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Originally published In Press as doi:10.1074/jbc.M313388200 on January 23, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14819-14827, April 9, 2004
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LeMPK3 Is a Mitogen-activated Protein Kinase with Dual Specificity Induced during Tomato Defense and Wounding Responses*

Maya Mayrose, Arale Bonshtien, and Guido Sessa{ddagger}

From the Dept. of Plant Sciences, Tel-Aviv University, 69978 Tel-Aviv, Israel

Received for publication, December 8, 2003 , and in revised form, January 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitogen-activated protein (MAP) kinase cascades are readily activated during the response of plants to avirulent pathogens or to pathogen-derived elicitors. Here we show that the tomato MAP kinase LeMPK3 is specifically induced at the mRNA level during elicitation of the hypersensitive response in resistant plants infected by avirulent strains of the phytopathogenic bacteria Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato, as well as upon treatment with the fungal elicitor ethylene-inducing xylanase. LeMPK3 gene expression was also induced very rapidly by mechanical stress and wounding much earlier than upon pathogen infection, but not in response to the defense-related plant hormones ethylene and jasmonic acid. Moreover, in resistant tomato plants infected by X. campestris pv. vesicatoria, transcript accumulation was followed by an increase in LeMPK3 kinase activity. Biochemical characterization of a glutathione S-transferase-LeMPK3 fusion protein revealed that the LeMPK3 MAP kinase autophosphorylates in vitro mainly on tyrosine and less so on threonine and serine, whereas it phosphorylates myelin basic protein on serine and threonine. In vitro phosphorylation of a poly-(Glu-Tyr) copolymer by LeMPK3 demonstrated its capability to phosphorylate tyrosine residues on substrates as well. By mutagenesis and phosphoamino acid analysis, Tyr-201 in the kinase activation domain was identified as the main LeMPK3 autophosphorylation site and as critical for kinase activity. Finally, LeMPK3 autophosphorylation showed a preference for Mn2+ cations and proceeded via an intramolecular mechanism with an estimated Km value for ATP of 9.5 µM. These results define LeMPK3 as a MAP kinase with dual specificity and strongly suggest that it represents a convergence point for different signaling pathways inducing the activation of defense responses in tomato.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plants resist pathogen challenge both with preformed defenses, such as physical barriers or antimicrobial secondary metabolites, and by inducing efficient defense responses. Typical defense responses observed in resistant plants include the elicitation of a rapid and localized cell death, known as hypersensitive response (HR),1 production of reactive oxygen species, and synthesis of pathogenesis-related proteins and antimicrobial phytoalexins (1, 2). The activation of defense responses is mediated by the specific recognition of the invading pathogen either by the binding of a non race-specific elicitor to its receptor or by a gene-for-gene interaction between a plant resistance gene (R) and a pathogen avirulence gene (avr) (3, 4). Successful pathogen recognition triggers the activation of various signaling pathways that lead to cellular responses (4).

In recent years the study of defense signaling events activated by general elicitors and Avr proteins revealed that MAP kinase modules are involved in the induction of defense responses (5, 6). In plants, as in yeast and mammals, MAP kinase signaling cascades enable the cell to transduce extracellular stimuli into intracellular responses. In a typical three-tiered MAP kinase cascade, a MAP kinase is activated by a MAP kinase kinase (MAPKK) through dual phosphorylation of threonine and tyrosine residues of a TXY motif located between subdomains VII and VIII of the kinase catalytic domain. In turn, MAPKK are activated by phosphorylation of an upstream MAP kinase kinase kinase. Such three-kinase modules function as molecular switches that turn on expression of specific sets of genes, resulting in the activation of cellular responses.

A comprehensive analysis of the Arabidopsis genome sequence identified a group of at least 20 genes encoding putative MAP kinases that were further classified into four distinct major homology groups (7, 8). Arabidopsis MAP kinases of group A and their homologs from different plant species have been implicated in the plant response to non-race-specific elicitors. Two members of this group, AtMPK3 and AtMPK6, are part of a recently dissected signaling cascade, which is activated by the bacterial-derived flagellin elicitor and induces general defense responses but not the hypersensitive response (9, 10). In this pathway, binding of flagellin to the leucine-richrepeat receptor kinase FLS2 induces MAP kinase cascades, which activate AtMPK3 and AtMPK6, and leads to the activation of transcription factors of the WRKY family (9). Similarly, in parsley cell cultures PcMPK3 and PcMPK6, homologs of AtMPK3 and AtMPK6, respectively, are activated by a Phytophtora sojae-derived oligopeptide elicitor, which binds to a plasma membrane receptor and induces a broad array of defense responses (11, 12). Activation of four different MAP kinases of group A has also been observed in alfalfa cell cultures in response to distinct pathogen-derived elicitors (13, 14).

The tobacco SIPK and WIPK, which are homologs of Arabidopsis AtMPK6 and AtMPK3, respectively, are the only MAP kinases to date that have been shown to be activated both by general elicitors and by the interaction of race-specific avirulence proteins and their cognate resistance proteins (6, 15–17). In tobacco plants activation of SIPK and WIPK was found to occur in an N-gene-dependent manner after tobacco mosaic virus inoculation and in a Cf9-dependent manner after inoculation with the fungal avirulence protein Avr9 (16, 17). Clear evidence for the central importance of MAP kinase cascades in R-gene-mediated plant disease resistance has been recently attained in gain-of-function and silencing experiments. Transient expression of constitutively active mutants of SIPK and its upstream MAPKK NtMEK2 resulted in elicitation of the hypersensitive response and of several associated defense-related responses, including induction of defense genes and ethylene biosynthesis (18–20). Conversely, simultaneous reduction in the expression of NtMEK2, SIPK and WIPK, or of SIPK and WIPK only by virus-induced gene silencing resulted in attenuation of resistance to tobacco mosaic virus and to Pseudomonas cichorii, respectively (21, 22). In addition, tomato resistance to Pseudomonas syringae pv. tomato was compromised by virus-induced gene silencing of genes homologous to the MEK1 and MEK2 MAPKKs and to the NTF6 and WIPK MAP kinases (23). Finally, virus-induced gene silencing of the MAPKK kinase NPK1 in Nicotiana benthamiana plants interfered with the function of several disease resistance genes (24).

