicited and nonelicited solubilized membrane extracts on an SDS gel, proteins between 63 and 80 kD, including the 68-/70-kD CDPK, and eluted them from cut gel slices (see Methods), and the eluates were incubated with histone and
Avr9/Cf-9–Dependent CDPK Interconversion and AOS Production To locate the identified CDPK in an intracellular signaling pathway and to investigate whether the enzyme might be required for the Avr9-induced AOS production, we challenged Cf9 cell cultures with IF(Avr9+), IF(Avr9–), or chemically synthesized Avr9 in the presence of various inhibitors, and samples were analyzed 15 min after elicitation (Fig 6, lanes 1 to 8). Elicitation with IF(Avr9+) or chemically synthesized Avr9 induced CDPK transition into the 70-kD form as well as AOS production (Fig 6, bars 1 and 3). No response could be detected with IF(Avr9–) (Fig 6, bars 2); also, no transition was observed 30 min (data not shown) or 60 min (Fig 6, bars 9) after the phosphatase inhibitor cantharidin was added in the absence of elicitor. La3+, an inhibitor of plasma membrane Ca2+ channels, and the protein kinase inhibitor staurosporine compromised both responses (Fig 6, bars 4 and 5), whereas 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of MAP kinase kinases, had no effect on either of them (bar 8).
Diphenyleneiodonium chloride abolished AOS production (
Function of 68-/70-kD CDPK in the Plant Defense Response
The elucidation of a potential cellular function of the 68-/70-kD CDPK requires identification of its in vivo substrates. The enzyme might phosphorylate transcription factors. A pathogen-induced kinase is responsible for the activation of a basic leucine zipper transcription factor G/HBF-1, enabling its binding to the chalcone synthase chs15 promoter from soybean (
Some in vivo substrates of the identified 68-/70-kD CDPK also might be located in the membrane. In vitro experiments with fava bean guard cells demonstrated that a 57-kD CDPK phosphorylated the KAT1 K+ channel (
Another potential target of the Avr9-induced 68-/70-kD CDPK might be the plasma membrane H+-ATPase. Elicitation of Cf9 tobacco cell cultures with Avr9 resulted in changes of H+ fluxes, detectable as media alkalinization, which could be observed within 20 min after elicitation. These fluxes supposedly are accomplished by way of inactivating an H+-ATPase (
Is the 68-/70-kD CDPK Involved in Activation of an NADPH Oxidase?
Previously, a CDPK was postulated to phosphorylate NADPH oxidase subunits in response to pathogen-related stimuli by way of an activation mechanism identical to that described for the animal system (
In Vivo Interconversion of the 68-/70-kD CDPK Forms Involves Phosphorylation According to the results of the in-gel kinase assay, both CDPK forms appeared to be equally active, as judged by the phosphorylation signal intensity in the in-gel kinase assay before and after the transition (Fig 1A). However, the experimental conditions chosen for the in-gel assay would be sufficient to induce an in vitro transition from the nonelicited to the elicited form, which is similar to what was demonstrated in Fig 3B, and subsequently did not allow the comparison of enzymatic parameters between the two CDPK forms. Protein kinase assays with gel-eluted CDPK revealed increased enzymatic activity of the elicited sample compared with the nonelicited sample. The elicited membrane extract also showed a higher in vitro kinase activity with syntide-2 as artificial CDPK substrate than did the nonelicited form. No difference in Ca2+-dependent and EGTA-inhibitable protein kinase activity between elicited and nonelicited extract was detectable, however, when the samples were preincubated with ATP (Fig 5A). These data suggest that Avr9-induced phosphorylation and subsequent interconversion of CDPK forms are accompanied by an increase in CDPK enzyme activity. However, only the cloning of the corresponding gene coding for the 68-/70-kD CDPK and subsequent experiments with either specific antibodies or tagged enzyme versions will allow investigators to differentiate the contribution of this isoenzyme to various biochemical parameters from those contributed by the other CDPK family members.
