The WRKY70 Transcription Factor: A Node of Convergence forJasmonate-Mediated and Salicylate-Mediated Signals inPlant Defense W

Jing Li, Gunter Brader, and E. Tapio Palva1

Department of Biosciences, Division of Genetics, University of Helsinki, FIN-00014 Helsinki, Finland

Cross talk between salicylic acid (SA)– and jasmonic acid (JA)–dependent defense signaling has been well documented in

plants, but how this cross talk is executed and the components involved remain to be elucidated. We demonstrate that the

plant-specific transcription factor WRKY70 is a common component in SA- and JA-mediated signal pathways. Expression

of WRKY70 is activated by SA and repressed by JA. The early induction of WRKY70 by SA is NPR1-independent, but

functional NPR1 is required for full-scale induction. Epistasis analysis suggested that WRKY70 is downstream of NPR1 in an

SA-dependent signal pathway. Modulation of WRKY70 transcript levels by constitutive overexpression increases resistance

to virulent pathogens and results in constitutive expression of SA-induced pathogenesis-related genes. Conversely,

antisense suppression of WRKY70 activates JA-responsive/COI1-dependent genes. The effect of WRKY70 is not caused by

subsequent changes in SA or JA levels. We suggest that WRKY70 acts as an activator of SA-induced genes and a repressor

of JA-responsive genes, integrating signals from these mutually antagonistic pathways.

INTRODUCTION

The need of plants to cope with potential pathogens has led to

the evolution of complex adaptive responses involving protective

physical barriers and production of a diverse array of antimicro-

bial metabolites and proteins. A key step in induced plant

resistance is the timely recognition of the invading pathogen.

Plants have the ability to recognize pathogen-derivedmolecules,

exogenous elicitors, or molecules released from the plant by the

action of the pathogen (Yang et al., 1997; Van der Biezen and

Jones, 1998; Montesano et al., 2003). This recognition can

trigger a local response (hypersensitive response), a form of

programmed cell death at the site of infection, whichmay contain

the invading pathogen. Hypersensitive response is often associ-

ated with the development of a plant immune response and

systemic acquired resistance (SAR) (Feys and Parker, 2000).

SAR is characterized by an increase in endogenous salicylic acid

(SA), transcriptional activation of pathogenesis-related (PR)

genes (e.g., PR1, BGL2 [PR2], and PR5), and enhanced

resistance to a broad spectrum of virulent pathogens (Uknes et

al., 1992; Ryals et al., 1996). SA is necessary for SAR, and a series

of studies demonstrated that SA signaling is mediated by an

ankyrin repeat protein, NPR1/NIM1 (Cao et al., 1994; Delaney

et al., 1995; Li et al., 1999; Fan and Dong, 2002), but also that an

NPR1-independent pathway does exist (Clarke et al., 1998,

2000; Shah et al., 1999).

In addition to SA, other signaling molecules such as jasmonic

acid (JA) and ethylene (ET) regulate various plant defense

responses through signaling pathways that are distinct from

the classic SA-mediated SAR pathway (Glazebrook, 2001;

Kunkel and Brooks, 2002; Turner et al., 2002). For example,

concomitant activation of the JA and ET response pathways is

required for expression of PDF1.2 encoding an antimicrobial

defensin (Penninckx et al., 1998). Arabidopsis thaliana mutants

that are impaired in JA production (e.g., fad3 fad7 fad8 triple

mutant) or perception (e.g., coi1 and jar1) exhibit enhanced

susceptibility to a variety of virulent fungal and bacterial

pathogens (Kunkel and Brooks, 2002).

In the complex network of regulatory interactions during plant

resistance responses, an antagonistic relationship between SA

and JA signaling pathways is evident (Kunkel and Brooks, 2002).

The inhibitory effect of SA on JA signaling in Lycopersicon

esculentum (tomato) is well established (Doares et al., 1995), and

recent studies using A. thaliana mutants provide genetic evi-

dence for this antagonism. The eds4 and pad4mutants deficient

in SA accumulation as well as the npr1 mutant with impaired

response to SA exhibit enhanced induction of JA-responsive

genes (Penninckx et al., 1996; Clarke et al., 1998; Gupta et al.,

2000). New evidence indicates that cellular localization of NPR1

might play a crucial role in modulating the SA-mediated sup-

pression of JA-responsive genes (Spoel et al., 2003). Moreover,

studies with Nicotiana tabacum (tobacco) and A. thaliana reveal

that JA- and SA-mediated defense responses can be mutually

antagonistic (Vidal et al., 1997; Norman-Setterblad et al., 2000).

