From the Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica-Consejo Superior de Investigaciones Científicas, Camino de Vera s/n, 46022-Valencia, Spain
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ABSTRACT |
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Screening of a genomic library from tomato plants
(Lycopersicon esculentum) with a cDNA probe encoding a
subtilisin-like protease (PR-P69) that is induced at the
transcriptional level following pathogen attack (Tornero, P., Conejero,
V., and Vera, P. (1996) Proc. Natl. Acad. Sci. U. S. A.
93, 6332-6337) resulted in the isolation of a cluster of genomic
clones that comprise a tandem of four different subtilisin-like
protease genes (P69A, P69B, P69C,
and P69D). Sequence analyses and comparison of the encoded proteins revealed that all are closely related (79 to 88% identity), suggesting that all are derived from a common ancestral gene. mRNA
expression analysis as well as studies of transgenic plants transformed
with promoter- Proteolysis is fundamental for the normal functioning of
multicellular organisms and plays key roles in a variety of processes such as development, physiology, defense and stress responses, and
adaptation to the changing environment. In plants, despite the
importance of all these processes and involvement of different classes
of proteinases (Refs. 1-5, and references therein), it still remains
to be defined more precisely what components and molecular mechanisms
are responsible for regulating specific aspects of protein
degradation/processing. A major task for research will be to determine
which pathway of proteolysis is responsible for the degradation of
particular proteins.
The serine proteases are one of the best characterized groups of
proteolytic enzymes in higher organisms. They can be grouped in six
clans, of which one of the largest is the subtilisin-like clan (EC
3.4.21.14) that includes over 200 different members. Despite this
wealth of knowledge, very little is know about subtilisin-like proteases in plants. Recently, we and others have shown the existence of members of this clan in plants, including Arabidopsis
(6), tomato (7, 8), melon (9), and Lilium plants (10).
According to a recent classification (11), the subtilisin-like
proteases from plants can be grouped within the Pyrolysin
subfamily, which is highly related to the Kexin subfamily of
proteases involved in the posttranslational processing of peptide
hormones (12, 13). Comparative molecular, biochemical, and cellular
studies indicate that the subgroup of plant subtilisin-like enzymes are characterized by the presence of a large polypeptide sequence insertion
preceding the reactive Ser residue and/or long C-terminal extensions
relative to all other subtilisin-like proteases. Furthermore, they were
found to be glycosylated and to be secreted to the plant extracellular
matrix (ECM) where they accumulated and presumably exert their
biochemical function(s) by recognizing and processing pericellular
substrates (7-11).
We here describe the isolation and characterization of a genomic
cluster comprised of four genes encoding different, but highly related,
members of subtilisin-like proteases (named as P69A, P69B, P69C, and P69D). While the four
clustered proteases exhibit a high degree of amino acid sequence
identity, we show that they are differentially regulated at the
transcriptional level, each showing a different expression pattern,
either during normal plant development or following pathogenic attack.
Plant Material, Growth Conditions,
Treatments--
Lycopersicon esculentum cv. Rutgers and
Arabidopsis thaliana (Col-0) plants were grown at 22 °C
in growth chambers programmed for a 14-h light and 10-h dark cycle.
Fully expanded leaves or rosette leaves were sprayed with SA (0.5 mM) or buffer alone (50 mM phosphate buffer, pH
7.2), and samples were taken for analysis after 48 h. Suspensions
of Pseudomonas syringae strains (0.09 O.D.) were infiltrated
locally in one part of the leaf. Control (mock) plants were injected
similarly with the solution containing no bacteria. Samples were
analyzed 24-48 h post-inoculation.