MAP kinase homologs were recently isolated from tomato plants, and their inducibility by different elicitors and stress signals were analyzed (25). LeMPK1 and LeMPK2, which are highly homologous to the Arabidopsis AtMPK6 and tobacco SIPK, were activated by UV-B radiation as well as in response to systemin and to several oligosaccharide elicitors. An additional MAP kinase, LeMPK3, which shows high homology to the Arabidopsis AtMPK3 and tobacco WIPK, was activated only by UV-B.

In this report we provide evidence that LeMPK3 may represent a convergence point of different signaling pathways that lead to elicitation of the hypersensitive response and which are mediated either by resistance proteins and their corresponding race-specific elicitors or by cultivar-specific receptors and their corresponding elicitors. Evidence suggesting the involvement of LeMPK3 in the tomato response to mechanical stress and wounding is also presented. Moreover, by a detailed in vitro characterization of the kinase activity of a glutathione S-transferase-LeMPK3 fusion protein, we provide important insights about LeMPK3 biochemical properties, which define it as a MAP kinase with dual specificity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Treatments—The following tomato (Lycopersicum esculentum) cultivars were used in the experiments Hawaii 7981 (26), Hawaii 7998 (27), Rio Grande PtoR (carrying the Pto resistance gene), and Rio Grande PtoS (lacking the Pto gene) (28). For pathogen treatments, 6-week-old plants were inoculated by vacuum infiltration for 1 min with bacterial cultures prepared as follows. Xanthomonas campestris pv. vesicatoria 110C strain T1 (Xcv T1) and 97–2 strain T3 (Xcv T3) were grown overnight at 28 °C in NYGB liquid medium (0.5% (w/v) Bacto Proteose Peptone, 0.3% (w/v) Yeast extract, and 4% (v/v) glycerol). Bacteria were pelleted by centrifugation, washed twice with 10 mM MgCl2, and diluted to a concentration of 108 cfu/ml in 10 mM MgCl2 and 0.005% (v/v) Silwett-L77. Bacterial cultures of P. syringae pv. tomato (Pst) strain T1 expressing avrPto (T1A) were grown overnight at 28 °C in King's B liquid medium with the addition of 100 mg/liter rifampicin and 25 mg/l kanamycin and prepared for inoculation as described for Xcv strains. For treatment with the ethylene-inducing xylanase (EIX) elicitor from Trichoderma viride (29), plants were vacuum-infiltrated with a solution containing 0.5 mg/l EIX (Sigma-Aldrich) and 0.005% (v/v) Silwett-L77. For ethylene treatment, plants were placed in a plexiglass chamber where ethylene was injected to a final concentration of 20 ppm. For jasmonic acid treatment plants were sprayed with a solution containing 100 µM jasmonic acid (Sigma-Aldrich) in 0.1% (v/v) ethanol and 0.005% (v/v) Silwett-L77. Wounding treatment was carried out by gently rubbing the surface of leaves with wet Carborundum powder.

Northern Blot Analysis—Tissues were harvested from treated plants at different time points as indicated in each experiment and used to extract RNA with the SV Total RNA Isolation kit (Promega). Total RNA (15 µg from each sample) was denatured at 60 °C, fractionated on a 1% formaldehyde-agarose gel, and blotted onto a Hybond-N membrane (Amersham Biosciences) in 10x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate) for 16 h. Blots were hybridized with probes labeled with [{alpha}-32P]dCTP (Amersham Biosciences) by using standard random priming methods. Hybridizations were performed at 60 °C for 16 h in a solution containing 50 mM NaH2PO4, pH 7.4, 0.75 M NaCl, 2% SDS, 0.1 mg/ml salmon sperm DNA, and 5 mM EDTA. After hybridization, blots were washed for 15 min in 2x SSC and 0.1% SDS at 25 °C and twice for 15 min in 1x SSC and 0.1% SDS at 60 °C and exposed to autoradiography for 48 h. The probes used were: LeMPK3, a gene-specific probe from position 872–1503 in the LeMPK3 nucleotide sequence (GenBankTM accession number AY261514 [GenBank] ); Lox2, a cDNA clone encoding lipoxygenase2 (GenBankTM accession number AW033547 [GenBank] ); GluB, a cDNA clone encoding a basic (1–3)-{beta}-glucanase (a gift from Gregory Martin).

Expression of Fusion Proteins in Escherichia coli—For expression in bacteria the LeMPK3 protein was fused in-frame to the C terminus of glutathione S-transferase (GST) or to the C terminus of the maltose-binding protein (MBP). To develop the GST-LeMPK3 and MBP-LeMPK3 fusions, the LeMPK3-coding region (25) was amplified by PCR using as a template the tomato EST cTOB17A11 (GenBankTM accession number AW624703 [GenBank] ; a gift from Gregory Martin) and the primers 5'-GGAATTCATGGTTGATGCTAATATGGGTG-3' and 5'-GCTCGAGTTAAGCATATTCAGGATTCAACG-3'. The PCR product was digested by EcoRI and XhoI and inserted at the corresponding sites into the pGEX-4T1 (Amersham Biosciences) and pMALc2X (New England BioLabs) vectors.

For expression of the LeMPK3 mutant forms GST-LeMPK3(K70R) and GST-LeMPK3(T199A/Y201P), mutations were generated in the pGEX-4T1 plasmid containing the GST-LeMPK3 fusion using the QuikChange kit (Stratagene). The sequence of the oligonucleotides used is as follows: for GST-LeMPK3(K70R), 5'-GAGATGGTTGCAGTTCGAAAAATCGCCAATGC-3' and 5'-GCATTGGCGATTTTTCGAACTGCAACCATC-3'; for GST-LeMPK3(T199A/Y201P), 5'-GAACGAGAATATGGCAGAATTCGTAGTAACCAGAT-3' and 5'-ATCTGGTTACTACGAATTCTGCCATATTCTCGTTC-3'. The insertion in the gene of the desired mutation and the absence of undesired mutations were confirmed by nucleic acid sequencing using an ABI Prism 3100 Genetic analyzer (PerkinElmer Life Sciences).