As shown by elegant biochemical studies with recombinant enzymes, in its resting state, CDPK is autoinhibited by an interaction of a pseudosubstrate site within its junction domain that blocks the active site of the kinase domain (
When we integrated our data obtained with the Avr9/Cf-9 gene-for-gene interaction system into that model for CDPK activation, the following picture could be envisaged. In nonelicited cells, CDPK remains basically in its 68-kD form. Challenge with Avr9 causes an Avr9/Cf-9–mediated increase in the cytosolic free Ca2+ concentration (M. Blatt, A. Grabov, A. Brearley, and J.D.G. Jones, unpublished data). The conformational change allows the enzyme to become a substrate for phosphorylation and within 5 min after elicitation to interconvert from its 68-kD into the 70-kD form. The fact that the Avr9-induced phosphorylation and subsequent transition into the 70-kD form are not inhibited by W-7, neither in vivo (Fig 6, bars 7) nor in vitro (Fig 3B), argues against CDPK autophosphorylation and suggests that this step is catalyzed by an unknown upstream protein kinase. Also, although CDPK autophosphorylation has been reported for recombinant and purified enzymes, we were unable to detect any 68-/70-kD phosphorylation signals in our in-gel kinase assays, even after prolonged exposure. The 70-kD CDPK then phosphorylates its target protein(s). Inactivation of the CDPK, which occurred 60 to 120 min after Avr9 elicitation, may be achieved by way of a protein phosphatase. The transition back to the 68-kD CDPK form was observed in the in-gel assay (Fig 1A) and during protein gel blot analysis (data not shown) in which the 68-kD protein band became detectable again. Alternately, the elicited CDPK became proteolytically degraded, and the increase in 68-kD signal after 90 min was attributable to newly synthesized CDPK. The presence of conserved motifs for protein degradation (PEST sequences; In an alternate scenario, the challenge with Avr9 and the subsequent signaling steps presumably could result in the inactivation of a protein phosphatase, which then would enable the phosphorylated 70-kD CDPK form to accumulate. However, the fact that no CDPK interconversion was observed after addition of the phosphatase inhibitor cantharidin renders this hypothesis less likely.
Function of CDPKs in Multiple Signaling Pathways
We have identified a 68-/70-kD CDPK that becomes activated in a gene-for-gene–dependent manner, suggesting that it participates in the Avr9/Cf-9–mediated signaling to activate the plant defense. According to preliminary reverse genetics experiments in which virus-induced gene silencing was applied for CDPK analysis, the Avr9/Cf-9–dependent hypersensitive response observed in the CDPK-silenced plants was less than in control plants (T. Romeis and J.D.G. Jones, unpublished data). However, only the cloning of the corresponding gene for this specific enzyme and subsequent rigorous biochemical and genetic analyses will allow definition of the specific role of the enzyme in activating the plant defense response. The reverse genetic analysis of CDPK isoforms was launched by identifying Arabidopsis mutant lines that carry T-DNA insertions (
Tobacco Cell Culture Conditions and Treatments
For elicitation, cells were challenged with 75 µL of intercellular fluid originating from transgenic tobacco that produces the Avr9 peptide apoplastically (IF[Avr9+]) or with control intercellular fluid lacking Avr9 (IF[Avr9-]) (
For studying the effect of various inhibitors, we added the compounds either 5 min before elicitation with Avr9 (0.5 mM lanthanum chloride, 0.8 µM diphenyleneiodonium chloride, or 25 µM staurosporine) or 10 min before elicitation (250 µM N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide [W-7] or 250 µM 2'-amino-3'-methoxyflavone [Calbiochem-Novabiochem, San Diego, CA]). Cantharidin was added at 5 µM, and cells were harvested after 60 min. If not otherwise mentioned, all compounds were purchased from Sigma Chemicals (Poole, UK). The synthetic Avr9 peptide was used at a concentration of 10 nM (
Determination of Active Oxygen Species
Preparation of Protein Extracts The protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Chester, UK) with BSA as a standard.
