From the Department of Stress and Developmental Biology, Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany
Received for publication, August 11, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Plants are continuously exposed to
attack by potential phytopathogens. Disease prevention requires
pathogen recognition and the induction of a multifaceted defense
response. We are studying the non-host disease resistance response of
parsley to the oomycete, Phytophthora sojae using a cell
culture-based system. Receptor-mediated recognition of P. sojae may be achieved through a thirteen amino acid peptide
sequence (Pep-13) present within an abundant cell wall
transglutaminase. Following recognition of this elicitor molecule,
parsley cells mount a defense response, which includes the generation
of reactive oxygen species (ROS) and transcriptional activation of
genes encoding pathogenesis-related (PR) proteins or enzymes involved
in the synthesis of antimicrobial phytoalexins. Treatment of parsley
cells with the NADPH oxidase inhibitor, diphenylene iodonium (DPI),
blocked both Pep-13-induced phytoalexin production and the accumulation
of transcripts encoding enzymes involved in their synthesis. In
contrast, DPI treatment had no effect upon Pep-13-induced PR
gene expression, suggesting the existence of an oxidative
burst-independent mechanism for the transcriptional activation of
PR genes. The use of specific antibodies enabled the
identification of three parsley mitogen-activated protein kinases
(MAPKs) that are activated within the signal transduction pathway(s)
triggered following recognition of Pep-13. Other environmental challenges failed to activate these kinases in parsley cells, suggesting that their activation plays a key role in defense signal transduction. Moreover, by making use of a protoplast
co-transfection system overexpressing wild-type and loss-of-function
MAPK mutants, we show an essential role for post-translational
phosphorylation and activation of MAPKs for oxidative burst-independent
PR promoter activation.
In most circumstances plants are able to defend themselves against
pathogen attack. This is primarily facilitated through recognition
mechanisms, which plants use to sense the presence of the pathogen
(1-3), and through triggering intrinsic defense mechanisms that either
kill the pathogen or limit its spread to the site of immediate
infection (4, 5). Parsley (Petroselinum crispum) exhibits a
non-host resistance response to attack by the oomycetes,
Phytophthora infestans and Phytophthora sojae (6, 7). Defense reactions are triggered through the recognition of an
abundant cell wall transglutaminase present and conserved in all but
one tested member of Phytophthora (8). This protein was
previously characterized as a 42-kDa glycoprotein purified from
P. sojae that was able to trigger phytoalexin accumulation when added to cultured parsley cells (9, 10). Within this protein
resides a conserved peptide sequence of 13 amino acids (Pep-13) that is
necessary and sufficient for its elicitor activity (11). The ability of
Pep-13 to trigger defense responses in parsley requires its interaction
with a 100-kDa receptor protein present in the plasma membrane of
parsley cells (12, 13), since all mutations made within the Pep-13
sequence that prevented binding to the receptor also inhibited the
elicitation of defense reactions (11, 14-17). The defense response
itself is multifaceted and involves the generation of reactive oxygen
species (ROS)1 (15), the
synthesis of antimicrobial furanocoumarin phytoalexins (10), and the
expression of defense-related genes including pathogenesis-related
(PR) genes (18). Pep-13-induced defense gene activation is
temporally regulated (18). Transcripts of immediate early genes,
including the WRKY1, -3, -4, and
-5 transcription factor genes, accumulate rapidly after
elicitation apparently without the requirement of de novo
protein synthesis (19). With a slight delay, transient activation of
another group of early genes is observed, among these are the
PR1 and PR2 genes (18, 20, 21). Many
PR-type defense-related genes appear to be regulated by WRKY
transcription factors (22, 23), which have been analyzed in particular
for the parsley PR1 promoter (21, 24). Transcripts encoding
enzymes implicated in phenylpropanoid metabolism and the synthesis of
the furanocoumarin phytoalexins, including phenylalanine ammonia-lyase
(PAL), 4-coumarate:CoA ligase (4CL), and
S-adenosyl-L-methionine:bergaptol O-methyltransferase (BMT) accumulate even later (20).
Treatment of parsley cells with diphenylene iodonium chloride (DPI)
blocks both the induction of the oxidative burst and phytoalexin
biosynthesis by elicited parsley cells (15). Moreover, it has been
shown that the generation of O Pharmacological and 32P-labeling studies have long since
indicated the importance of protein phosphorylation and protein kinase activities in bringing about pathogen defense responses both in parsley
and other systems (25, 26). Among the many implicated protein kinases,
the activation of MAPKs has been shown to be a consistent and common
response of plant cells following infection and exposure to microbial
elicitors (27, 28). Based upon analysis of the fully sequenced
Arabidopsis thaliana genome, plants appear to contain more
putative MAPKs than any other known organism, including humans (29).
Arabidopsis possesses at least 20 MAPK-encoding genes that
fall into a minimum of four subgroups (30, 31). In all systems whereby
MAPK activity has been studied with respect to elicitor responses,
activation of members of the AtMPK6 subgroup has been described (1,
27). This includes the responses of tobacco SIPK to general elicitors,
such as Harpin and elicitins, TMV infection, and race-specific
elicitation (32-36); alfalfa SIMK to chitin, ergosterol, and
Evidence indicating the importance of MAPK activation for the
elicitation of defense reactions has recently emerged from
gain-of-function experiments whereby MAPKs themselves, or
constitutively active forms of their upstream activators, MAPK kinases
(MAPKKs), were transiently overexpressed in tobacco and
Arabidopsis leaves (45-47). This resulted in a
hypersensitive response-type phenotype in leaves in addition to
activation of genes implicated in the biosynthesis of defense-related
antimicrobial compounds. These observations have recently been
supported by the identification of a complete MAPK cascade from
A. thaliana that is triggered through recognition of flg22
(48). This resulted not only in the accumulation of transcripts of a
group of defense-related genes, but also in increased resistance to
attack by both fungal and bacterial pathogens (48). In addition to
these functions in defense, AtMPK6 homologues have been shown to be
activated in response to various abiotic stresses including osmotic
stresses, ozone exposure, oxidative stress, cold stress, drought, and
treatment with salicylic acid (32, 49-58). It has therefore been
suggested that members of this class of MAPKs may function as points of
cross-talk between various stress signaling pathways in plants (3, 27,
30).