For the construction of the GST-LeMPK2 fusion, the LeMPK2-coding region (25) was amplified by PCR using as a template the tomato EST cLEF44J1 (GenBankTM accession number AW931285 [GenBank] ; a gift from Gregory Martin) and the primers 5'-CGGGATCCATGGATGGTTCAGCTCCGCAA-3' and 5'-GGCTCGAGTCACATGTGCTGGTATTCGGGATT-3'. After digestion by BamHI and XhoI, the PCR product was inserted at the corresponding sites into the pGEX-4T1 vector. The resulting constructs were used to transform the E. coli strain BL21 (DE3) cells. Expression of recombinant proteins was induced by 0.5 mM isopropyl {beta}-D-thiogalactopyranoside for 3 h at 28 °C. GST and MBP fusion proteins were affinity-purified by using glutathione-agarose beads (Sigma-Aldrich) and amylose resin (New England Biolabs), respectively.

Kinase Activity Assays—Autophosphorylation activity of fusion proteins was assayed at different protein concentrations ranging from 0.6 to 29 µM in 20 µl of kinase reaction buffer (50 mM Tris-HCl, pH 7.0, 1 mM dithiothreitol, 10 mM MnCl2, and 20 µM ATP) containing 1 µCi of [{gamma}-32P]ATP (3000 Ci/mmol; Amersham Biosciences). Reactions were incubated for 10 min at room temperature and stopped by the addition of EDTA to a final concentration of 10 mM. At this time phosphate incorporation was found to be linear for the highest amount of enzyme used in the reaction. Proteins were then fractionated by 10% SDS-PAGE and stained by Coomassie Brilliant Blue R-250, and the dried gel was analyzed by phosphorimaging (Fujifilm FLA-2000) or exposed to autoradiography. For the ATP dependence assay of autophosphorylation, GST-LeMPK3 (2 µg) was incubated with different concentrations of cold ATP ranging from 2 to 50 µM in 20 µl of reaction buffer containing 1 µCi of [{gamma}-32P]ATP. Reactions were performed as described above, and ATP incorporation was calculated based on the radioactivity counts and the concentration of cold ATP. Values for the Vmax and Km parameters were estimated according to the double-reciprocal plot and linear regression. For the divalent cation dependence assay, 2.5 µg of GST-LeMPK3 was incubated with different concentrations of MnCl2 or MgCl2, ranging from 0.1 to 20 mM, or with 10 mM CaCl2 in 20 µl of kinase reaction buffer containing 1 µCi of [{gamma}-32P]ATP. To test intermolecular autophosphorylation, MBP-LeMPK3 (1.5 µg) was incubated with the kinase-deficient fusion GST-LeMPK3(K70R) (1.5 µg) in 20 µl of reaction buffer containing 1 µCi of [{gamma}-32P]ATP. For phosphorylation of substrates, the fusion proteins GST-LeMPK3, GST-LeMPK3(K70R), or GST-LeMPK3(T199A/Y201P) (1 and 2 µg for phosphorylation of myelin basic protein and of poly(Glu-Tyr), respectively) were mixed with 4 µg of myelin basic protein or with 8 µg of poly(Glu-Tyr) 4:1 copolymer (Sigma-Aldrich) in 20 µl kinase reaction buffer containing 1 µCi of [{gamma}-32P]ATP. Reactions were performed and analyzed as described above.

Phosphoamino Acid Analysis—Autophosphorylated GST-LeMPK3, GST-LeMPK3(T199A/Y201P) fusion proteins, or myelin basic protein phosphorylated by GST-LeMPK3 were fractionated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. The 32P-labeled proteins were visualized by India ink (1 µl/ml) staining, and the pertinent regions of the membrane were excised. Proteins were then hydrolyzed in 200 µlof6 M HCl for 1 h at 110 °C. The hydrolysates were dried in a Speedvac concentrator and resuspended in 20 µl of first dimension buffer (2.2% (v/v) formic acid and 7.8% (v/v) acetic acid, pH 1.9) containing 2 µg each phosphoamino acid standards (phosphoserine, phosphothreonine, and phosphotyrosine; Sigma-Aldrich). Each sample was analyzed by two-dimensional electrophoresis on thin layer chromatography (TLC) cellulose plates (pH 1.9, 1.5 kV for 30 min, and pH 3.5, 1.3 kV for 20 min) by using a HTLE7000 Hunter thin layer electrophoresis system (C.B.S. Scientific Co., Del Mar, CA), as described by van der Geer et al. (30). After spraying with 0.25% (w/v) ninhydrin in acetone to visualize the phosphoamino acid standards, the TLC plates were subjected to autoradiography for the detection of labeled phosphoamino acids.

Antibody Production, Immunoprecipitation, and Immunocomplex Kinase Assay—The peptide MVDANMGAAQFPDFP, corresponding to the N terminus of the LeMPK3 protein, was synthesized and conjugated to keyhole limpet hemocyanin carrier. Polyclonal antiserum was obtained from rabbits immunized with 100 µg of the above-conjugated peptide for 4 times at 20-day intervals (Genemed Synthesis, Inc., South San Francisco, CA). For immunoprecipitation, leaf samples were homogenized in extraction buffer (20 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM NaVO3, 10 mM {beta}-glycerophosphate, 1 mM phenylmethanesulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5% Nonidet P-40, and 1% Triton X-100), and total extracts (100 µg) were rotated for 2 h at 4 °C with 20 µl of anti-LeMPK3 antiserum. Protein A immobilized on Sepharose 4 fast flow beads (25 µl packed volume; Amersham Biosciences) prewashed with extraction buffer was added to samples, and incubation was continued for an additional 2 h. The beads were precipitated by a brief centrifugation, washed at 4 °C 3 times with extraction buffer and 6 times with kinase reaction buffer (without ATP). Immunocomplexes were tested for kinase activity in 20 µl of reaction buffer containing 1 µCi of [{gamma}-32P]ATP and 4 µg of myelin basic protein. After 25 min reactions were stopped by the addition of EDTA to a final concentration of 10 mM. Proteins were then fractionated on a 15% (w/v) SDS-PAGE, and the dried gels were exposed to autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LeMPK3 Is Induced at Multiple Levels during the Tomato Resistance Response to Xcv—For the identification of plant genes involved in tomato resistance to Xcv, we have used the suppression subtractive hybridization techniques (31) to construct a cDNA library enriched for Xcv-induced genes. Leaf material used for preparation of the library was derived from plants of the resistant tomato line Hawaii 7981, which develops a HR upon infection by Xcv T3 strains (26). In the cDNA library enriched for genes induced by Xcv T3 we identified the LeMPK3 gene, which was recently shown to be involved in the UV-B response of Lycopersicon peruvianum suspension cells (25). LeMPK3 encodes a MAP kinase highly homologous to MAP kinases from different plant species that have been implicated in the plant defense response (8, 25).

