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Receptor-Mediated Activation of a MAP Kinase in Pathogen Defense of Plants

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Science  27 Jun 1997:
Vol. 276, Issue 5321, pp. 2054-2057
DOI: 10.1126/science.276.5321.2054

Abstract

Parsley cells recognize the fungal plant pathogenPhytophthora sojae through a plasma membrane receptor. A pathogen-derived oligopeptide elicitor binds to this receptor and thereby stimulates a multicomponent defense response through sequential activation of ion channels and an oxidative burst. An elicitor-responsive mitogen-activated protein (MAP) kinase was identified that acts downstream of the ion channels but independently or upstream of the oxidative burst. Upon receptor-mediated activation, the MAP kinase is translocated to the nucleus where it might interact with transcription factors that induce expression of defense genes.

Plants react to pathogen attack with a variety of defense responses, including transcriptional activation of defense genes, accumulation of phytoalexins and pathogen-related (PR) proteins, and impregnation of the cell wall with phenolic substances and specific proteins (1). Infection of parsley leaves with spores from the soybean pathogen Phytophthora sojae leads to small necrotic lesions resulting from hypersensitive cell death, incorporation of phenolic compounds into, and apposition of callose onto, cell walls at the infection site, as well as local and systemic activation of defense-related genes and secretion of fouranocoumarin phytoalexins into the infection droplet (2, 3). Cultured parsley cells show most of these defense reactions when treated with elicitor preparations from the fungus and have been used as a model system to study the plant-pathogen interactions (4-7). An extracellular 42-kD fungal glycoprotein was identified in these preparations as the principal elicitor of the multicomponent defense response in parsley cells (6). An oligopeptide fragment of 13 amino acids in length (Pep13) within this glycoprotein is necessary and sufficient to induce the same reactions as the intact glycoprotein (7, 8). Pep13 specifically interacts with a plasma membrane target site in the plant and initiates a signal transduction cascade leading to the transient activation of plant defense genes and the accumulation of phytoalexins (7).

Elicitor signal transduction in parsley cells involves Ca2+-dependent transient changes in protein phosphorylation, suggesting the participation of protein kinases in defense gene activation (9). To detect specific protein kinases that catalyze such reactions, we treated cultured parsley cells with Pep25, a larger fragment of the elicitor that includes the Pep13 sequence and induced an identical response but was more stable in the culture medium than Pep13 (7). A protein kinase that phosphorylated myelin basic protein (MBP) was activated within 5 min after elicitor treatment (Fig.1A). From its relative mobility on SDS-polyacrylamide gels, the apparent molecular mass of this enzyme was estimated to be ∼45 kD, similar to that of known plant mitogen-activated protein (MAP) kinases (10-15).

Figure 1

A specific MAP kinase is activated by elicitor. Suspension cultured parsley cells were treated with the synthetic peptide elicitor, Pep25 (175 nM), or water alone. Cell extracts were prepared at 0, 1, 3, 5, 10, 20, and 40 min after initiation of elicitor treatment in extraction buffer [25 mM tris-HCl (pH 7.5), 15 mM MgCl2, 15 mM EGTA, 75 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% NP-40, 15 mMp-nitrophenylphosphate, 60 mM β-glycerophosphate, 0.1 mM NaVO3, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml each of leupeptine and aprotinin]. After centrifugation at 100,000g for 1 hour, the cleared supernatant was used. (A) In-gel protein kinase assay. Each lane contained 20 μg of total protein from cell extracts, which was separated by SDS–polyacrylamide gel electrophoresis (PAGE). MBP (0.5 mg/ml) was used as a substrate and was polymerized in the polyacrylamide gel. After protein renaturation, the kinase reactions were carried out in the gel with [γ-32P]adenosine 5′-triphospate (ATP) as described (12). (B) Immunoprecipitation of an elicitor-responsive MAP kinase. Cell extracts containing 100 μg of total protein were immunoprecipitated with 5 μg of protein A–purified M7, M11, and M14 antibodies that were produced against synthetic peptides encoding the COOH-terminal 10 amino acids of the alfalfa MMK4, MMK2, and MMK3 MAP kinases, respectively (12). The immunoprecipitated proteins were washed three times with wash buffer I (20 mM tris-HCl, 5 mM EDTA, 100 mM NaCl, 1% Triton X-100), once with the same buffer but containing 1 M NaCl, and once with kinase buffer [20 mM Hepes (pH 7.5), 15 mM MgCl2, 5 mM EGTA, 1 mM DTT]. Kinase reactions of the immunoprecipitated proteins were performed in 15 μl of kinase buffer containing MBP (0.5 mg/ml), 0.1 mM ATP, and 2 μCi of [γ-32P]ATP at room temperature for 30 min. The reactions were stopped by the addition of SDS sample buffer. The phosphorylation of MBP was analyzed by autoradiography after SDS-PAGE.