Library Screening and DNA Sequence Analysis--
A tomato
genomic DNA library constructed in RT-PCR--
cDNA synthesis, quantification of the products,
and reverse transcriptase-mediated
PCR1 were conducted as
described (8). The oligonucleotide primer pairs (50 pmol each), a1 + a2
(ATGGGATTCTT GAAAATCCTT + TCAACAAAAGTGCAATTGGACTTC), b1 + b2 (ATGGGATT
CTTGAAAATCCTT + CCTAGGCAGACACAACTGCAAT), c1 + c2 (ATGGGAT
TCTTGAAAATCCTT + TCATATCAATGTCCTCTCAAAGAG) and d1 + d2
(ATGGGATTCTTGAAAATT + TTATTCAGCAGACACTCTAACTGC), specific for the
amplification of P69A, P69B, P69C, and
P69D sequences, respectively, were used to amplify by PCR
the in vitro synthesized single-stranded cDNA from the
different mRNA sources in a Perkin-Elmer/Cetus DNA Cycler. PCR
amplification was programmed as described before (8). The amplified DNA
fragments were visualized in agarose gels, or alternatively, they were
hybridized with a radiolabeled p26 cDNA probe. The inability of
each combination of primers to amplify the closely related P69
sequences was confirmed in control PCR reactions that included 10 ng of
plasmid DNA containing each of the four P69 ORFs as template.
Promoter-GUS Fusion, Plant Transformation, and Analysis of
Transgenic Plants--
Oligonucleotides GEN69a
(5'-GCCCGGGGGCTTGCAAATGGTATAG-3'), GEN69b
(5'-GCCCGGGGGCTAGCTAATACAACAAGTG-3'), GEN69c
(5'-GCCCGGGGGCTGCAAATACAAGAAG-3'), and GEN69d (5'-GCC
CCGGGTTGCTGGTATAGAGTAATTGG-3') in combination with the T7
oligonucleotide served as primers for the incorporation of a
synthetic SmaI restriction site in each promoter by
site-directed mutagenesis (15). These primers introduced the
SmaI site at positions Characterization of a Genomic Cluster Containing Four Genes
Encoding Highly Related P69 Proteases--
A DNA fragment encoding the
signal sequence and propeptide for the previously identified P69
protease was obtained from plasmid p26 (7) and used as a radiolabeled
probe to screen a tomato genomic library constructed in
Computer-aided comparison of the amino acid sequence at the
NH2 termini (Fig. 2) along with the hydropathy profiles
(data not shown) identified within the cluster indicated the existence of a preprosequence in all cases for the four P69 proteases. The prosequence comprises a hydrophobic signal peptide at the extreme N
terminus which, accordingly to von Heijne (17), is cleaved C-terminal
of the conserved Ser-22 residue. In all cases, the signal peptide is
followed by a 92-amino acid prosequence, which is a typical feature of
proteases of the subtilisin family, and its cleavage is mandatory for
the generation of the active protease from the inactive zymogen (18).
The putative N-terminal amino acid of the mature proteins is the
conserved Thr-115, identified also by comparison with other plant
subtilisin-like proteases (6-11). The predicted mature enzymes thus
contain 631, 631, 552, and 632 amino acids for the P69A, P69B, P69C,
and P69D isoforms, respectively. Within the four mature proteins, the
amino acid residues Asp-146, His-203, and Ser-532 (or Ser-531 for P69B
and p69C), corresponding to the catalytic site (catalytic triad)
essential for the enzymatic activity of subtilisin-like members to
function as proteases, were identified (Fig. 2). Also the proteases of the four P69s in the cluster have an Asn residue (Asn-306, or Asn-305
for P69B and P69C) that has been found to be highly conserved in this
position and that is catalytically important in the subtilisins (12,
13). However, sequences close to this Asn-306 are highly variable
within the four P69s (Fig. 2). In all cases there is also an insertion
of a long sequence (226 amino acids) between the stabilizing Asn-306
and the reactive Ser-532, relative to all other subtilisin-like
proteases (11), in which these two residues are separated by much
shorter distances. This displacement has also been observed in the
three other subtilisin-like proteinases recently identified from plants
(6, 9, 10) and could represent a characteristic signature of the
subtilisin enzymes from plants (Fig. 3).
Expression Analysis of P69 Genes--
The differential expression
pattern of the four P69 genes was initially determined by gene-specific
RT-PCR reactions combined with Southern blot analyses. In
vitro synthesized single-stranded cDNAs from mRNA samples
of fully grown leaves from healthy and Pseudomonas syringae
pv. syringae-infected tomato plants were assayed by PCR
using sets of primers which were specific for each P69 gene
member. Primer specificity was demonstrated in pilot experiments using
each of the individually cloned gene as template in the PCR reaction
(Fig. 4). Whereas P69A
mRNAs accumulate to detectable levels in fully expanded leaves of
healthy tomato plants, the P69D mRNAs accumulate in
marginal amounts in the same leaf samples. Neither P69A nor
P69D were found to be induced over basal levels in leaves
that were infected with P. syringae. Conversely, similar
RT-PCR analysis with primers specific for the P69B gene and
the P69C gene indicated that while the corresponding
mRNAs were nearly undetectable in leaves from healthy control
plants, a dramatic accumulation of the transcripts occurred in P. syringae-infected leaves (Fig. 4).