In this article we demonstrate the existence of parallel pathways that
operate to induce the transcriptional activation of particular sets of
defense-related genes in parsley. One pathway is triggered downstream
of the oxidative burst and controls genes implicated in phytoalexin
biosynthesis. The second pathway is independent of the oxidative burst,
but is dependent on MAPK activity. The MAPKs involved are activated in
parsley cells through receptor-mediated recognition of the Pep-13
elicitor and other elicitors of defense reactions, but appear largely
insensitive to abiotic stresses, suggesting that their activation is
primarily associated with pathogen defense. Furthermore, by utilizing a
protoplast transient transfection system employing loss-of-function
MAPK mutants, we demonstrate a requirement of MAPK activity for the
elicitor-mediated oxidative burst-independent activation of
PR genes, which represent classical markers for pathogen
defense responses in plants.
Elicitor Preparations--
The Pep-13 elicitor was chemically
synthesized as previously described (13). Pseudomonas
syringae HrpZ was expressed and purified as a recombinant protein
from Escherichia coli (59). Synthetic N-acetyl
chitoheptaose was provided by Naota Shibuya (University of Tsukuba,
Tsukuba, Japan).
Cell Culture Handling, Treatment, and Protoplast
Isolation--
Cultured parsley cells were maintained in modified
Gamborg's B5-Medium containing 1 mg/liter of 2,4-D as
previously described (60). Protoplasts were isolated 5 days following
transfer of the culture to fresh medium according to previously
described methods (61). Cells were treated by addition of the stimulus to cells previously washed and allowed to equilibrate for 30 min in
fresh medium. All treatments were performed by direct application from
appropriate stock solutions, or in the case of hypo-osmotic treatment,
following dilution in four volumes of medium lacking the osmoticum
(sucrose-free). Following appropriate time points cells were collected
by vacuum filtration, quickly frozen in liquid N2, and
stored at RNA Isolation and Reverse Transcription (RT)-PCR
Analysis--
Total RNA was extracted from parsley cells at different
times after elicitor treatment by using the TRIzol reagent (Invitrogen) according to the manufacturer's guidelines. For RT-PCR analysis cDNA was synthesized from 2 µg of total RNA by using reverse
transcriptase and oligo(dT) or 18Sr primers. The cDNA was
amplified by PCR with the following gene-specific primers: PR2f
(5'-AGGGCTTTCTTCTTGACAT-3'), PR2r (5'-CTTCGATTGACTTTATTATTCTTA-3'),
BMTf (5'-CAAAGCTGGCCCTGGTAACTATT-3'), BMTr
(5'-GGCGTCTCCTTTTGGCACAC-3'), WRKY1f (5'-AATCATAACCATCCAAAGC-3'), WRKY1r (5'-CATATTTCAAACAAGGTACACT-3'), PAL2f (5'-TG
AAATTGCGATGGCTAG-3') PAL2r (5'-TTTAAGTAGCAAGAGCCTT-3'), 18Sf
(5'-GATGGTAGGATAGTGGCCTA-3'), and 18Sr (5'-TGGTTCAGACTAGGACGATA-3').
PCR was performed in a 50-µl reaction volume containing 1×
TaqPCR buffer (Promega, Madison, WI), 0.25 mM
dNTPs, 0.5 units of Taq, and 0.5 µM
concentrations of each primer. The PCR cycle consisted of 2 min at
94 °C, 18 cycles of 30 s at 94 °C, 45 s at 50 °C,
80 s at 72 °C, and one final extension step of 7 min at
72 °C. The products were analyzed by agarose gel electrophoresis.
Acquisition and Analysis of MAPK cDNAs--
A
Site-directed Mutagenesis--
Single point mutations were
introduced into MAPK sequences present within vector pGEM-T (Promega,
Mannheim, Germany) by PCR-based site-directed mutagenesis using the
GeneEditor system (Promega, Mannheim, Germany) and the following
5'-phosphorylated oligonucleotides: PcMPK6Y214F,
5'-GATTTTATGACAGAATTTGTTGTTACAAGATGG; PcMPK6D348N, 5'-CTGCACGACATCAGTAACGAGCCTGTATGTG; PcMPK4Y200F,
5'-GATTTTATGACAGAATTTGTTGTTACTCGCTGG. The manufacturer's
guidelines were followed, and the resulting mutants were verified by sequencing.
Generation and Bacterial Expression of Glutathione S-Transferase
Fusion Proteins--
MAPK-encoding open reading frames were cloned as
BamH1/XhoI PCR fragments into vector pGEX 2T-2
(Amersham Biosciences) for expression in E. coli (strain
BL-21) as fusion proteins containing an N-terminal GST moiety.
Recombinant proteins were subsequently purified using
glutathione-Sepharose 4B according to the manufacturer's guidelines.
Protoplast Transfection and Co-transfection--
For MAPK
activity measurements wild-type or mutated open reading frames were
cloned as NcoI/BamH1 fragments into vector pRT100 (64) behind an introduced c-Myc-encoding sequence and the
35S-cauliflower mosaic virus promoter. 30 µg of each construct were
then used to transfect 2 × 106 protoplasts (~200
µl). Protoplasts and DNA were mixed before the addition of 600 µl
of 25% (w/v) polyethylene glycol (PEG) 6000, pH 9.0, containing 100 mM Ca(NO3)2 and 45 mM
mannitol. Following a 20-min incubation, the protoplasts were collected
by centrifugation after the addition of 7 ml of 0.275 M
Ca(NO3)2, pH 6.0, then resuspended in 4 ml of
B5-sucrose solution (0.28 M sucrose, 1 mg/ml 2,4-D, 3.2 mg/ml B5 medium (solid)) and divided into two Petri dishes. Following 24 h of incubation, the dishes were treated with either 100 nM Pep-13 or water for 15 min. The protoplasts were
then collected by centrifugation following the addition of 25 ml of
0.24 M CaCl2 and quickly frozen in liquid
N2. Co-transfection experiments were performed as already
described with the following modifications: 20 µg of MAPK constructs
were transfected in combination with 5 µg of PR2-promoter-GUS
( Protein Extraction--
Proteins were extracted by grinding
frozen cells in extraction buffer (25 mM Tris-HCl, pH 7.8, 75 mM NaCl, 15 mM EGTA, 15 mM
glycerophosphate, 15 mM 4-nitrophenylphosphate, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM NaF, 0.5 mM Na3VO4,
0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
10 µg/ml aprotinin, 0.1% (v/v) Tween 20) followed by centrifugation
(23,000 × g) for 10 min at 4 °C. Protoplasts were
extracted in the same buffer by vortexing for 30 s. For studies
involving luciferase (LUC) and GUS measurements protoplasts were
extracted in
K2HPO4/KH2PO4, pH 7.5, containing 1 mM dithiothreitol.