To determine whether the elicitor-activated protein kinase might belong to the class of MAP kinases, we incubated the same cell extracts used for activity assays with three different antisera—M7, M11, and M14—that were raised against synthetic peptides representing the COOH-terminal 10 amino acids of the alfalfa MMK4, MMK2, and MMK3 MAP kinases, respectively (12). Elicitor treatment exclusively activated a protein kinase that was immunoprecipitated by the M7 antiserum (Fig. 1B). The similarity of the activation kinetics in the kinase and immunoprecipitation assays indicate that elicitor treatment activates a specific MAP kinase pathway in parsley cells.

Because the M7 antiserum specifically recognized the elicitor-responsive MAP kinase from parsley, a radiolabeled fragment of the alfalfa MMK4 gene was used to screen a cDNA library prepared from RNA isolated from cultured parsley cells. A 1.6-kb cDNA fragment was isolated that contained an open reading frame of 1113 nucleotides potentially encoding a protein of 371 amino acids and a molecular mass of 43 kD. The deduced amino acid sequence is most similar to those of the MAP kinases from Arabidopsis (MPK3, 83%) (11), alfalfa (MMK4, 81%) (12), and tobacco (WIPK, 83%) (13). The overall structure of the parsley, tobacco, Arabidopsis, and alfalfa kinases is highly conserved (Fig. 2). DNA gel blot analysis of parsley cells with the radiolabeled kinase cDNA fragment under high-stringency hybridization conditions revealed the parsley kinase to be present as a single-copy gene (16). RNA gel blot analysis of cultured parsley cells with radiolabeled fragments, containing either the coding region or the 3′-untranslated region of the kinase cDNA, showed a severalfold increase of kinase transcript levels within 30 min after elicitor treatment (16). When the parsley cDNA was expressed as a fusion protein with glutathione-S-transferase (GST) inEscherichia coli, the recombinant protein catalyzed its autophosphorylation and phosphorylated MBP. In immunoblots, the GST-MAP kinase fusion protein was exclusively recognized by the M7 antiserum that recognized the elicitor-responsive protein kinase from cultured parsley cells. In contrast, the M11 and M14 antisera did not decorate the parsley kinase fusion protein. These results suggest that the cDNA isolated from parsley cells indeed encodes the elicitor-activated kinase detected in the activity and immunocomplex assays, which we therefore denote ERM kinase for elicitor-responsive MAP kinase.

Figure 2

Primary structure of an elicitor-responsive MAP (ERM) kinase from parsley. The nucleotide and predicted amino acid sequence of the ERM kinase has been deposited with GenBank, DNA Data Base Japan, and European Molecular Biology Laboratory databases (accession number Y12875). The primary sequence of ERM kinase was deduced from the sequence of a cDNA clone isolated by standard methods (28) from a parsley cDNA library constructed in the λ-ZAP vector (Stratagene), by use of a 1.1-kb random primed 32P-labeled DNA probe (megaprime labeling kit, Amersham), representing the near full-length open reading frame of MMK4 (12). The positive clone thus isolated is aligned with its closest homologs, MPK3 from Arabidopsis thaliana (11), MMK4 from Medicago sativa(12), and WIPK from Nicotiana tabacum(13). Shaded areas represent identical sequences. Roman numerals indicate kinase subdomains (29). Conserved phosphorylation sites are marked with an asterisk.

To investigate whether Pep13 activates this kinase through the same receptor that is used for the induction of the other defense responses, we determined ERM kinase activation upon treatment of parsley cells with four different but structurally related elicitor oligopeptides. Pep13 and Pep25, both corresponding to the wild-type sequence of the 42-kD P. sojae glycoprotein elicitor, activated the elicitor-responsive MAP kinase in an identical manner (Figs. 1 and3). Two Pep13 derivatives in which the second (Pep13A2) or the fifth (Pep13A5) amino acid had been replaced by alanine did not activate ERM kinase, whereas a derivative with an alanine substitution in position 12 (Pep13A12) was as active as Pep13 and Pep25 (Fig. 3). These results correlate well with binding and elicitor studies (7) with the same Pep13 derivatives, which showed that Pep13A12competes with binding of Pep13 to its receptor and elicits a normal pattern of defense reactions. In contrast, Pep13A2 and Pep13A5 were inactive in both assays, indicating that the Pep13 receptor that initiates the multicomponent defense response is also engaged in ERM kinase activation.

Figure 3

ERM kinase is exclusively activated by active peptide elicitor. Suspension-cultured parsley cells were treated with the synthetic peptide elicitor Pep13 (50 nM), and with inactive (Pep13A2, 50 nM, and Pep13A5, 50 nM) and active (Pep13A12, 50 nM) derivatives. Cell extracts were prepared at 0, 1, 3, 5, 10, 20, 40, and 60 min after elicitor treatment. Cell extracts containing 100 μg of total protein were immunoprecipitated with M7 antibody. The kinase activity of the immunocomplexes was determined by in vitro kinase assays with MBP as substrate as described in Fig. 1B.