To investigate the possible involvement of LeMPK3 in tomato disease resistance to Xcv, we first analyzed its inducibility at the mRNA level during two different tomato-Xcv-incompatible interactions. The tomato lines used in these experiments are Hawaii 7981, which is resistant to Xcv T3 and susceptible to Xcv T1, and Hawaii 7998, which is susceptible to Xcv T3 and resistant to Xcv T1. Resistance of Hawaii 7981 to Xcv T3 is specified by a typical gene-for-gene interaction (26, 32), whereas resistance of Hawaii 7998 to Xcv T1 is mediated in a quantitative fashion by three genes in the plant and by a single gene in the bacterium (33, 34). Leaves of these two lines were infiltrated by vacuum with a suspension of either Xcv T3 or Xcv T1 bacteria at a titer of 108 cfu/ml. A mock-inoculation treatment was also included as a control for changes in gene expression resulting from vacuum infiltration. A typical hypersensitive response was observed only in the incompatible interactions of Hawaii 7981 and Hawaii 7998 with Xcv T3 and Xcv T1, respectively. Tissues were sampled at different time points after inoculation and used for total RNA extraction. A partial LeMPK3 cDNA clone, which represents a gene-specific probe, as established by Southern blot analysis (data not shown), was used for monitoring LeMPK3 expression levels in the treated plants. As shown in Fig. 1, LeMPK3 expression was significantly induced during the resistance response of the two tomato lines to Xcv as early as 8 h after inoculation. Accumulation of LeMPK3 transcripts was slightly faster during the incompatible interaction of Hawaii 7981 with Xcv T3 than in the interaction of Hawaii 7998 with Xcv T1. This correlated well with the earlier HR observed in Hawaii 7981 as compared with Hawaii 7998, taking place ~18 and 24 h after inoculation, respectively. A very low increase in LeMPK3 mRNA levels was observed in the compatible interactions, and no effect of the mock-inoculation treatment was detected at the time points included in these experiments.



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  		FIG. 1.
Gene expression of LeMPK3 during Xcv infection. Hawaii 7981 and Hawaii 7998 plants were inoculated with suspensions of Xcv T1 or Xcv T3 bacteria (108 cfu/ml) or with a 10 mM MgCl2 control solution (Mock). Total RNA was extracted from leaf samples collected at the indicated time points and subjected to Northern blot analysis using as probe a partial LeMPK3 cDNA. Equal loading of RNA was confirmed by hybridizing blots with a ribosomal RNA probe (rRNA).

 
To determine whether the accumulation of LeMPK3 mRNA correlated with higher protein and activity levels of the encoded MAP kinase, we first raised antibodies against a 15-amino acid peptide corresponding to the unique N terminus of LeMPK3. Comparison of the sequence of this peptide with proteins in the data base did not show any significant homology. To test the specificity of the antiserum, LeMPK3 and the closely related LeMPK2 MAP kinase were expressed in E. coli as GST fusion proteins and affinity-purified (Fig. 2A, left panel). The anti-LeMPK3 antibodies specifically recognized the GST-LeMPK3 kinase and did not show any cross-reactivity with GST-LeMPK2 or GST alone (Fig. 2A, central panel). Moreover, the addition to the immuno-reaction of competitor peptide completely blocked binding of the anti-LeMPK3 antibodies to the GST-LeMPK3 protein (Fig. 2A, right panel). Attempts to use anti-LeMPK3 antibodies to monitor LeMPK3 protein levels in treated plants by Western blot analysis were unsuccessful, presumably due to low LeMPK3 expression. However, these antibodies and the universal MAP kinase substrate myelin basic protein were successfully employed to detect changes in LeMPK3 kinase activity in tomato plants upon Xcv infection. Hawaii 7981 plants were inoculated as described above with bacterial suspensions of Xcv T1 and Xcv T3 or mock-treated. Protein extracts were prepared from leaves sampled at various times after inoculation and subjected to an immuno-complex kinase assay (Fig. 2B). Interestingly, two activities that phosphorylated myelin basic protein with distinct induction patterns were detected when including Mn2+ or Mg2+ ions in the kinase assay. The Mg2+-dependent activity was detected in both mock-inoculated and pathogen-infected plants (data not shown). Conversely, the Mn2+-dependent activity was barely detectable in mock-inoculated plants and strictly regulated by pathogen challenge (Figs. 2, B and C). During the incompatible interaction between Hawaii 7981 and Xcv T3, the Mn2+-dependent activity dramatically increased 8 h after inoculation, peaked at 12 h (~9-fold), and gradually decreased. These induction kinetics correlated well with the accumulation of LeMPK3 mRNA in Hawaii 7981 plants, which reached a maximum at 8 h post-inoculation and, thus, preceded the increase in kinase activity. Induction of the Mn2+-dependent activity was also observed during the Hawaii 7981-Xcv T1 compatible interaction but at a lesser extent and with shorter time-amplitude, reaching back basal levels 12 h after inoculation (Figs. 2, B and C). Its correlation with LeMPK3 mRNA induction kinetics and dependence on Mn2+ ions, which is characteristic of the bacterially expressed LeMPK3 kinase (see below), strongly suggest that the Mn2+-dependent activity immunoprecipitating with anti-LeMPK3 antibodies originates from the endogenous LeMPK3 MAP kinase. Taken together, these results indicate that LeMPK3 is dramatically and specifically induced at multiple levels during the race-specific resistance response of tomato to Xcv bacteria.



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  		FIG. 2.
Immune-complex kinase assays using LeMPK3-specific antibodies. A, specificity of anti-LeMPK3 antibodies. Fifty ng each of GST-LeMPK3 (lane 1), GST-LeMPK2 (lane 2), and GST (lane 3) were separated by SDS-PAGE and blotted. Proteins were analyzed for cross-reactivity with antibodies raised against a 15-amino acid peptide corresponding to the N terminus of LeMPK3 in the presence (right panel) or absence of 2 µg/ml competitor peptide (central panel). On a separate gel 1 µg of the recombinant proteins was visualized by Coomassie staining (left panel). B, immuno-complex kinase assays using anti-LeMPK3 antibodies. Hawaii 7981 plants were inoculated with Xcv T1 or Xcv T3 bacteria (108 cfu/ml) or with a 10 mM MgCl2 control solution (mock). Protein extracts were prepared from samples collected at the indicated time points and reacted with anti-LeMPK3 antibodies. The antigen-antibody complex was precipitated with protein A-Sepharose beads and added to a kinase assay mixture containing [{gamma}-32P]ATP, myelin basic protein, and MnCl2. The reaction mixture was then separated by 15% SDS-PAGE, and the dried gel was exposed to autoradiography. C, induction kinetics of LeMPK3 activity in Xcv-infected Hawaii 7981. LeMPK3 kinase activities were quantified using phosphorimaging, and fold-induction relative to activities of samples from mock-inoculated plants was calculated for activities derived from Xcv-infected plants. The experiment was repeated three times with similar kinetics.