Several genetic studies provide evidence that JA signaling

negatively regulates the expression of SA-responsive genes inA.

thaliana (Petersen et al., 2000; Kachroo et al., 2001; Kloek et al.,

1 To whom correspondence should be addressed. E-mail tapio.palva@helsinki.fi; fax 358-9-19159076.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: E. Tapio Palva(tapio.palva@helsinki.fi).WOn-line version contains Web-only data.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.016980.

The Plant Cell, Vol. 16, 319–331, February 2004, www.plantcell.orgª 2004 American Society of Plant Biologists

2001). It is likely that these two regulatory networks controlling

plant defense signaling contain several nodes of interaction, but

the identity of those nodes remains to be elucidated. One of the

open questions is whether transcription factors are involved in

cross talk between SA and JA signaling.

Transcriptional regulation of defense gene expression appears

central in induced disease resistance, and[1500 transcription

factors (Riechmann et al., 2000) may be involved in plant

defense. These include the plant-specific WRKY family of tran-

scription factors defined by a DNA binding domain that contains

the highly conserved amino acid sequence WRKYGQK (Eulgem

et al., 2000). WRKY factors have been implicated in plant

defense, plant senescence, and response to various environ-

mental stresses (Yang et al., 1999; Du and Chen, 2000; Robatzek

and Somssich, 2001; Yu et al., 2001; Chen andChen, 2002; Chen

et al., 2002) as well as in developmental processes (Tepperman

et al., 2001; Johnson et al., 2002). The involvement of WRKY

factors in plant defense is well documented; some of these

factors have been shown to confer disease resistance (De-

slandes et al., 2002), trigger expression of defense-related genes

in SAR (Eulgem et al., 1999; Maleck et al., 2000; Robatzek and

Somssich, 2002), and control A. thaliana innate immunity

activated by bacterial flagellin (Asai et al., 2002).

In this study, we identified WRKY70 as a key factor controlling

plant response to Erwinia carotovora subsp carotovora, a bac-

terial necrotroph that can trigger both SA- and JA-dependent

defense signaling (Kariola et al., 2003). Resistance to this

pathogen can be induced by activation of either SA-mediated

(Palva et al., 1994; Kariola et al., 2003) or JA/ET-mediated

(Norman-Setterblad et al., 2000; Kariola et al., 2003) defense

pathways. We characterized the expression of WRKY70 in re-

sponse to plant hormones and pathogen elicitors and generated

WRKY70 transgenic plants to identify target genes controlled by

WRKY70. Expression profiling in transgenic plants suggests that

WRKY70 can function either as a positive or a negative regulator

of PR gene expression, and we identified subsets of genes

divergently controlled by WRKY70. Our studies indicate that

WRKY70 is an important node of convergence integrating SA

and JA signaling during plant defense responses.

RESULTS

WRKY70, an Uncommon Member of the WRKY

Transcription Factor Family

To elucidate the spectrum of plant gene activation that occurs

during infection by the plant pathogenic enterobacterium E. c.

carotovora, we identified A. thaliana genes that increase in

expression in response to elicitor preparations (culture filtrates

[CF]) from E. c. carotovora SCC3193 using suppressive sub-

tractive hybridization. A subtracted cDNA library enriched for

sequences preferentially expressed in CF-treated leaves was

established (Brader et al., 2001). One of the isolated clones,

corresponding to a CF-induced gene, showed complete match

with the putative coding region fragment (437 to 768 bp

downstream of ATG) from ATT5P19_50 (accession number

AL163972). Using PCR, a 956-bp full-length fragment for this

gene encoding a DNA binding protein of the WRKY family was

acquired from a cDNA library of CF-treated A. thaliana plants.

Recently, the gene was defined as WRKY70 (accession

number AF421157). The deduced amino acid sequence shares

the closest similarity to that of WRKY54 (�53% identity),

suggesting that the protein is not very closely related to other

members of this transcription factor family. The two proteins

have been classified into group III of the WRKY family (Kalde

et al., 2003).