GUS and LUC Determinations--
For LUC activities, 10 µl of
protoplast extracts were mixed with 90 µl of LUC substrate (20 mM Tricine, pH 7.8, 2.5 mM MgSO4, 1 mM
(MgCO3)4/Mg(OH)2·5H2O,
0.1 mM EDTA, 30 mM dithiothreitol, 300 µM coenzyme A, 500 µM ATP, 500 µM luciferin) and measured for 5 s in a luminometer
(Luminoscan Ascent plate reader, Labsystems, Frankfurt, Germany). For
GUS activities, 10 µl of protoplast extract was mixed with 40 µl of
substrate (50 mM
Na2HPO4/NaH2PO4, pH 7, 10 mM mercaptoethanol, 2 mM
4-methylumbelliferyl In-gel Protein Kinase Assays--
Cell extracts containing 20 µg of protein per lane were separated on 10% PAGE gels containing
0.1 mg/ml myelin basic protein (MBP) (Sigma). All subsequent
denaturation, renaturation, kinase activity, and washing steps were
performed as previously described (66). Protein kinase activity was
visualized by phosphorimaging (Molecular Dynamics, Krefeld, Germany).
Antibody Production--
Peptides were synthesized corresponding
to amino acid sequences 2-15 in PcMPK6 (DGSTQPSDTVMSDAC); 1-11 in
PcMPK3b (MANPGDGQYDC); and 360-374 in PcMPK4 (CEQHALTEEQMRE). The
peptides were then coupled to keyhole limpet hemocyanin and used to
raise antiserum following immunization of rabbits (Eurogentec, Seraing, Belgium).
Western Blotting--
SDS-PAGE gels were semidry-blotted onto
nitrocellulose membrane (Porablot-NCL, Machery-Nagel, Düren,
Germany). Membranes were blocked at 4 °C overnight in either TBS (20 mM Tris-HCl, 150 mM NaCl), 0.1% (v/v) Tween 20 (TBST) containing 5% (w/v) skimmed milk powder or 5% (w/v) bovine
serum albumin. Primary antibody solutions were prepared in blocking
solution at the following dilutions: 1:10,000 anti-PcMPK6, 3, or 4;
1:500 monoclonal anti-c-Myc (Sigma); 1:15,000 anti-ACTIVETM
MAPK (Promega, Mannheim, Germany). Secondary antibodies coupled to
either horseradish peroxidase or alkaline phosphatase were also
prepared in blocking solution. All washes were performed in TBST. Blots
were developed using either enhanced chemiluminescence (Amersham
Biosciences) or nitro blue terazolium/5-bromo-4-chloro-3-indolyl phosphate precipitate formation.
Immunoprecipitation/Protein Kinase Assays--
Cell or
protoplast extracts containing 100 µg of protein were
immunoprecipitated for 1 h at 4 °C with either MAPK-specific or
c-Myc antibodies coupled to protein A- or protein G-Sepharose (Amersham
Biosciences). Subsequent washing and in vitro MBP
phosphorylation reactions were as described previously (16). Reactions
were stopped by the addition of SDS sample buffer and boiling. The proteins were then separated by SDS-PAGE, and MBP phosphorylation was determined by phosphorimaging.
Differential Activation of Defense Genes through Oxidative
Burst-dependent and -independent Pathways--
We sought
to identify genes whose transcriptional activation occurred
independently of the oxidative burst signaling pathway, by performing
RT-PCR analysis of defense transcript accumulation in Pep-13- and
DPI-treated parsley cells. Prior to RNA isolation, treated cells were
tested to ensure that 10 µM DPI had effectively blocked
Pep-13-induced phytoalexin production, measured 24 h after elicitation (not shown). Transcripts were examined belonging to each of
the "immediate early," "early," and "late" responses in addition to an 18 S rRNA control, and typical results were seen as
illustrated by the duplicate treatments shown in Fig.
1A. The WRKY1
transcription factor and PR2 genes are characteristic
immediate early and early elicitor-responsive genes, respectively (24, 67). As illustrated in Fig. 1A, the transcriptional
activation of each gene, measured at 1 and 4 h post-elicitation,
respectively, was unaffected by DPI treatment. This was in contrast to
genes characteristic of the late response, including PAL2
and BMT genes (20), whose activation was inhibited by DPI
treatment at all time points tested (Fig. 1 displays the 8-h
PAL2 and 24-h BMT). In addition to the genes
shown in Fig. 1A, other genes were examined that were either
sensitive, such as the
S-adenosyl-L-methionine:caffeoyl-CoA O-methyltransferase gene, or insensitive to DPI, such as the
PR1-3 gene. In principle, those genes encoding enzymes of
phenylpropanoid metabolism were most strongly affected (data not
shown). The only unexpected variation to this theme was 4CL whose
transcript accumulation reproducibly showed no inhibition by DPI under
the conditions tested. However, as illustrated by Fig. 1A,
which clearly and consistently showed inhibition by DPI of
PAL gene expression, transcript accumulation of the
PR2 gene was not affected, suggesting that this gene is
regulated by an oxidative burst-independent pathway. To further test
this hypothesis we performed additional experiments aimed at studying
PR2 promoter activity in Pep-13- and DPI-treated transfected
protoplasts. Parsley protoplasts were co-transfected with a plasmid
containing a PR2 promoter-GUS construct (24, 67)
in addition to a 35S-promoter-LUC construct for
normalization. Twenty-four hours after elicitation, the protoplasts
were first tested for phytoalexin synthesis prior to their harvesting
and the determination of GUS and LUC activities in extracts. Fig. 1C shows that 10 µM DPI effectively blocked
phytoalexin synthesis by the transfected protoplasts, which is in
agreement with the responses seen in cells. However, this treatment had
no effect upon the elicitor responsiveness of the PR2
promoter (Fig. 1B), whose activation was indistinguishable
to that seen in solvent-treated cells in response to Pep-13. These data
support the hypothesis that there exist parallel signaling pathways
leading to defense gene expression in parsley cells, one being mediated
through the oxidative burst, while the other appears independent of
this response and results in the activation of PR2 and
WRKY1 genes.