 
LeMPK3 Transcripts Accumulate during Elicitation of the HR and Abiotic Stress but Not in Response to Ethylene and Jasmonic Acid—To assess whether induction of LeMPK3 gene expression is restricted to Xcv resistance or represents a convergence point for tomato defense responses activated by different biotic and abiotic stress, we first tested LeMPK3 inducibility by the bacterial pathogen Pst and by the fungal elicitor EIX. To test LeMPK3 inducibility by Pst, the near-isogenic lines used were Rio Grande PtoR (RG-PtoR), which carries the Pto resistance gene and is resistant in a gene-for-gene manner to Pst T1 strain expressing the avrPto gene (T1A), and Rio Grande PtoS (RG-PtoS), which does not carry the Pto gene and is susceptible to T1A (28). RG-PtoR and RG-PtoS plants were infiltrated by vacuum with a suspension of T1A bacteria at a titer of 108 cfu/ml or mock-inoculated. A typical hypersensitive response was observed in resistant RG-PtoR plants 6 h after inoculation, whereas at this time no symptoms were yet detected in the susceptible RG-PtoS plants. As shown in Fig. 3A, LeMPK3 expression was strongly induced in the Pst-tomato incompatible interaction with a peak at 3 h after treatment, whereas in the compatible interaction only a slight increase in LeMPK3 expression was observed as compared with mock-inoculated plants. Next, LeMPK3 inducibility by the cultivar-specific EIX elicitor from T. viride was tested in the EIX-responding tomato line M82 and in the EIX-insensitive line IL7–5. The response of M82 plants to EIX is modulated by a single dominant gene and typified by elicitation of ethylene biosynthesis and localized cell death (35). Leaves of M82 and IL7–5 plants were infiltrated with EIX by vacuum, and a typical HR was observed 36 h after treatment only in the EIX-responding M82 plants (Fig. 3B). The appearance of the HR was preceded by the accumulation of increasing levels of LeMPK3 transcripts starting 4 h after inoculation. These results indicate that LeMPK3 may participate in the elicitation of the HR mediated by distinct plant-pathogen recognition events, including those between bacterial effectors and the corresponding race-specific resistance genes and those between a cultivar-specific elicitor and its receptor protein.



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  		FIG. 3.
Inducibility of LeMPK3 transcript levels in response to Pst infection and to the fungal elicitor EIX. A, Rio Grande PtoR (RG-PtoR) and Rio Grande PtoS (RG-PtoS) plants were inoculated with suspensions of P. syringae pv. tomato T1 strain expressing avrPto (Pst T1A) (108 cfu/ml) or with a 10 mM MgCl2 control solution (Mock). B, M82 and IL7–5 plants were infiltrated with a solution containing 0.5 mg/l EIX or with a control solution (Mock). Total RNA was extracted from leaf samples collected at the indicated time points, subjected to Northern blot analysis, and hybridized with a LeMPK3 gene-specific probe. Equal loading of RNA was confirmed by hybridizing blots with a ribosomal RNA probe (rRNA).

 
Plant MAP kinases closely related to LeMPK3 have been reported to be induced at different levels by abiotic environmental stimuli (7). To investigate a possible link between LeMPK3 and abiotic stress, we monitored changes in LeMPK3 gene expression as a result of wounding caused by rubbing the leaf surface with Carborundum and of mechanical stress determined by vacuum infiltration. A rapid and transient accumulation of LeMPK3 mRNA was observed in Hawaii 7981 plants upon both treatments (Fig. 4A). The increase in LeMPK3 expression became evident within 15 min, and maximal accumulation was reached at 30–60 min before returning to basal levels 2–4 h after treatment. It should be noted that there was no overlap in time between the early and transient response of LeMPK3 to mechanical stress and its later response to avirulent bacteria, which were infiltrated in the plants by vacuum. This indicates that LeMPK3 may be induced with distinct activation kinetics by different stimuli. Together, these results suggest that LeMPK3 may also be involved in signaling events that mediate tomato defense response to stress that not necessarily leads to elicitation of the HR.



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  		FIG. 4.
Gene expression of LeMPK3 in response to abiotic stress and defense-related plant hormones. Total RNA was extracted from leaf samples collected at the indicated time points from Hawaii 7981 plants that were subjected to the following treatments. A, left panel, gentle rubbing with wet Carborundum powder. A, right panel, vacuum infiltration of a 10 mM MgCl2 solution. B, left panel, spraying with a 100 µM jasmonic acid solution. B, right panel, spraying with control solution. C, left panel, exposure to 20 ppm ethylene in a closed chamber. C, right panel, exposure to air in a closed chamber. RNA samples were subjected to Northern blot analysis using the following probes: LeMPK3, lipoxygenase 2 (Lox2), basic-type (1–3)-{beta}-glucanase (GluB), and ribosomal RNA (rRNA).

 
Finally, the interplay between the LeMPK3 MAP kinase and hormones that are involved in the plant defense response was investigated. Hawaii 7981 tomato plants were exposed to ethylene in a closed chamber or sprayed with jasmonic acid, and leaf samples were collected at different time points. The efficiency of the treatments was tested by monitoring the induction of lypoxigenase2 and (1–3)-{beta}-glucanase, which are known defense-related genes responsive to jasmonic acid and ethylene, respectively. As shown in Figs. 4, B and C, ethylene and jasmonic acid enhanced the expression of their respective marker genes. However, the two hormones did not induce accumulation of LeMPK3 transcripts. Based on its induction pattern, we conclude that the LeMPK3 MAP kinase may represent a convergence point of different signaling pathways, including those activated by avirulent pathogens, cultivar-specific elicitors, wounding, and mechanical stress but not by the defenserelated hormones ethylene and jasmonic acid.