In-Gel Kinase Assay
The reaction was stopped by transferring the gel into a solution of 5% trichloroacetic acid (w/v) and 1% phosphoric acid (v/v). Unincorporated
In Vitro Interconversion Experiments To induce transition from the nonelicited into the elicited CDPK form, we incubated solubilized membrane extracts (20 µg) from untreated cells resuspended in elution buffer (see above) for 5 min at room temperature with 0, 1, 2.5, 5, 7.5, or 10 µM ATP or with 10 µM ATP plus 2 mM EGTA, 250 µM W-7, or 250 µM N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5). Reactions were stopped by the addition of sample buffer and analyzed by the in-gel kinase assay.
Cell Fractionation
In Vitro Kinase Assays
For kinase assays with gel-purified CDPK, membrane extracts were separated on an SDS gel as in the in-gel assay protocol (above) except that no kinase substrate was incorporated. After incubation in buffer E, gel slices containing proteins between 63 and 80 kD were cut from the rest of the gel, macerated with a pestle, and incubated with two volumes of buffer E for 15 min at room temperature. After centrifugation for 5 min at 15,000g and room temperature in a microcentrifuge, the supernatant was used for kinase assays. To determine kinase activity, we incubated 40 µL of reaction mixture in buffer E containing 30 µL of enzyme supernatant (corresponding originally to 16 µg of solubilized membrane extract), 100 µg/mL histone, and 0.19 MBq (5 µCi) of
Immunoblotting
We thank Alice Harmon (Gainesville, FL) for providing the CDPK antibody. We are grateful to Matthew Smoker for the propagation of cell cultures and to Alan Cavill for assistance with the figures. This work was supported by the Gatsby Charitable Foundation and the Calcium and Activated Oxygens as Signals for Stress Tolerance project (Grant No. BIO-96-0101). T.R. was supported by a European Molecular Biology fellowship. Received November 29, 1999; accepted March 8, 2000.
Blatt, M.R., Grabov, A., Brearley, J., Hammond-Kosack, K., and Jones, J.D.G. (1999) K+ channels of Cf-9-transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9. Plant J. 19:453-462[CrossRef][Web of Science][Medline]. Blumwald, E., Aharon, G.S., and Lam, B.C.-H. (1998) Early signal transduction pathways in plant–pathogen interactions. Trends Plant Sci. 3:342-346[CrossRef]. Bögre, L., Oláh, Z., and Dudits, D. (1988) Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation. Plant Sci. 85:135-144. Bolwell, G.P. (1999) Role of active oxygen species and NO in plant defense responses. Curr. Opin. Plant Biol. 2:287-294[CrossRef][Web of Science][Medline]. Camoni, L., Harper, J.H., and Palmgren, M.G. (1998a) 14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK). FEBS Lett. 430:381-384[CrossRef][Web of Science][Medline]. Camoni, L., Fullone, M.R., Marra, M., and Aducci, P. (1998b) The plasma membrane H+-ATPase from maize roots is phosphorylated in the C-terminal domain by a calcium-dependent protein kinase. Physiol. Plant. 104:549-555[CrossRef].
Chandra, S., and Low, P.S. (1997) Measurement of Ca2+ fluxes during elicitation of the oxidative burst in aequorin-transformed tobacco cells. J. Biol. Chem. 272:28274-28280 Chung, H.-J., Sehnke, P.C., and Ferl, R.J. (1999) The 14-3-3 proteins: Cellular regulators of plant metabolism. Trends Plant Sci. 4:367-371[CrossRef][Web of Science][Medline]. Després, C., Subramaniam, R., Matton, D.P.R., and Brisson, N. (1995) The activation of the potato PR-10a gene requires the phosphorylation of the nuclear factor PBF-1. Plant Cell 7:589-598[Abstract]. De Wit, P.J.G.M. (1997) Pathogen avirulence and plant resistance: A key role for recognition. Trends Plant Sci. 2:452-458[CrossRef]. Dröge-Laser, W., Kaiser, A., Lindsay, W.P., Halkier, B.A., Loake, G.J., Doerner, P., Dixon, R.A., and Lamb, C. (1997) Rapid stimulation of a soybean protein-serine kinase which phosphorylates a novel bZIP DNA-binding protein, G/HBF-1, during the induction of early transcription-dependent defenses. EMBO J. 16:726-738[CrossRef][Web of Science][Medline]. Durrant, W.E., Rowland, O., Piedras, P., Hammond-Kosack, K.E., and Jones, J.D.G. (2000) cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. Plant Cell 12, in press.. Ellard-Ivey, M., Hopkins, R.B., White, T.J., and Lomax, T.L. (1999) Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.). Plant Mol. Biol. 39:199-208[CrossRef][Web of Science][Medline]. Ellis, J., and Jones, D. (1998) Structure and function of proteins controlling strain-specific pathogen resistance in plants. Curr. Opin. Plant Sci. 1:288-293. Flor, H.H. (1971) Current status of gene-for-gene concept. Annu. Rev. Phytopathol. 9:275-296[CrossRef][Web of Science].