Binding of Pep13 to its receptor induces phytoalexin synthesis, defense gene activation, in vivo phosphorylation of proteins, and the production of an oxidative burst, which all depend on the integrity of specific ion channels that mediate rapid ion fluxes across the cell membrane in response to elicitor (7, 9,17, 18). To investigate whether ERM kinase activation also depended on the activity of these ion channels, we incubated parsley cells with the ion channel blocker, anthracene-9-carboxylate (A9C), which inhibits the elicitor-stimulated ion fluxes, thereby blocking all subsequent defense responses (17). Under these conditions, Pep13 activation of the ERM kinase was completely inhibited, indicating that ion channel activation was also necessary for this reaction (Fig. 4). Amphotericin B, which mimics elicitor-induced ion fluxes and thereby induces the full set of defense responses (17), also activates the ERM kinase in the absence of elicitor (Fig. 4). Activation of ERM kinase (Fig. 4), ion fluxes, and the oxidative burst (17) by amphotericin B all occur after a delay of about 30 min. Thus, ERM kinase activation depends on the state of specific ion channels, and activation of these channels is necessary and sufficient for ERM kinase activation as it is for the induction of the other elicitor responses in this system.

Figure 4

ERM kinase activation depends on elicitor-stimulated ion-channel activity but not on an oxidative burst. Suspension-cultured parsley cells were preincubated with 100 μM of the ion-channel blocker anthracene-9-carboxylate (A9C), with 50 μM of the polyene antibiotic, amphotericin B (Amph), or with 50 μM of diphenylene iodonium (DPI), an inhibitor of the oxidative burst, followed by addition of Pep13 (50 nM) to A9C- and DPI-treated cells 30 min later. After the indicated periods of treatment the cells were harvested, and total protein was extracted and analyzed by (A) in-gel MBP kinase assays and (B) M7 antibody–precipitated immunocomplex kinase assays as described in Fig. 1.

The elicitor-stimulated production of reactive oxygen species is thought to be catalyzed by an NADH [nicotinamide adenine dinucleotide (reduced)] or NADPH [nicotinamide adenine dinucleotide phosphate (reduced)] oxidase that is inhibited by diphenylene iodonium (DPI) (19). In elicitor-treated parsley cells DPI blocked the oxidative burst, defense gene activation, and phytoalexin accumulation without affecting ion fluxes (17). Together with the results from gain-of-function experiments with KO2, which stimulated phytoalexin production in the absence of elicitor, this placed the oxidative burst downstream of the ion channels within the elicitor signal transduction cascade (17). Pep13 activation of the ERM kinase was not inhibited by DPI, indicating that this kinase acts either upstream or independently of the oxidative burst (Fig. 4).

Certain MAP kinases are translocated into the nucleus upon activation, where they may catalyze phosphorylation of transcription factors and thereby regulate gene transcription (20-22). The subcellular location of ERM kinase was determined with M7 antiserum in immunofluorescence microscopy before and after treating parsley cells with Pep25 elicitor. Within 3 to 10 min after Pep25 treatment, ERM kinase was translocated into the nucleus (Fig. 5C). Because no nuclear localization signal is present in the ERM kinase, translocation of the activated kinase into the nuclear compartment may be initiated by its interaction with another protein, perhaps a transcription factor. In parsley, several elicitor-responsive genes have been identified and have led to the identification of cis elements and transcription factors that may be involved in mediating pathogen-induced transcription (1,23). Although it has not yet been shown that phosphorylation of these transcription factors is responsible for elicitor-induced transcription of PR genes, the elicitor-induced relocation of ERM kinase into the nucleus might link cytosolic signal transduction to nuclear activation of plant defense genes.

Figure 5

ERM kinase is translocated to the nucleus upon elicitor activation. Cultured parsley cells were treated with Pep25 (175 nM) and harvested before (A and B) or 5 min after initiation of treatment (C and D). Sections (2 μm) across cell clusters were fixed with 4% formaldehyde, embedded in polyethylene glycol (30), and either stained with the M7 antiserum (A and C), specifically recognizing ERM kinase, or with 4′,6′-diamidino-2-phenylindole (DAPI) (B and D) to visualize nuclei. Biotinylated secondary antibody, streptavidin–horseradish peroxidase, and fluorescein tyramid reagent were used to visualize the primary antibody bound to ERM kinase according to the manufacterer's instructions (Tyramid Signal Amplification Systems, TSA-Direct-Green, Du Pont, NEN, Boston, Massachusetts). After treatment with Pep25 most nuclei were decorated by the M7 antibody (B), whereas no or little staining was detectable in untreated cells (A), in cells treated with water instead of Pep25, or when the M7 antibody was replaced by preimmune serum. Bar (D), 50 μm.

MAP kinases were first found in yeast and animals, where they participate in signaling cascades linking plasma membrane receptors that perceive extracellular signals to a variety of cellular response mechanisms (24, 25). The MAP kinases known in plants are activated by environmental stresses and plant hormones (26, 27). Our results demonstrate posttranslational and transcriptional activation of a plant MAP kinase within a signal transduction pathway that mediates the response to a pathogen. Activation of ERM kinase follows input from receptor-regulated ion channels of the plasma membrane and precedes or parallels the formation of O2 radicals, which in turn activate defense genes and phytoalexin synthesis (17).

  • * To whom correspondence should be addressed. E-mail: hehi{at}gem.univie.ac.at

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