LeMPK3 Encodes a Dual Specificity Protein Kinase—The deduced amino acid sequence of LeMPK3 presents the typical structural characteristics of a plant MAP kinase of the A1 subgroup (8, 25). To study the biochemical properties of this MAP kinase, its coding region was expressed in E. coli as a GST fusion and affinity-purified. The purified GST-LeMPK3 protein was tested in vitro for autophosphorylation and phosphorylation of substrates. Figs. 5, A and B, show that GST-LeMPK3 was able to autophosphorylate and to phosphorylate the universal MAP kinase substrate myelin basic protein. Phosphoamino acid analysis revealed that LeMPK3 autophosphorylation occurred predominantly on tyrosine and to a much lower extent on serine and threonine residues (Fig. 5D), whereas myelin basic protein was phosphorylated by GST-LeMPK3 mainly on threonine, less on serine, but not on tyrosine residues (Fig. 5E). To test whether GST-LeMPK3 would phosphorylate exogenous substrates on tyrosine, a copolymer of glutamic acid and tyrosine, poly(Glu-Tyr) 4:1, was used as a substrate. As shown in Fig. 5C, the GST-LeMPK3 fusion efficiently phosphorylated tyrosine residues of the poly(Glu-Tyr) copolymer. To verify that the observed kinase activity was indeed derived from GST-LeMPK3 and not from a contaminating bacterial protein, the invariant lysine of the kinase subdomain II of LeMPK3 was substituted by site-directed mutagenesis for an arginine. As expected, the introduction of a mutation at Lys-70, which is highly conserved in all protein kinases and required for activity, completely abolished the capability of GST-LeMPK3 to autophosphorylate and to phosphorylate substrates (Figs. 5, A–C).



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  		FIG. 5.
Analysis of LeMPK3 phosphorylation activity. A–C, assay of GST-LeMPK3 autophosphorylation and phosphorylation of substrates. GST-LeMPK3, GST-LeMPK3(K70R), and GST-LeMPK3-(T199A/Y201P) were tested in vitro for autophosphorylation activity (A) and phosphorylation of myelin basic protein (B). In C, GST-LeMPK3 and GST-LeMPK3(K70R) were tested for phosphorylation of poly(Glu-Tyr) 4:1, which is a mixture of heteropolymers of 20–50 kDa. Fusion proteins (in A and B, 1 µg each; in C, 2 µg each) were fractionated by 10% SDS-PAGE, and the dried gels were exposed to autoradiography. Autoradiograms in A (left panel), B, and C show the phosphorylated proteins, whereas the Coomassie-stained gel in A (right panel) shows the protein species present in the reaction mixtures. D–F, phosphoamino acid analysis of autophosphorylated GST-LeMPK3 (D) of myelin basic protein phosphorylated by GST-LeMPK3 (E) and of autophosphorylated GST-LeMPK3(T199A/Y201P) (F). After phosphorylation in vitro, proteins were hydrolyzed, and amino acids were separated by two-dimensional electrophoresis on a TLC plate along with phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) standards. Phosphoamino acid standards were visualized with ninhydrin, and the 32P-labeled phosphoamino acids were detected by autoradiography. A dashed circle in E represents the location of the phosphotyrosine standard on the TLC plate. wt, wild type.

 
Dual phosphorylation in a conserved TXY motif, which is located in the activation loop between kinase subdomains VII and VIII, represents a universal activation mechanism of MAP kinases. To examine the requirement of the TXY motif for LeMPK3 kinase activity, Thr-199 and Tyr-201 residues were mutated to Ala and Pro, respectively. The mutant GST-LeMPK3(T199A/Y201P) fusion was then tested for kinase activity. As shown in Figs. 5, A and B, the introduction of mutations in the LeMPK3 TXY motif significantly decreased LeMPK3 autophosphorylation activity and its capability to phosphorylate substrates. Remarkably, phosphoamino acid analysis of the autophosphorylated protein revealed a marked reduction in the relative abundance of phosphorylated tyrosine compared with serine and threonine (Fig. 5F), suggesting that in vitro Tyr-201 was the main site of GST-LeMPK3 autophosphorylation. Taken together these results define LeMPK3 as a MAP kinase with dual specificity having the potential to phosphorylate serine, threonine, and tyrosine residues.

Enzymatic Characteristics of LeMPK3 Phosphorylation—To further characterize LeMPK3 enzymatic properties, we examined the kinetics of GST-LeMPK3 autophosphorylation at different ATP concentrations. As shown in Fig. 6A, GST-LeMPK3 autophosphorylation activity showed typical Michaelis-Menten kinetics with respect to ATP. The apparent Km and Vmax values for ATP were estimated by a double reciprocal plot and linear regression as 9.5 µM and 40 nm/min/mg, respectively.



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  		FIG. 6.
Enzymatic characteristics of LeMPK3 autophosphorylation. A, kinetics of GST-LeMPK3 autophosphorylation. Kinetics were tested by monitoring the initial velocity of autophosphorylation at different ATP concentrations ranging from 2 to 50 µM. A double reciprocal plot and linear regression were used to estimate a Km value 9.5 for ATP of µM and a Vmax value of 40 nmol/min/mg. B–D, dependence of GST-LeMPK3 activity on divalent cations. Autophosphorylation assays were carried out in the presence of 10 mM concentrations of the indicated divalent cations (B) or with increasing concentrations of MnCl2 (C) or MgCl2 (D). Proteins were then fractionated by 10% SDS-PAGE, stained with Coomassie (lower panel), and exposed to autoradiography (upper panel).

 
To investigate the dependence of LeMPK3 kinase activity on different divalent cations, GST-LeMPK3 autophosphorylation assays were performed in the presence of Mn2+, Mg2+, or Ca2+ ions. Fig. 6B shows a marked preference of LeMPK3 for Mn2+ rather than Mg2+, whereas no activity was detected when Ca2+ was included in the reaction. GST-LeMPK3 autophosphorylation activity was strongly stimulated by increasing concentrations of MnCl2 up to 5 mM, whereas when including MgCl2 in the reaction, maximal activity was observed at a concentration of 10 mM (Figs. 6, C and D).