Treatment of Cultured Parsley Cells with the Pep-13 Elicitor
Induces the Activation of At Least Three MAPKs--
We reported
previously that in parsley cells a MAPK is activated in a
receptor-mediated manner following treatment with the Pep-13 elicitor
peptide (16). This activation was shown to be DPI-insensitive,
suggesting that these activities are located upstream or independent of
the oxidative burst and may be involved in the oxidative
burst-insensitive pathway leading to PR gene expression. By using a
modified MBP in-gel assay we found that in fact three MBP kinases were
rapidly activated in response to Pep-13 treatment (Fig.
2, lower panel). The largest
kinase had an apparent molecular weight of 46 kDa and showed a
sustained activation lasting for up to 240 min, while two other
proteins (44 and 42 kDa in size) showed a more transient activation
profile. As the MBP in-gel kinase assay is a sensitive detection method for activated MAPKs, and as the size of the detected kinases are in
agreement with those of this class of protein (31, 68), we hypothesized
that all the elicitor-responsive MBP kinases are indeed MAPKs. To
verify this we used an antiserum that recognizes the dually
phosphorylated TPEYP motif, that is present in
the activation loop of most MAPKs from mammals and yeast (68), and also
from plants (31). The phosphorylation of this motif is mediated by dual
specificity upstream MAPKKs, and leads to the activation of the kinase
activity of the MAPKs (30, 68). In Western blot experiments with
protein extracts from elicitor-treated cells, this
anti-TPEYP antiserum detected three bands of
sizes identical to those seen in the in-gel kinase assay (Fig. 2,
upper panel). In contrast to this, no signals were detected
in protein extracts from non-treated cells, confirming that the
elicitor-responsive MBP kinases are MAPKs. The activation
characteristics of the 46- and 44-kDa kinases matched the pattern seen
in the in-gel assays. In contrast to this, the 42-kDa MAPK gave a
relatively stronger signal in the Western blot experiments and was
detectable up to 240 min after initiation of elicitor treatment.
Cloning of Parsley MAPK cDNA Clones--
In order to identify
the MAPKs detected in the Western blotting and in-gel kinase assays,
and to address the question of their function in elicitor signal
transduction, we initiated efforts to clone a variety of different
MAPK-encoding cDNAs. Screening of a parsley cDNA library
generated from a mixture of elicited and un-elicited cells with a DNA
probe derived from the alfalfa SIMK/MMK1 cDNA (52, 55) was
performed. This resulted in the identification of five independent
cDNAs, out of which four contained complete open reading frames.
Comparison of the deduced amino acid sequences of the encoded kinases
(Fig. 3) indicated that they fall into
three characteristic subgroups. One cDNA encoded a 46-kDa MAPK with
strongest homology to a subclass of enzymes containing AtMPK6 from
A. thaliana, and thus we refer to this sequence as PcMPK6.
Additionally, two parsley cDNAs showed high sequence homology to
one another (89% identity) and encode proteins of indistinguishable
molecular weight (~44 kDa). These proteins exhibit closest homology
to a subgroup of plant MAPKs containing Arabidopsis AtMPK3
and we thus refer to them as PcMPK3a (formerly described as ERMK, Ref.
16) and PcMPK3b. The final cDNA encodes a MAPK of 44 kDa with
closest homology to AtMPK4 and is therefore named PcMPK4.
Use of Specific Antisera Reveal Pep-13-induced Activation of PcMPK6
and 3a/b--
We next wished to determine, whether any of the parsley
MAPK cDNAs we had cloned encoded one of the elicitor-responsive
enzymes seen in the in-gel assay and Western blotting experiments. For this purpose antibodies discriminating between the different MAPK subgroups were produced by immunizing rabbits with synthetic peptides corresponding to the extreme N-terminal amino acid sequences of PcMPK6
and 3b and the extreme C terminus of PcMPK4, respectively. The
specificity of the obtained antisera was tested in Western blot
experiments with recombinant MAPKs produced as GST fusion proteins in
E. coli (Fig. 4A).
The antiserum generated against the peptide sequence of PcMPK6 only
detected MPK6, while anti-PcMPK4 peptide antiserum only cross-reacted
with MPK4. As predicted from the amino acid sequence conservation, the
antiserum generated against the N-terminal peptide of PcMPK3b detected
both PcMPK3b and 3a recombinant kinases with equal affinity but did not
recognize either of the other tested MAPKs (Fig. 4A).
The antisera were then used in coupled immunoprecipitation/in
vitro MBP kinase assays. Immunoprecipitations performed with anti-PcMPK6 and 3b sera precipitated MBP kinase activity from extracts
of Pep-13-treated cells (Fig. 4B). These activities
increased rapidly, within 5 min of elicitor treatment, and persisted at high levels for up to 240 min thereafter. To further test the specificity of the antisera in the immunoprecipitation experiments we
performed competition studies using the peptides to which the antisera
were generated. Fig. 4C demonstrates that the addition of a
large excess of the peptide corresponding to the N terminus of PcMPK6
(6-N) prevented the immunoprecipitation of the activated PcMPK6 from
elicited extracts. In contrast, addition of the peptide corresponding
to the N terminus of the PcMPK3 proteins did not affect the
immunoprecipitation of PcMPK6 by this antibody. Fig. 4C also
shows the same pattern for the immunoprecipitation of the activated
PcMPK3(s), whose immunoprecipitation was only blocked by addition of
the N-terminal peptide of PcMPK3b (3-N). In contrast, the antiserum
specific for PcMPK4 failed to immunoprecipitate an activated protein
kinase from Pep-13-treated cells. These observations demonstrate that
PcMPK6 and PcMPK3a and/or 3b are activated following Pep-13 treatment
while PcMPK4 is not.
Transient Protoplast Transformation Confirms Pep-13-induced
Activation of both PcMPK 3a and 3b--
As described above and shown
in Fig. 4A, the antiserum that immunoprecipitates activated
PcMPK3 is unable to discriminate between the PcMPK3a and 3b homologues.