Fuglsang, A.T., Visconti, S., Drumm, K., Jahn, T., Stensballe, A., Mattei, B., Jensen, O.N., Aducci, P., and Palmgren, M.G. (1999) Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947). J. Biol. Chem. 274:36774-36780 Gelli, A., Higgins, V.J., and Blumwald, E. (1997) Activation of plant plasma membrane Ca2+-permeable channels by race-specific fungal elicitors. Plant Physiol. 113:269-279[Abstract]. Hammond-Kosack, K.E., and Jones, J.D.G. (1997) Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607[CrossRef][Web of Science].
Hammond-Kosack, K.E., Tang, S., Harrison, K., and Jones, J.D.G. (1998) The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the complementary fungal avirulence gene product Avr9. Plant Cell 10:1251-1266 Harmon, A.C. (1997) The calcium connection. Trends Plant Sci. 1:121-122[CrossRef].
Harmon, A.C., Putnam-Evans, C., and Cormier, M.J. (1987) A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol. 83:830-837 Hong, Y., Takano, M., Liu, C.-M., Gasch, A., Chye, M.L., and Chua, N.-H. (1996) Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana. Plant Mol. Biol. 30:1259-1275[CrossRef][Web of Science][Medline]. Hrabak, E.M., Dickmann, L.J., Satterlee, J.S., and Sussman, M.R. (1996) Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana. Plant Mol. Biol. 31:405-412[CrossRef][Web of Science][Medline]. Huang, J.-F., Teyton, L., and Harper, J.F. (1996) Activation of a Ca2+-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain. Biochemistry 35:13222-13230[CrossRef][Medline].
Iwata, Y., Muriyama, M., Nakakita, M., Kojima, H., Ohto, M., and Nakamura, K. (1998) Characterization of a calcium-dependent protein kinase of tobacco leaves that is associated with the plasma membrane and is inducible by sucrose. Plant Cell Physiol. 39:1176-1183
Jones, D.A., Thomas, C.M., Hammond-Kosack, K.E., Balint-Kurti, P.J., and Jones, J.D.G. (1994) Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging. Science 266:789-793
Keller, T., Damude, H.G., Werner, D., Doerner, P., Dixon, R.A., and Lamb, C. (1998) A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs. Plant Cell 10:255-266
Krysan, P.J., Young, J.C., Tax, F., and Sussman, M.R. (1996) Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport. Proc. Natl. Acad. Sci. USA 93:8145-8150 Lamb, C., and Dixon, R.A. (1997) The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:251-275[CrossRef][Web of Science]. Lee, J.-Y., Yoo, B.-C., and Harmon, A.C. (1998) Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean. Biochemistry 37:6801-6809[CrossRef][Medline].
Li, J., Lee, Y.-R., and Assmann, S.M. (1998) Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel. Plant Physiol. 116:785-795
Ligterink, W., Kroj, T., zur Nieden, U., Hirt, H., and Scheel, D. (1997) Receptor-mediated activation of a MAP kinase in pathogen defense of plants. Science 276:2054-2057 Majumdar, S., Kane, L.H., Rossi, M.W., Volpp, B.D., Nauseef, W.M., and Korchak, H.M. (1993) Protein kinase C isotypes and signal-transduction in human neutrophils: Selective substrate specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC. Biochim. Biophys. Acta 1176:276-286[Medline]. Martin, G.B. (1999) Functional analysis of plant disease resistance genes and their downstream effectors. Curr. Opin. Plant Biol. 2:273-279[CrossRef][Web of Science][Medline].