Developmental and Tissue-specific Regulation of the Different P69
Promoters in Transgenic Plants--
To investigate in detail the
spatial pattern of expression of the four P69 genes, each of the 5'
flanking promoter regions was fused to the
To study comparatively the distribution of GUS activity in
planta, the initial transgenic plants generated for each
construct were selfed, and the kanamycin-resistant progeny was analyzed in situ using the chromogenic substrate X-Gluc (Fig.
6). Expression of GUS activity driven by
the P69A promoter was detected in the seedlings as well as
in fully grown plants. As deduced from the tissue staining pattern, it
seems as if the P69A gene is expressed in a general fashion
in all organs of the plant, except in roots and in flower organs, where
no GUS activity could be detected in any plant.
Likewise, transgenic plants in which GUS expression was driven by the
P69D promoter revealed that this is active predominantly in
expanding cotyledons and leaves (Fig. 6). This expression was transient
because it disappeared once the leaves or cotyledons had enlarged and
matured. Interestingly, the P69D gene is also observed to be
expressed in inflorescences, and more particularly, in stigmas. We
still do not know whether or not this expression pattern in flowers is
also transient.
Conversely, no constitutive expression of the GUS gene driven by the
P69B or P69C promoters could be detected in any
of the transgenic plants generated (Fig. 6). However, in some of the P69C::GUS transgenic lines, we could detect GUS activity in
discrete groups of cells (islands) that appeared sporadically, and with an unpredictable location, either in stems, roots or leaves (not shown).
Induced expression was also analyzed in rosette leaves from transgenic
plants before and after inoculation with P. syringae pv.
tomato (Pst) strain DC3000 carrying the avrRpm1
avirulence gene, which is recognized by the corresponding resistance
gene in A. thaliana ecotype Col-0. Pst
DC3000(avrRpm1) and causes a macroscopic hypersensitive
response (HR) at the inoculation site. These studies revealed that GUS
expression driven by either the P69B and P69C
promoters was induced in the infected leaves. Induction occurred
throughout the inoculated leaves and was not restricted to the site of
inoculation where the HR became apparent (Fig. 7). Conversely, in transgenic plants
carrying P69A::GUS or
P69D::GUS constructs, GUS expression was not
induced during the course of infection.
Because salicylic acid (SA) has been demonstrated to be a master
regulatory molecule mediating most of the plant defense responses to
challenging pathogens (19), we tested whether or not SA could act as
inducer of any of these genes. Spraying of healthy transgenic Arabidopsis plants with a 0.5 mM solution of SA
resulted in the induction of GUS activity only in plants harboring the
P69B::GUS and P69C::GUS
transgenes, and in a manner similar to that observed following bacteria
inoculation (Fig. 7). These results further reinforce our consideration
that the P69B and P69C gene pair is involved in
pathogenic response of the plant to challenging pathogens, whereas the
P69A and P69D pair is more related to development or to a basic biochemical function.
In this work, we provide structural and functional information on
a genomic cluster comprising four different members of a family of
plant genes, which on the basis of amino acid sequence conservation and
structural organization are related to the subtilisin-like protease
clan (EC 3.4.21.14) (11). The genomic cluster was identified by
screening of a genomic DNA library from tomato plants with a partial
cDNA probe for the previously identified pathogenesis-related PR-P69 protease (7) (here renamed as P69A). The predicted primary structure of the four P69 proteases designated P69A, P69B, P69C, and
P69D, indicate that all of them are synthesized as precursor proteins
(preproenzyme) composed of three distinct domains: a 22-amino acid
signal peptide, a 92-amino acid propolypeptide, and a mature
polypeptide of variable length for each protease in the cluster. Within
the mature polypeptides, the amino acid sequences surrounding Asp-146,
His-203, and Ser-531 are the most salient features of all these
proteases, which are identical to those of the catalytic sites
(catalytic triad) of subtilisins (11). Interesting also is the
conservation of an insertion of a long sequence (226 amino acids)
between the conserved Asn residue and the reactive Ser residues of the
catalytic triad, relative to all other subtilisin-like proteases. The
meaning of such a conserved displacement remains unknown, but its
conservation suggests it may subserve important functions in regulating
the properties of this subgroup of subtilases in plants.