Peptides that diverge in the highly homologous MPK3a and MPK3b proteins
were found to be unsuitable for antibody production. We therefore
decided to test, whether both these kinases were activated during the
elicitor response by employing a protoplast transient expression
system. N-terminal c-Myc-tagged PcMPK3a, 3b or 4 were overexpressed
through the activity of the 35S-promoter in parsley protoplasts. The
protoplasts were then treated with Pep-13, and immunoprecipitations
were performed on cell extracts using c-Myc antibodies. The kinase
activities of the immunoprecipitated epitope-tagged MAPKs were then
determined by MBP phosphorylation. Equal expression of the constructs
was verified by Western blotting with the c-Myc antiserum. As shown in
Fig. 4D, both c-Myc-PcMPK3a and c-Myc-PcMPK3b were activated following Pep-13 treatment, suggesting that both kinases are activated in the parsley elicitor response, and make up together one of the
activated MAPKs seen in the initial in-gel and Western blot experiments. In contrast to this, and in agreement with the
immunoprecipitation experiments performed with the kinase-specific
antibodies, c-Myc-PcMPK4 was not activated following treatment with
Pep-13 (Fig. 4B).
Responses of PcMPK6 and PcMPK3 to Biotic and Abiotic Stress
Stimuli--
Studies performed in other plant systems have
demonstrated that MAPK activation occurs as a common feature of many
plant stress responses (30). In order to determine whether any of the
parsley MAPKs plays a more general role in plant stress adaptation, we tested whether a selection of commonly studied stress treatments would
induce activation of PcMPK6 and PcMPK3a/b. A range of treatments was
applied to parsley cell cultures based upon conditions shown to
activate MAPK signaling in cell cultures or protoplasts of alfalfa,
tobacco, and Arabidopsis (32, 49-58).
Immunoprecipitation/MBP phosphorylation assays were then performed and
kinase activities were expressed against that seen in response to
treatment with 100 nM Pep-13, which reproducibly gave the
strongest kinase activation. The results of these investigations are
presented in Fig. 5. No significant
activation of either the PcMPK6 or PcMPK3a/b kinases were observed
following treatments of parsley cells with 1 µM N-acetyl chitoheptaose (chitin), 250 µM
salicylic acid, 250 mM NaCl, 500 nM sorbitol,
or 4 volumes of hypotonic buffer (hypo-osmotic). These treatments did
also not stimulate phytoalexin synthesis in parsley cells (not shown).
Treatment of cells with 500 nM recombinant HrpZ from
P. syringae pv. phaseolicola activated both
PcMPK6 (~80% of Pep-13 response) and the PcMPK3 kinases (~45% of
Pep-13 response). HrpZ also acted as an elicitor of parsley cells and
induced phytoalexin synthesis with an EC50 in the nanomolar
range.2 The concentration of
500 nM HrpZ used here gave maximal responses with respect
to phytoalexin synthesis by parsley cells (not shown). Only two
treatments were able to separate the activation of the different
elicitor-responsive MAPKs. Treatment with 20 mM
H2O2 induced the activation of PcMPK6 (~35%
of Pep-13 response), but did not activate the PcMPK3 kinases. PcMPK6
was found to be activated by H2O2
concentrations of between 2 and 20 mM in a
dose-dependent manner, whereas concentrations up to 1 mM had no effect (not shown). PcMPK6 was also activated in
the absence of PcMPK3a/b activity following addition of a combination
of heavy metals. This suggests that under some circumstances the
elicitor-responsive MAPKs can be activated independently of one
another, possibly during oxidative or heavy metal stress signaling. All
activity measurements were performed in triplicate 15 min following the
application of the treatment. All treatments were also analyzed after
30 min (data not shown) and yielded identical results to those shown
for 15 min (Fig. 5). Neither H2O2 nor heavy
metals stimulated phytoalexin accumulation 24 h after elicitation
(not shown).
PcMPK6 Activation through Phosphorylation of Tyrosine 214 Is
Required for PR Gene Promoter Activity Following Pep-13 Treatment of
Parsley Protoplasts--
Previous work had suggested that activated
MAPKs might play roles in the control of elicitor-responsive gene
expression in parsley (16). In order to directly test this, we
performed transient expression experiments using dominant negative MAPK
mutants to address, through a loss-of-function approach, the
involvement of MAPKs in the activation of Pep-13-induced defense gene
activation. Single point mutations predicted to influence kinase
activity were introduced into the elicitor-responsive PcMPK6 and the
un-responsive PcMPK4. The conserved tyrosine residue present within the
TEY activation loop motif was mutated to phenylalanine in both PcMPK6 (6Y214F) and PcMPK4 (4Y200F). These mutations were predicted to render
the protein kinases incapable of activation by upstream MAPKK-type
activities (69). Both constructs contained an N-terminal c-Myc tag that
enabled the determination of expression levels via Western blotting in
addition to kinase activities through immunoprecipitation/MBP kinase
assays on protoplast extracts. As shown in Fig.
6A, wild-type c-Myc-PcMPK6 was
activated following treatment of transfected protoplasts with Pep-13.
This supported the previous data that demonstrated activation of this
MAPK in parsley cells. However, we were unable to detect any activation of the PcMPK6 Y214F mutant in Pep-13-treated transfected protoplasts. Given that the expression levels were equal to those of the wild-type construct (Fig. 6A, lower panel), it appears that
the Y214F mutation renders PcMPK6 incapable of activation through
upstream MAPKK activities. For this reason the PcMPK6Y214F construct
provided us with an ideal dominant negative form of the MAPK for
further analysis of its influence on defense gene expression. As
expected, and also shown in Fig. 6A, the PcMPK4 wild-type
and 4Y200F kinases were again seen to be un-responsive to Pep-13
elicitor treatment.
The parsley PR2 gene promoter has been studied in much
detail (67, 70), and we have already demonstrated that its activation, like MAPK activation, occurs independently of the Pep-13-triggered oxidative burst response (Fig. 1, A and B).
Therefore, we selected this promoter for use in co-transfection assays,
to determine whether overexpression of an inactive MAPK impairs
PR2 promoter activation. Plasmids of the
PR2-GUS construct (24) were co-transfected along
with the constructs shown in Fig. 6A and an
35S-promoter-LUC construct for normalization purposes.
Following 8 h of incubation, the protoplasts were treated with 100 nM Pep-13 and then left for a further 14 h.
Protoplasts were then harvested, and GUS and LUC activity
determinations were performed upon protein extracts. A typical data set
for these co-transfection experiments is shown in Fig. 6B.