Martin, G.B., Brommonschenkel, S.H., Chunwongse, J., Frary, A., Ganal, M.W., Spivey, R., Wu, T., Earle, E.D., and Tanksley, S.D. (1993) Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262:1432-1436 Mithöfer, A., Fliegmann, J., and Ebel, J. (1999) Isolation of a French bean (Phaseolus vulgaris L.) homolog to the beta-glucan elicitor-binding protein of soybean (Glycine max L.). Biochim. Biophys. Acta 1418:127-132[Medline]. Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497[CrossRef]. Nennstiel, D., Scheel, D., and Nürnberger, T. (1998) Characterization and partial purification of an oligopeptide elictor receptor from parsley (Petroselinum crispum). FEBS Lett. 431:405-410[CrossRef][Medline]. Nürnberger, T., Nennstiel, D., Jabs, T., Sacks, W.R., Hahlbrock, K., and Scheel, D. (1994) High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses. Cell 78:449-460[CrossRef][Web of Science][Medline]. Pei, Z.-M., Ward, J.M., Harper, J.F., and Schroeder, J.I. (1996) A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK. EMBO J. 15:6564-6574[Web of Science][Medline]. Perkins, R.S., Lindsay, M.A., Barnes, P.J., and Giembycz, M.A. (1995) Early signalling events implicated in leukotriene B4-induced activation of the NADPH oxidase in eosinophils: Role of Ca2+, protein kinase C and phospholipases C and D. Biochem. J. 310:795-806. Piedras, P., Hammond-Kosack, K.E., Harrison, K., and Jones, J.D.G. (1998) Rapid, Cf-9 and Avr9-dependent, production of active oxygen species in tobacco suspension cultures. Mol. Plant-Microbe Interact. 11:1155-1166[Web of Science]. Rechsteiner, M., and Rogers, S.W. (1996) PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21:267-271[CrossRef][Web of Science][Medline]. Richberg, M.H., Aviv, D.H., and Dangl, J.L. (1998) Dead cells do tell tales. Curr. Opin. Plant Biol. 1:480-485[CrossRef][Web of Science][Medline]. Roberts, D.M., and Harmon, A.C. (1992) Calcium-modulated proteins: Targets of intracellular calcium signals in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414[CrossRef][Web of Science].
Roberts, M.R., and Bowles, D. (1999) Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants. Plant Physiol. 119:1243-1250
Romeis, T., Piedras, P., Zhang, S., Klessig, D.F., Hirt, H., and Jones, J.D.G. (1999) Rapid Avr9- and Cf-9–dependent activation of MAP kinases in tobacco cell cultures and leaves: Convergence of resistance gene, elicitor, wound, and salicylate responses. Plant Cell 11:273-287 Satterlee, J.S., and Sussman, M.R. (1998) Unusual membrane-associated protein kinases in higher plants. J. Membr. Biol. 164:205-213[CrossRef][Web of Science][Medline].
Schaller, A., and Oecking, C. (1999) Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11:263-272 Schaller, G.E., Harmon, A.C., and Sussman, M.R. (1992) Phosphorylation of the plasma membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase. Biochemistry 31:1721-1727[CrossRef][Medline]. Scheel, D. (1998) Resistance response physiology and signal transduction. Curr. Opin. Plant Biol. 1:305-310[CrossRef][Web of Science][Medline]. Schwacke, R., and Hager, A. (1992) Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+ and protein-kinase activity. Planta 187:136-141. Sehnke, P.C., and Ferl, R.J. (1996) Plant metabolism: Enzyme regulation by 14-3-3 proteins. Curr. Biol. 6:1403-1405[CrossRef][Web of Science][Medline].