LeMPK3 Autophosphorylation Occurs through an Intramolecular Mechanism—The dual specificity character of LeMPK3 kinase activity and MAP kinase activation by dual phosphorylation prompted us to test a possible LeMPK3 autoactivation mechanism by intermolecular autophosphorylation. To this aim, we tested whether LeMPK3 autophosphorylation mechanism is intramolecular (first order with respect to enzyme concentration) or intermolecular (second order with respect to enzyme concentration). For this purpose, phosphate incorporation was monitored in autophosphorylation reactions carried out with increasing concentrations of GST-LeMPK3. Fig. 7A shows a linear increase in the autophosphorylation rate with respect to enzyme concentration, indicating that LeMPK3 autophosphorylation follows first order rather than second order reaction kinetics. In addition, the phosphate incorporation per molecule varied by only 1.4 when GST-LeMPK3 concentration varied by 49-fold (Fig. 7B). Finally, the van't Hoff plot of autophosphorylation (logarithm of phosphorylation rate versus logarithm of enzyme concentration), whose slope indicates the order of the reaction, had a slope of 1.07 and a correlation coefficient for linear regression of 0.992 (Fig. 7C). These data suggest that GST-LeMPK3 autophosphorylation occurs via an intramolecular mechanism. To confirm that intermolecular autophosphorylation does not occur, the kinase-deficient mutant GST-LeMPK3(K70R) was used as a potential substrate for the wild type LeMPK3 fused to MBP. As shown in Fig. 7D, the MBP-LeMPK3 protein was able to autophosphorylate but failed to phosphorylate the inactive GST-LeMPK3(K70R) fusion. These results combined with the data presented above strongly support the notion that LeMPK3 autophosphorylation proceeds in vitro through an intramolecular rather than intermolecular mechanism.



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  		FIG. 7.
Effect of enzyme concentration on GST-LMPK3 autophosphorylation. A–C, test of in vitro GST-LeMPK3 autophosphorylation activity at different enzyme concentrations ranging from 0.6 to 29.2 µM. A, plot of phosphate incorporation rate versus enzyme concentration. B, specific activity of GST-LeMPK3 presented as phosphate incorporation rate per pmol of GST-LeMPK3. C, van't Hoff plot of GST-LeMPK3 autophosphorylation. Linear regression of the data estimated a slope of 1.07 and a correlation coefficient of 0.992. Data in A–C are the mean ± S.E. (n = 4). D, intermolecular autophosphorylation assay of MBP-LeMPK3 and kinase-deficient GST-LeMPK3(K70R). Kinase activity was tested in reactions containing 1.5 µg of the following fusion proteins: lane 1, MBP-LeMPK3; lane 2, MBP-LeMPK3 and GST-LeMPK3(K70R); lane 3, GST-LeMPK3(K70R). Proteins were fractionated by 10% SDS-PAGE and Coomassie-stained (left panel), and the dried gel was exposed to autoradiography (right panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the recent years it has become clear that MAP kinase modules are implicated in plant disease resistance and participate in signaling pathways leading to elicitation of the HR and related defense responses (4, 21–24). In this report we investigated the induction pattern of the tomato LeMPK3 MAP kinase in response to pathogens and abiotic stress and extensively characterized its biochemical properties in vitro.

Expression of the LeMPK3 gene was transiently induced during the tomato resistance response to phytopathogenic bacteria. Remarkably, LeMPK3 inducibility was dictated by genetic interactions of different kind. These include both quantitative resistance to Xcv T1 strains, which requires the expression of three R genes in the plant and of the avrRxv gene in the pathogen (33, 34), and classical gene-for-gene interactions mediating resistance to Xanthomonas T3 strains expressing avrXv3 (32) as well as resistance to Pst strains expressing avrPto (28). Immune-complex kinase assays demonstrated that during the tomato resistance response to Xcv T3, mRNA accumulation of LeMPK3 in leaf tissue is followed by an increase of LeMPK3 kinase activity. A similar induction pattern at the mRNA and activity levels was observed for the tobacco LeMPK3 homolog WIPK during elicitation of the HR in an N-gene or Cf9-dependent manner after inoculation with tobacco mosaic virus and the Cladosporium fulvum avirulence protein Avr9, respectively (16, 17). The observed increase in LeMPK3 kinase activity may be determined by higher protein levels present in the cell as a result of higher transcription rate and of de novo protein synthesis. However, this hypothesis could not be tested due to undetectable LeMPK3 protein levels in Western blot analysis. Alternatively, the increase in LeMPK3 kinase activity may reflect the activation of preexisting LeMPK3 pools or a combination of transcriptional and post-translational regulation.

The R-gene(s) dependent induction of LeMPK3 during several incompatible interactions suggests that this MAP kinase participates in signaling pathways generated by different recognition events between tomato R proteins and pathogen race-specific effectors and is, therefore, involved in the activation of the plant defense response. This possibility is strongly supported by the recent finding that attenuation of LeMPK3 expression by virus-induced gene silencing impairs tomato resistance to Pst (23). Similarly, the tobacco LeMPK3-homolog WIPK is required along with other components of the same MAP kinase pathway for full disease resistance to tobacco mosaic virus (21).

A slight but significant increase in LeMPK3 mRNA was also observed during the compatible interaction of tomato with Xcv or with Pst virulent strains. In addition, a 5-fold induction of LeMPK3 kinase activity was observed 8 h after inoculation of susceptible tomato with a virulent Xcv strain. This is in agreement with the concept that a main difference between resistance and susceptible plants lies in the amplitude of the defense response. In support of this notion, a large scale gene expression profiling study in Arabidopsis recently revealed that changes in gene expression during compatible and incompatible interactions are qualitatively similar but quantitatively more robust during the incompatible interaction (36).

LeMPK3 mRNA level was also induced during the response of tomato to the cultivar-specific elicitor EIX, to mechanical stress and to wounding. As observed during pathogen infection, LeMPK3 transcript accumulation upon treatment with EIX was strictly dictated by the plant genetic background and correlated with the elicitation of the HR. Induction by general elicitors and abiotic stress represents a common characteristic of LeMPK3 homologs from several plant species (Ref. 37 and references therein). However, LeMPK3 activity has been recently reported to be induced by UV-B but not in response to several general elicitors (25). It should be noted that Holley et al. (25) in their experiments performed kinase assays in the presence of Mg2+ as a cofactor for ATP, which, based on our results, is at least 10-fold less efficient than Mn2+. For this reason the effect of general elicitors on LeMPK3 activation might have been underestimated in these experiments.