Transfection with the PcMPK6 wild-type construct led to little or no
reduction (~20%) in Pep-13-induced promoter activity compared with
the co-transfections performed with the empty vector controls. However,
co-transfection of the dominant negative form (6Y214F) of PcMPK6 led to
a dramatic reduction in Pep-13-induced PR2 promoter activity
(~80% inhibition). In addition to this, the basal (non-elicited)
levels of activity were also reduced, suggesting that the PcMPK6Y214F
construct has a strong negative effect on both, activity and Pep-13
responsiveness of this promoter. Importantly, Fig. 6B also
shows that co-transfection with either wild-type or Y200F forms of
PcMPK4 had no effect on the Pep-13 responsiveness of the promoter. This
agrees well with the fact that PcMPK4 is not activated in response to
the Pep-13 elicitor and is therefore unlikely to trigger downstream
events of the defense response.
Receptor-mediated perception of plant pathogens results in the
activation of intracellular signaling pathways that function in
triggering downstream defense reactions (3, 4). Defense reactions
themselves are characterized by large-scale transcriptional activation
of genes, whose products are believed to be actively involved in
resisting pathogen attack (20, 71). Our studies have demonstrated that
particular signaling pathways are responsible for the transcriptional
activation of distinct subsets of defense genes. It is clear that both
oxidative burst-dependent and -independent pathways play
roles in this response. Previous studies, and those presented here,
have demonstrated that the generation of O Changes in protein phosphorylation have long been known to occur as a
consequence of treatment of plant cells with microbial elicitors (25,
26). Among the many protein kinases believed to be involved in these
events, members of the MAPK family are becoming increasingly recognized
as playing important roles in defense signaling (27, 28). In the
present study we have shown that in parsley cells at least four
different MAPKs are activated in a receptor-dependent
manner by the Phytophthora-derived elicitor peptide, Pep-13.
Three of these MAPKs could be identified by molecular cloning,
immunoprecipitation, and transient transformation assays, and they were
found to be homologous to MAPKs implicated in defense signaling in
other plant species (3, 27, 31, 48). The initial in-gel and Western
blotting experiments also suggest that at least one elicitor-responsive
MAPK remains to be identified. Based upon the activation profile seen
for each of the kinases with these methods, and compared with the
activities determined through specific immunoprecipitation/kinase
assays, this remaining kinase would appear to be activated more
transiently than the PcMPK6 and PcMPK3 kinases. Given the lack of any
cross-reacting antisera we have as yet been unable to identify this
additional activity.
The MAPKs we have identified as Pep-13-responsive have homology to
those seen to be implicated in elicitor signaling in other systems,
i.e. homologues of the AtMPK6 and AtMPK3 MAPKs from
Arabidopsis (27). In addition, we isolated a parsley
homologue of AtMPK4, a MAPK shown to be a negative regulator of
disease-resistance responses in Arabidopsis (40). This MAPK
was not responsive to elicitors (Pep-13 or HrpZ) in parsley cells, and
we cannot say whether it is functionally homologous to the
Arabidopsis MAP kinase 4, which was previously described as
being activated in response to Harpin treatments (39). We also isolated
two parsley MAPKs belonging to the AtMPK3 class and have shown that
both become activated following Pep-13 treatment. Whether these two
kinases share a common function remains to be determined. One might
suppose that they could have, despite their high degree of sequence
identity, slight differences with respect to substrate specificities
and interaction with activators and deactivators, or even that their expression profile in planta might differ. In
Arabidopsis quite a number of such highly homologous MAPK
pairs have been identified (29, 31), and it will be interesting in the
future to learn to what extent their functions are redundant.
The other Pep-13-responsive MAPK was shown to be PcMPK6, a homologue of
the AtMPK6, SIPK, and SIMK MAPKs from Arabidopsis, tobacco,
and alfalfa, respectively, each of which has been shown to be activated
following elicitation (32, 35, 39). As reflected in their nomenclature,
many of these kinases have also been shown to become activated
following abiotic stress treatments including salicylic acid (54),
salt, or hyper-osmotic (52), hypo-osmotic, and oxidative stresses (51,
58). It was therefore surprising that no significant increases in
PcMPK6 or PcMPK3 activities were observed when cultured parsley cells
were placed under conditions described to activate MAPKs in other
systems. The exceptions, from the conditions tested, were
H2O2 and heavy metal treatments that activated
PcMPK6 alone. This may reflect a role for this class of MAPKs in
responses to oxidative stress, which has been suggested, with respect
to treatment with millimolar concentrations of
H2O2, by the activation of AtMPK6 in
Arabidopsis (51, 58). It has also been shown that treatment
of plants with micromolar concentrations of heavy metals, including
copper, results in the transcript accumulation of many oxidative
stress-protective and -responsive genes (72). AtMPK6 class MAPKs may
therefore operate as components of signal cascades initiated by these
environmental stimuli. In this case the specificity of the outcome may
be determined by the relative duration of the kinase activation (as in
our hands, the oxidative stress PcMPK6 activation was more transient
than that seen in response to elicitors, not shown) or in the
contribution made by parallel signaling pathways. Perhaps
significantly, none of the abiotic treatments described resulted in the
activation of PcMPK3a or 3b. Even with respect to PcMPK6, the highest
and most persistent levels of activity strictly correlated with
treatments that induce phytoalexin synthesis in parsley cells,
i.e. elicitors. P. syringae HrpZ and the NPP1
protein from P. parasitica effectively and strongly
activated both PcMPK6 and PcMPK3, although to levels not quite that
seen following Pep-13 treatment (not shown)
(73).3 However, these
activities also remained significantly higher than the activity of
PcMPK6 during the oxidative stress responses. This alone suggests that
these MAPKs play important roles in plant defense signaling. The use of
different elicitors also highlights the way in which different
perception mechanisms can and do converge upon these kinases, as has
also been reported in other systems (37). The identification of the
sequential upstream components of these MAPK cascades, and the
determination of the initial convergence points will be of significant
interest in the future.