Sheen, J. (1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274:1900-1902
Song, W.-Y., Wang, G.-L., Chen, L.-L., Kim, H.-S, Pi, L.-Y., Holsten, T., Gardner, J., Wang, B., Zhai, W.-X., Zhu, L.-H., Fauquet, C., and Ronald, P. (1995) A receptor kinase-like protein encoded by the rice disease resistance gene Xa21. Science 270:1804-1806
Staskawicz, B.J., Ausubel, F.M., Baker, B.J., Ellis, J.G., and Jones, J.D.G. (1995) Molecular genetics of plant disease resistance. Science 268:661-667 Subramaniam, R., Després, C., and Brisson, N. (1997) A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato. Plant Cell 9:653-664[Abstract]. Tähtiharju, S., Sangwan, V., Monroy, A.F., Dhindsa, R.S., and Borg, M. (1997) The induction of kin genes in cold-acclimating Arabidopsis thaliana. Evidence for a role for calcium. Planta 203:442-447[CrossRef][Web of Science][Medline]. Takahashi, K., Isobe, M., and Muto, S. (1997) An increase in cytosolic calcium ion concentration precedes hypoosmotic shock-induced activation of protein kinases in tobacco suspension culture cells. FEBS Lett. 401:202-206[CrossRef][Web of Science][Medline]. Torres, M.A., Onouchi, H., Hamada, S., Machida, C., Hammond-Kosack, K.E., and Jones, J.D.G. (1998) Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J. 14:365-370[CrossRef][Web of Science][Medline].
Umemoto, N., Kakitani, M., Iwamatsu, A., Yoshikawa, N., Yamaoka, N., and Ishida, I. (1997) The structure and function of a soybean ß-glucan-elicitor-binding protein. Proc. Natl. Acad. Sci. USA 94:1029-1034 Urao, T., Katagiri, T., Mizoguchi, T., Yamaguchi-Shinozaki, K., Hayashida, N., and Shinozaki, K. (1994) Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana. Mol. Gen. Genet. 244:331-340[Web of Science][Medline]. Van den Ackerveken, G.F.J.M., Van Kan, J.A.L., and De Wit, P.J.G.M. (1992) Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis. Plant J. 2:359-366[Web of Science][Medline]. Xing, T., Higgins, V.J., and Blumwald, E. (1996) Regulation of plant defense response to fungal pathogens: Two types of protein kinases in the reversible phosphorylation of the host plasma membrane H+-ATPase. Plant Cell 8:555-564[Abstract]. Xing, T., Higgins, V.J., and Blumwald, E. (1997) Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH-oxidase to the plasma membrane of tomato cells. Plant Cell 9:249-259[Abstract].
Yang, Y., Shah, J., and Klessig, D.F. (1997) Signal perception and transduction in plant defense responses. Genes Dev. 11:1621-1639 Yoo, B.-C., and Harmon, A.C. (1996) Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain. Biochemistry 35:12029-12037[CrossRef][Medline]. Yoon, G.M., Cho, H.S., Ha, H.J., Liu, J.R., and Pai Lee, H.-S. (1999) Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. Plant Mol. Biol. 39:991-1001[CrossRef][Web of Science][Medline].
Zhang, S., and Klessig, D.F. (1998) N resistance gene–mediated de novo synthesis and activation of a tobacco MAP kinase by TMV infection. Proc. Natl. Acad. Sci. USA 95:7433-7438
Zhang, S., Du, H., and Klessig, D.F. (1998) Activation of the tobacco SIP kinase by both a cell wall–derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp. Plant Cell 10:435-449
Zimmermann, S., Nürnberger, T., Frachisse, J.-M., Wirtz, W., Guern, J., Hedrich, R., and Scheel, D. (1997) Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense. Proc. Natl. Acad. Sci. USA 94:2751-2755
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ABSTRACT
INTRODUCTION
phosphatase for the times indicated, and samples were analyzed using the in-gel kinase assay.
revealed that both the 68- and 70-kD enzymes cross-react with the antibody (
-32P-ATP for 5 min in the presence of the previously mentioned inhibitors. Histone was chosen as the substrate because gel eluate supernatants prevented binding of the syntide-2 peptide to phosphocellulose paper. Phosphorylation of histone was analyzed by SDS gel electrophoresis and autoradiography, and equal loading was confirmed by protein staining (