Comparative studies of the mode of expression by RT-PCR and by analysis
of transgenic plants harboring independent promoter-GUS fusions for the
four P69 genes indicate that they are regulated differently.
P69A is transcribed at all stages of plant growth and in all
organs except roots and flowers where the expression is absent.
Likewise, P69D is also transcribed under resting conditions in emerging leaves, but its expression is transient. This transient expression pattern, which has been observed also in transgenic tobacco
plants (data not shown), is presumably associated with the elongation
processes in these organs because transcription is repressed once the
leaves cease elongation. Although P69D is not expressed in
roots, transcription is specifically recovered in flowers, and in the
stigma in particular. Neither P69A nor P69D gene
expression is induced over basal levels during pathogenesis.
Conversely, although neither P69B or P69C show
constitutive expression in any organ of plants grown under resting
conditions, both are transcriptionally activated upon infection with
avirulent bacteria, either in transgenic Arabidopsis plants
or in tomato plants. This latter result is consistent with our previous
observations that one member of this family, now identified as P69B on
the basis of sequence identity, is coordinately induced with a set of
pathogenesis-related (PR) proteins associated with the defense response
in tomato plants (7, 20, 21).
Interestingly, the observation that the induced transcriptional
activation of P69B and P69C is not restricted to
the point of inoculation with the avirulent bacteria (Fig. 7), where
the HR cell death and the induction of protective genes occur (22), but
rather is extended throughout the afflicted leaf blade suggests that
their induction is mediated by a long distance signaling process.
Because the benzoic acid-derivative SA is one of the candidate signal
molecules mediating long distance activation of plant defense reactions
(19), we tested the ability of SA to induce expression of these two
pathogen-inducible genes. The result presented in Fig. 7 demonstrates
that local application of SA to leaves promotes the transcription of
both genes de novo. Thus, these results reconcile with the
idea that proteases, and in particular members of the subtilisin-like
family, are components of the general plant response to attacking pathogens.
The differential expression profile found for these four P69
genes indicate that different regulatory mechanisms have evolved to
control the expression of these genes either during normal growth or
under pathological situations. It remains to be demonstrated whether or
not these differential expression patterns also imply different roles
for the protein products. In this regard, it is tempting to speculate
that the permanent expression of the P69A gene at the
different stages of growth of the plant may suggest a housekeeping
function for the P69A protein and that this function may be
advantageously enhanced during processes of plant cell elongation
and/or pathogenesis by supplementing with the other isoforms that may
serve backup functions to the principal protease. It has been
demonstrated in animal systems, where natural substrates for
subtilisin-like proteases have been identified (12, 13), that when the
different protease members of a family are removed from their
biological context and assayed in vitro, many of these proteases are able to process the same substrates. This observation again raises the question of whether such a functional redundancy exists among the family members in vivo and how this might
be regulated. One insight into defining functionality has been provided by examining the expression of the individual protease members (23).
From such analysis, it has been shown that protease substrate specificity in vivo is influenced by restricting expression
to particular tissues and also by compartmentalization of the
individual enzymes to specific intracellular locations (24). Thus, it
may be the case that a similar regulation also applies for the
differentially regulated P69 protease isoforms under consideration as a
likely explanation for delimiting redundant functions in
vivo. Alternatively, we cannot disregard the possibility that each
member functions separately by recognizing different substrate(s) and
thus implies that both function and gene expression patterns evolved
coordinately, but separately, for each P69 protease.
Whatever the meaning of such complexity is, the diversification of
either the regulation of gene expression or function for each member of
the P69 clan would be in agreement with the polymorphism found for
other unrelated gene families in higher plants, which arise from gene
duplication events of a common ancestral gene (25, 26).