What functions do MAPKs have in plant defense responses? Recent
gain-of-function experiments in tobacco and Arabidopsis
leaves overexpressing constitutively active MAPKK or wild-type SIPK
resulted in the formation of hypersensitive response-like necrotic
lesions (45-47). In addition, accumulation of transcripts associated
with defense responses was observed. This clearly shows that
SIPK/AtMPK6 homologues or their upstream MAPKK activities when
overexpressed can trigger defense-related reactions. The mechanism by
which this is achieved remains, however, unclear and corresponding
loss-of-function approaches were not presented. The recent complete
functional identification of a MAPK cascade from Arabidopsis
that is sufficient to provide increased resistance to pathogen attack
has now confirmed the importance of MAPK signaling for plant defense
(48). We chose to investigate the importance of MAPK activity for the
induction of downstream defense responses that occurred independently
of the oxidative burst, using a loss-of-function approach. Our studies have shown that PR gene expression (this study) and MAPK
activation (16) in parsley cells occurred upstream or independently of the oxidative burst. It was therefore of interest to see whether one
response was linked to the other. Overexpression of PcMPK6 in parsley
protoplasts followed by Pep-13 treatment resulted in activation of the
kinase in a manner indistinguishable to that observed in cells. However
a Y-F activation loop mutant could not be activated in this system,
confirming this tyrosine phosphorylation reaction as essential for
kinase activation during the elicitor response that likely results from
activation of an upstream MAPKK activity. Moreover, in co-transfection
experiments, this Y-F mutant gave a strong inhibition of the elicitor
responsiveness of the PR2 promoter activity. As
PR gene expression is regarded as a classical marker for
plant defense, we can conclude that PcMPK6 plays an essential role in
the induction of these defense reactions. It is unlikely that the
kinase is solely responsible for this activity, since, as we
demonstrated here with respect to oxidative stress, it can be activated
by treatments that do not trigger typical defense reactions. We
therefore believe PcMPK6 activation to be a necessary, but not
sufficient component for PR2 gene expression during defense.
Interestingly, co-transfection experiments using a Y-F activation loop
mutant of PcMPK3b also showed a degree of inhibition of the
PR2 promoter, although not to the levels shown for the
PcMPK6 mutant construct (not shown). This may perhaps represent some
redundancy in MAPK signaling pathways during defense, where in almost
all cases studied to date, activity of the MPK3 class kinases is seen
in addition to the MPK6 class. However, hypotheses of this type need to
be addressed with the use of specific knockouts, which is seen as
difficult in plants, or the identification of specific substrates. It
is most likely that the overexpression of the PcMPK6Y214F construct
blocks the correct activation of the endogenous wild-type activity and
results in a reduced level of phosphorylation of a protein(s) that
regulates PR2 promoter activity. WRKY-type transcription
factors were first identified in parsley as proteins that bind to
response elements in these promoters (24), which are not present in
promoters of Pep-13-responsive parsley genes encoding phytoalexin
biosynthetic enzymes activated via the oxidative burst (74).
WRKY transcription factors have since been implicated in disease
resistance responses of Arabidopsis, occurring downstream of
MAPK signaling (48). Future studies should address the link between
MAPK and WRKY activities and will require the identification of MAPK
substrates, which at present remain unknown. The identification of
these unknown proteins represents a major future challenge for research
in plant MAPK signaling and function in mediating plant defense.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucans (37); A. thaliana AtMPK6 to bacterial elicitors
including the flg22 peptide from flagellin (38) and Harpin (39). It was
recently demonstrated for A. thaliana that MAPKs can also
act as negative regulators of defense responses, as shown for AtMPK4
mutants (40); however, this would appear to be contradictory to the
activation of this kinase described in response to Harpin (39). Members
of a second closely related class of MAPKs, initially characterized in
tobacco as being activated following wounding (WIPK) (41, 42), and
having homology to AtMPK3, have also been implicated in pathogen
defense signaling (34, 43, 44). Our previous studies demonstrated the
activation of such a homologue, described as ERM kinase,
following treatment of parsley cells with the Pep-13 elicitor (16).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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80 °C until use.
-ZAPTM II (Stratagene, Heidelberg, Germany) phage
cDNA library was prepared from mRNA of elicited and un-elicited
cultured parsley cells according to the manufacturer's guidelines and
screened with radioactively labeled probes corresponding to the open
reading frames of the MAPK-encoding genes MMK1 (62, 63) and
MMK4 from alfalfa (55). Each probe was used to screen 6 × 105 plaques and resulted in the acquisition of 8 cDNA clones encoding 4 different full-length open reading frames.
Sequence analysis of the cDNAs and their encoded proteins were
performed using the DNASIS 2.1 software (Hitachi, Tokyo, Japan).
-glucuronidase) construct (24) and 5 µg of the normalization
plasmid, pRTLUC (65). Following an 8-h incubation in B5-sucrose medium,
the protoplasts were treated either with water or 100 nM
Pep-13 and incubated for a further 14 h. Protoplasts were then
collected and stored as described.
-D-glucopyranoside, 0.1 mM EDTA, 0.1% (v/v) Triton X-100) and incubated at
37 °C for 1 h. Following addition of 200 µl of 0.4 M Na(CO3)2 fluorescence was
measured at 360 nm excitation/440 nm emission using the Cytofluor II
apparatus (Biosearch, Bedford, MA).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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View larger version (26K):
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Fig. 1.
Differential activation of parsley
defense-related genes through oxidative burst-dependent and
-independent pathways in response to the Pep-13 elicitor.
A, RT-PCR analysis of transcript accumulation demonstrates
the existence of parallel independent pathways leading to defense gene
expression. Parsley cells were pretreated for 30 min with either 10 µM DPI (+) or an equivalent volume of Me2SO
( ) prior to addition of 100 nM Pep-13 (+). Cells were
then harvested at the following time points: WRKY1, 1 h
post-elicitation; PR2, 4 h; PAL2, 8 h;
and BMT, 24 h. RNA was isolated and used for RT-PCR
analysis in order to determine defense gene transcript levels. The
transcript level of 18 S rRNA was also determined for each time point
for normalization purposes, and each treatment is shown in duplicate.
B, promoter activity studies confirm oxidative
burst-independent transcriptional activation of the PR2
gene. Parsley protoplasts were transfected with a PR2
promoter fused to the gene encoding GUS in addition to an
35S-promoter-driven LUC construct (35S-LUC). Protoplasts
were then treated with 10 µM DPI or a corresponding
volume of Me2SO 30 min prior to addition of 100 nM Pep-13. Following a further 14-h incubation, the
protoplasts were harvested, extracts were generated, and GUS and LUC
activity determinations were performed. The data are expressed as
GUS/LUC activities for each treatment (n = 4).
C, fluorescence of the culture medium was also measured
prior to protoplast harvesting to confirm the inhibitory effect of 10 µM DPI upon phytoalexin production.
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Fig. 2.