LeMPK3 gene expression was not affected by treating tomato plants with jasmonic acid or ethylene, which are plant hormones known for their involvement in defense responses (38). It has been recently shown, by monitoring bacterial growth in tomato mutants defective in jasmonic acid or ethylene signaling, that these two hormones do not affect resistance to Xcv, but they do affect the extent of cell death (39). Interestingly, activation in tobacco of MAP kinase cascades, which include the NtMEK2 MAPKK and the SIPK and WIPK MAP kinases, has been reported to cause an increase of ethylene levels (18). Based on this evidence and assuming a functional conservation for the LeMPK3 and WIPK homologs, ethylene is more likely to act downstream of LeMPK3 during the resistance response to Xcv. Taken together, our results strongly suggest that LeMPK3 represents a convergence point of signaling pathways activated either by race- and cultivar-specific elicitors, wounding, or mechanical stress but not by defense-related hormones.

By a detailed analysis of the kinase activity of a GST-LeMPK3 fusion we pinpointed important biochemical properties of this MAP kinase. Recombinant GST-LeMPK3 was found to be an active protein kinase in vitro, with a Km for ATP of 9.5 µM, which is comparable with values obtained for other characterized plant kinases (e.g. Refs. 40 and 41). In addition, the activity of LeMPK3 showed a marked preference for Mn2+ over Mg2+ as a cofactor for ATP, which is not uncommon for plant kinases (e.g. Refs. 40 and 41). GST-LeMPK3 autophosphorylated mainly on Tyr-201, which is located in the kinase activation domain and is required along with Thr-199 for phosphorylation of substrates. This pattern of autophosphorylation and its capability to use a poly(Glu-Tyr) 4:1 copolymer as a substrate define LeMPK3 as a dual specificity MAP kinase. Several dual specificity protein kinases have been identified in plants (e.g. Refs. 42 and 43). However, with the exception of MAPKKs, the biological significance of their dual phosphorylation remains unexplored. The evidence that LeMPK3 autophosphorylates with dual specificity along with the notion that activity of MAP kinases is modulated by dual phosphorylation opened the possibility that LeMPK3 is activated by intermolecular autophosphorylation. However, this kind of activation mechanism was excluded by the finding that LeMPK3 autophosphorylation proceeds exclusively by an intramolecular mechanism.

Because MAP kinases are typically activated by dual phosphorylation of the kinase activation domain, in vitro autophosphorylation of GST-LeMPK3 at Tyr-201 raises questions regarding its in vivo mechanism of activation. A first possibility is that, as observed for the mammalian ERK1 and ERK2 MAPKs (44), autophosphorylation is involved in LeMPK3 activation, but it may not be sufficient for its full activation, which requires an upstream MAPKK. This is reminiscent of the activation mechanism proposed for the Arabidopsis AtMPK4 MAP kinase that autophosphorylates in vitro on tyrosine residues and is activated by the AtMEK1 MAPKK through phosphorylation of threonine residues (45). In addition, it is possible that in the plant LeMPK3 autophosphorylation is antagonized by the activity of a protein phosphatase that keeps LeMPK3 in an inactive form. Remarkably, a MAP kinase phosphatase that physically interacts with a set of MAPKs, including the LeMPK3 homolog AtMPK3, has been recently identified in Arabidopsis (46). Moreover, overexpression in transgenic tobacco plants of a dual specificity protein phosphatase inhibited wound-induced activation of the SIPK and WIPK MAP kinases (47). Finally, it is also possible that LeMPK3 is fully activated in vivo by autophosphorylation and that its activity in the cell is solely regulated at the mRNA level. This mode of activation for LeMPK3 activity is less likely based on the multiple regulation levels observed for the LeMPK3 homolog WIPK in response to avirulent pathogens and abiotic stress (16, 17, 48). In conclusion, LeMPK3 inducibility by different biotic and abiotic stimuli and its biochemical properties suggest a pivotal function for this MAP kinase in signaling pathways leading to the activation of tomato defense responses.


   FOOTNOTES
 
* This research was supported in part by BARD, the United States-Israel Binational Agricultural Research and Development Fund Research Grant IS-3237-01 and in part by Israel Science Foundation Grant No. 713/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 972-3-640-9766; Fax: 972-3-640-9380; E-mail: guidos{at}post.tau.ac.il.

1 The abbreviations used are: HR, hypersensitive response; R, resistance gene; avr, avirulence gene; MAPK, mitogen-activated protein (MAP) kinase; MAPKK, MAPK kinase; GST, glutathione S-transferase; MBP, maltose binding protein; EIX, ethylene-inducing xylanase; Xcv, X. campestris pv. vesicatoria; RG-PtoR, Rio Grande carrying the Pto gene; RG-PtoS, Rio Grande without the Pto gene; Pst, P. syringae pv. tomato. T1A, T1 strain of Pst expressing the avrPto gene; cfu, colony-forming units. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Gregory B. Martin for sharing cDNA clones, Dr. John W. Scott for providing Xcv-resistant tomato lines, Boris Fichtman for assistance with phosphoamino acid analysis, and Dr. Shaul Yalovsky and members of the laboratory for critical comments on the manuscript.



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Tomato Mitogen-Activated Protein Kinases LeMPK1, LeMPK2, and LeMPK3 Are Activated during the Cf-4/Avr4-Induced Hypersensitive Response and Have Distinct Phosphorylation Specificities
Plant Physiology, July 1, 2007; 144(3): 1481 - 1494.
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 Plant Physiol.Home page
M. Shoresh, A. Gal-On, D. Leibman, and I. Chet
Characterization of a Mitogen-Activated Protein Kinase Gene from Cucumber Required for Trichoderma-Conferred Plant Resistance
Plant Physiology, November 1, 2006; 142(3): 1169 - 1179.
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 Plant Physiol.Home page
D. Lieberherr, N. P. Thao, A. Nakashima, K. Umemura, T. Kawasaki, and K. Shimamoto
A Sphingolipid Elicitor-Inducible Mitogen-Activated Protein Kinase Is Regulated by the Small GTPase OsRac1 and Heterotrimeric G-Protein in Rice
Plant Physiology, July 1, 2005; 138(3): 1644 - 1652.
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 J. Biol. Chem.Home page
K. F. Pedley and G. B. Martin
Identification of MAPKs and Their Possible MAPK Kinase Activators Involved in the Pto-mediated Defense Response of Tomato
J. Biol. Chem., November 19, 2004; 279(47): 49229 - 49235.
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