So far, only two protein substrates have been identified for these
plant subtilisin-like proteases. One is systemin, the traveling peptide
hormone mediating signaling processes during wound response in plants
(27), the other is LRP (28), an extracellular matrix associated
leucine-rich repeat (LRR) protein that is part of a family of proteins
that mediate molecular recognition and/or protein interaction processes
(29). Subtilisin-like enzymes have been shown to be secreted to the
plant extracellular matrix (ECM) (30). Thus, the consideration that the
P69s may be mediating in pericellular processing/degradation events may
indicate a role, similar to those in some animal systems (31, 32), in
modulating the interaction of the plant cell surface with the
extracellular environment.
-glucuronidase fusions for each of these genes
revealed that the four genes exhibit differential transcriptional
regulation and expression patterns. P69A and
P69D are expressed constitutively, but with different
expression profiles during development, whereas the P69B
and P69C genes show expression following infection with
Pseudomonas syringae and are also up-regulated by salicylic
acid. We propose that these four P69-like proteases, as members of a
complex gene family of plant subtilisin-like proteases, may be involved
in a number of specific proteolytic events that occur in the plant
during development and/or pathogenesis.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-EMBL3 was screened at 65 °C
as described (14) with the radiolabeled p26 cDNA encoding the
prepro sequence of the PR-P69 protein described previously (7). The
positive clones were isolated and characterized with the routine
described previously (14).
1 relative to the translation
initiation sites in each gene. SmaI-BamHI
fragments encompassing each of the P69 promoter regions were cloned
upstream of the
-glucuronidase gene in pBI101.1 (16) to
generate plasmids pP69A::GUS,
pP69B::GUS, pP69C::GUS, and
pP69D::GUS. The resulting transcriptional fusions
were verified by nucleotide sequence analysis using specific primers.
The constructs were introduced into Arabidopsis plants by
Agrobacterium tumefaciens mediated transformation.
Transformants were selected on MS agar medium containing kanamycin,
transferred to soil, and allowed to self. The transgenic lines were
assayed for GUS activity by a fluorimetric assay or by an in
situ assay using the colorigenic substrate X-gluc (16).
RESULTS
-EMBL3, and
different clones were isolated. After a third round of screening and
purification, three
clones (
-5,
-2, and
-3') were finally
selected for restriction analysis and sequencing. These analyses
revealed that the genomic DNA inserts of the three
clones were
overlapping clones encompassing ~41 kb of genomic DNA. Alignment of
the genomic sequences revealed the presence of a tandem of four
transcription units that were highly similar (Fig.
1). The first one, from here on
designated as P69A, was identical to the previously
identified P69 cDNA contained in plasmid p26. The last one in the
row, and designated as P69B, was identical to the previously
reported cDNA clone p9 (8). Between the P69A and
P69B transcription units, two additional ones, designated
P69C and P69D, were identified. The four genes were intronless. While the nucleotide sequence homology for the four
open reading frames was quite high (in the range of 75 to 85%
identical), the comparison of the 5' promoter regions (preceding the
ATG initiation codon) or the 3' region after the polyadenylation signal
of each gene revealed no homology between them. However, in all cases,
putative TATA boxes and CAAT boxes shortly upstream of the ATG
initiation codon were observed (data not shown). As deduced from the
nucleotide sequences of the open reading frames, the P69A,
B, C, and D genes encode polypeptides
of 745 aa (78,990 Da; pI 6.17), 745 aa (78,990 Da; pI 6.71), 666 aa
(70,680 Da; pI 5.26), and 747 aa (79,260 Da; pI 7.38), respectively
(Figs. 2 and
3).
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Fig. 1.
P69 genomic cluster. Four P69-like open
reading frames sequences (boxes) are arranged in a tandem
array (named as P69A, P69D, P69C, and
P69D). clones that span the array are shown below. The
EcoRI (E) restriction sites are indicated in each
clone. The distances are only approximate. Arrows indicate
direction of transcription.
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Fig. 2.
Amino acid sequence alignment for the
predicted P69 gene products. The sequence of P69A is shown in full
and compared with predicted open reading frames for P69B, P69C, and
P69D. Dashes represent sequence identity. The catalytically
important Asp, His, Asn, and Ser residues are shown in bold
with an asterisk. The propeptide domains are
dashed. Dots were introduced to maximize
alignment. Amino acid residues of each protease are numbered
from the precursor sequence.
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Fig. 3.