The Pep-13 elicitor activates at least three
independent MAPKs in cultured parsley cells. Cells treated with
100 nM Pep-13 were harvested after various time periods and
cell extracts were prepared. Proteins (20 µg/lane) were then
separated by SDS-PAGE, blotted, and probed with antibodies
cross-reacting with activated MAPKs
(anti-TPEYP, upper
panel), or separated on SDS-PAGE gels containing 0.1 mg/ml MBP to
test in-gel kinase activities (lower panel). Both techniques
revealed the activation of at least three MAPKs
(Mr ~46, 44, and 42) in Pep-13-treated
cells.
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Fig. 3.
Sequence alignments of the encoded proteins
of four parsley MAPK cDNA clones. Four MAPK encoding cDNA
clones were isolated from a library generated from a mixture of
Pep-13-treated and untreated parsley cells. Based upon the homology to
A. thaliana MAPKs the parsley MAPK are referred to as
PcMPK6, 3a, 3b, and 4. Alignments between the encoded amino acid
sequences are shown, and fully conserved residues are indicated in
black boxes.
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Fig. 4.
Use of MAPK-specific antisera and
immunoprecipitation/protein kinase assays identify PcMPK6, 3a, and 3b
as Pep-13-responsive. A, specificity of peptide
antibodies raised against peptide sequences contained within PcMPK6,
3b, and 4. Antisera cross-reactivity was tested by Western blotting
against each of the recombinant MAPKs (100 ng/lane). B,
immune complex-protein kinase assays. Cultured parsley cells were
elicited with 100 nM Pep-13, and cell extracts containing
200 µg of protein were immunoprecipitated with the indicated
antiserum coupled to protein A-Sepharose. The immune complexes were
then tested for kinase activity by measuring incorporation of
32P into MBP visualized following separation by SDS-PAGE.
C, specificity of antisera in immune kinase assays. MAPKs
were immunoprecipitated from extracts (200 µg of protein) of
Pep-13-elicited parsley cells with PcMPK6 and PcMPK3b sera in the
presence or absence of competitor peptides (20 µg/ml) corresponding
to the N termini of PcMPK6 (6-N) and PcMPK3a and 3b (3-N). Kinase
activity of the immune complexes was again determined using MBP as
substrate. D, Pep-13 activates both PcMPK3a and PcMPK3b.
PcMPK3a and 3b possessing an N-terminal c-Myc epitope tag were
transiently expressed in parsley protoplasts through the activity of
the 35S-promoter. Protoplasts were elicited with Pep-13 for 15 min
24 h after transfection. Proteins (100 µg) were extracted and
immunoprecipitated with an antibody to the c-Myc tag. The kinase
activity of the immune complexes was then determined using MBP as
substrate.
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Fig. 5.
The responses of PcMPK6 and 3a/b to a range
of biotic and abiotic stresses. The responses of the PcMPK6 and
PcMPK3a/b in parsley cells treated with various abiotic and biotic
stress stimuli were determined by immunoprecipitation/protein kinase
assays using MBP as substrate. All treatments were applied for 15 min.
The metal mix contained 100 µM CdCl2, 250 µM ZnCl2, and 250 µM
CuCl2. Proteins (100 µg) were extracted and
immunoprecipitated with either PcMPK6- or PcMPK3a/b-specific antisera.
Kinase activities in response to each treatment were determined in
triplicate by phosphorimage analysis and plotted against the maximum
measurable response seen following treatment of cells with 100 nM Pep-13. The kinase activity of PcMPK6 is represented by
the white bars and for PcMPK3a and 3b by the black
bars.
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Fig. 6.
PcMPK6 activation through phosphorylation of
Tyr-214 is required for Pep-13-induced PR2 promoter
activity. A, activity of MAPK mutants in transfected
protoplasts treated with 100 nM Pep-13. PcMPK6 and PcMPK4
wild-type and mutant constructs containing single point mutations in
the activation loop motif, TEY (6Y214F, 4Y200F), were generated and
transfected into protoplasts as c-Myc-tagged versions. Following
24 h expression under the control of the 35S-promoter the
protoplasts were treated with either water (control) or 100 nM Pep-13 for 15 min. Proteins (100 µg) were extracted
and MAPKs immunoprecipitated with an anti-c-Myc antibody. Kinase
activities present in the immune complexes were then determined by MBP
phosphorylation (upper and middle panels).
Correct and equal expression of all constructs was tested by Western
blotting of 10 µg of protein with c-Myc antibody (lower
panel). B, transient expression of a dominant negative
form of PcMPK6 (6Y214F) blocks the elicitor responsiveness of the
parsley PR2 promoter. Parsley protoplasts were
co-transfected with a PR2 promoter construct fused to
GUS, together with the MAPK constructs shown in A
or empty vector (control), and a 35S-promoter-LUC
construct for normalization. Eight hours after transfection the
protoplasts were treated with either water ( ) or 100 nM
Pep-13 (+). Following another 14-h incubation, the protoplasts were
harvested, and total extracts were prepared and assayed for GUS and LUC
activities. The influence of each co-transfected MAPK construct on the
PR2 promoter activity was determined in triplicate and
plotted against the effect of co-transfection with the empty vector
(control).
DISCUSSION
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DISCUSSION
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Heribert Hirt for providing the
MMK1 and MMK4 cDNAs and Dr. Naoto Shibuya for
a gift of N-acetyl chitoheptaose. Advice provided by
Christoph Ve and Dr. Stephan Clemens for heavy metal treatment of
cultured parsley cells is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Sche 235/3-1-235/3-4), European Commission Grant HPRN-CT-2000-00093, CRISP, and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) Y12875, AY173415, AY173414, and AY17343.
Present address: Laboratoire de Biologie Moleculaire des Relations
Plantes-Microorganismes, UMR CNRS/INRA 215, BP 27, 31326 Castanet-Tolosan cedex, France.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 49-345-5582-1400; Fax: 49-345-5582-1409; E-mail: dscheel@ipb-halle.de.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M208200200
2 J. Lee and T. Nürnberger, personal communication.
3 T. Nürnberger, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ROS, reactive oxygen
species;
PR, pathogenesis-related;
MAPK, mitogen-activated protein
kinase;
GST, glutathione S-transferase;
GUS, -glucuronidase;
LUC, luciferase;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MBP, myelin basic protein;
DPI, diphenylene iodonium;
Me2SO, dimethyl sulfoxide;
BMT, S-adenosyl-L-methionine:bergaptol
O-methyltransferase;
ERM kinase, elicitor-responsive MAPK;
2, 4-D,2,4-dichlorophenoxy acetic acid.
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