Schematic representation of the preproenzyme
structures and homology of the four P69s. A,
characteristic regions (signal peptide, propeptide, and mature peptide)
are shown by marked areas. Numbers
indicate positions of amino acid residues from the N terminus. The
amino acids forming the catalytic triad in the active site
(D, aspartate; H, histidine; S,
serine) and the conserved N (asparagine) residue are marked.
B, identity percentages between the different P69
proteases.
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Fig. 4.
RT-PCR detection of P69 genes
expression. Southern blot of DNA products derived from the PCR
amplification of reverse transcribed mRNA from mock inoculated
leaves (H) or Pseudomonas syringae pv.
syringae-infected (I) tomato plants using
specific sets of oligonucleotides (see "Experimental Procedures").
The blots were hybridized with a radiolabeled cDNA probe for the
P69A isoform. PCR products derived from amplification of plasmids
containing either the P69A, P69B,
P69C, or P69D open reading frames with the same
set of primers is shown on the left for comparison. A
EcoRI/HindIII digest of DNA is included as a
reference of molecular size markers.
-glucuronidase
(GUS) reporter gene in plasmid pBI101.1 to generate
constructs pP69A::GUS, pP69B::GUS,
pP69C::GUS, and pP69D::GUS (Fig.
5). These constructs were introduced
separately into Arabidopsis plants by transformation with
A. tumefaciens, and a minimum of ten independent
kanamycin-resistant transformants were generated for each construct.
GUS activity was initially analyzed in plants grown under normal growth
conditions and also after inoculation with the avirulent bacteria
P. syringae DC3000 (AvrRpm1) by a fluorimetric assay (16)
(data not shown). These assays revealed constitutive expression of GUS
activity in the transgenic plants generated with construct
P69A::GUS and P69D::GUS, whereas those harboring the P69B::GUS or the
P69C::GUS cassette expressed GUS activity only
upon bacterial infection (see below). These results were, to some
extent, coincident with the RT-PCR studies shown above (Fig. 4).
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Fig. 5.
Schematic representation of the different
P69:: -glucuronidase (GUS) gene
fusions. The diagonally striped box represents the
GUS gene. The white box at the right
represents the 3' nopaline synthase terminator. The length of each of
the promoter regions is shown above each construct in kb. The ATG codon
represent the first translation initiation codon which resides in the
GUS gene.
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Fig. 6.
GUS staining patterns of whole tissue and at
different stages of growth, including inflorescences, of transgenic
Arabidopsis (Col-0) plants harboring either
P69A::GUS, P69B::GUS,
P69C::GUS, or P69D::GUS
gene fusions. Top panel, 10-day-old GUS-stained
intact seedlings; middle panel, 17-day-old GUS-stained
intact seedlings; bottom panel, GUS staining of intact
inflorescences.
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Fig. 7.
GUS staining patterns in rosette leaves of
transgenic Arabidopsis (Col-0) plants carrying the
P69A::GUS, P69B::GUS,
P69C::GUS, and P69D::GUS
transgenes. Left panel, GUS staining pattern in
leaves from plants that have been sprayed with a 0.5 mM
salicylic acid solution; middle panel, GUS staining pattern
in leaves from plants that have been inoculated with P. syringae pv.tomato DC3000 carrying the avirulent
avrRpm1 gene. The site of inoculation and the area
undergoing the HR are indicated with an arrow in each case. Right
panel, GUS staining pattern in full expanded leaves from healthy
mock-inoculated control plants.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Jeff Dangl and Pablo Tornero for providing the Pst DC3000 strain.
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FOOTNOTES |
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* This work was supported in part by the Spanish Ministry of Science and Education.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 and DDBJ Data Banks with accession number(s) Y17275 (P69A), Y17276 (P69B), Y17277 (P69C), and Y17278 (P69D).
Supported by a Fellowship from the Conselleria de Educación
y Ciencia de la Generalitat de Valencia.
§ To whom correspondence should be addressed. Tel.: 34-96-3877864; Fax: 34-96-3877859; E-mail: vera{at}ibmcp.upv.es.
The abbreviations used are:
PCR, polymerase
chain reaction; RT, reverse transcription; Pst, Pseudomonas
syringae pv. tomato; SA, salicylic acid; GUS, -glucuronidase; aa, amino acid; HR, hypersensitive response; aa, amino acids.
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REFERENCES |
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