WRKY70 Expression Is Induced by Defense Signals

Plant response to pathogens is regulated by multiple signal

transduction pathways, in which SA, JA, and ET function as key

signaling molecules (Glazebrook, 2001). To elucidate the

potential involvement of WRKY70 in plant defense, we charac-

terized expression of the WRKY70 gene by RNA gel blot

hybridization in A. thaliana wild-type plants in response to the

exogenous signal molecules SA, 1-aminocyclopropane-1-car-

boxylic acid ([ACC], a natural precursor of ET), methyl jasmonate

(MeJA), and E. c. carotovora elicitors (CF). To monitor the treat-

ment procedure, we examined, in parallel, expression of the SAR

marker PR1 and a JA-inducible/COI1-dependent gene AtCOR1

(Benedetti et al., 1998). In accordance with previous results

(Norman-Setterblad et al., 2000), CF induced a delayed accu-

mulation of PR1 transcripts (Figure 1A), whereas AtCOR1 was

rapidly induced with a maximal transcript accumulation 2 h after

CF treatment. The antagonistic effect of SA on JA signaling was

evident in the following transient decrease ofAtCOR1 expression

after SA treatment (Figure 1A). WRKY70 displayed a basal

expression that showed some temporal variation (Figure 1A).

WRKY70 was strongly induced by CF and SA but not by ACC or

MeJA (Figure 1A). By contrast, MeJA treatment appeared to

repress WRKY70 expression at 8 h post-treatment (hpt). To

correlate the changes in CF-induced gene expression with

possible alterations in hormone levels, we characterized accu-

mulation of SA and JA in CF-treated leaves (Figure 1B). JA

content in untreatedmature leaves was low, whereas CF-treated

leaves exhibited rapid but transient accumulation of JA. By

contrast, there was a significant and stable increase in free SA

levels in response to CF after an initial lag period. These data

indicate that CF-induced expression of WRKY70 is correlated

with elevated levels of endogenous SA and suggest that the

increased expression is initially repressed by the transient

increase in JA.

WRKY70 Expression Involves SA Signaling

To elucidate plant defense signal transduction networks involved

in expression ofWRKY70, we employed transgenic NahG plants

that fail to accumulate SA and mutants that affect the SA-

dependent pathway (npr1-1), the ethylene-dependent pathway

(ein2-1), or the JA-dependent pathway (coi1-1). ET signaling

does not appear to be required for WRKY70 expression. By

contrast, altering of SA (NahG and npr1) or JA (coi1) signaling led

to significant changes in WRKY70 transcript levels (Figure 1C).

The complete block in basalWRKY70 expression inNahG plants

but only a reduction in npr1 plants suggested that endogenous

320 The Plant Cell

SA but not functional NPR1 is essential for the basal expression

of WRKY70. This is contrasted by enhanced accumulation of

WRKY70 transcripts in coi1 plants, supporting the hypothesis

that JA signaling represses WRKY70 expression.

SA signaling also appears essential for elicitor-induced

WRKY70 expression, as shown by the lack of WRKY70 tran-

scripts in CF-treated leaves of NahG plants and drastically

reduced levels of these transcripts in npr1 plants (Figure 1D).

Similarly, SA-induced expression of WRKY70 was completely

abolished inNahG plants (Figure 1D). However, the SA induction

of WRKY70 in npr1 plants (Figure 1D) was only abolished at the

later timepoints, suggesting that induction ofWRKY70 is partially

Figure 1. Differential Response of WRKY70 to Defense Signals in A. thaliana.

(A) Accumulation ofWRKY70 transcripts in fully expanded leaves of A. thaliana wild-type plants treated with 5 mM SA, 100 mMMeJA, 100 mM ACC, or

Erwinia elicitor preparation (CF). The ethidium bromide–stained rRNA is shown for loading control. hpt, hours post-treatment.

(B) Accumulation of free SA (squares) and JA (triangles) content was determined in A. thaliana in response to CF treatment. The values represent the

average of three replicates 6SD. FW, fresh weight.

(C) Basal expression of WRKY70 in transgenic NahG or mutant plants.

(D) Induced expression of WRKY70 in A. thaliana plants treated with 5 mM SA or CF. The RNA gel blots were hybridized with a WRKY70-specific DNA

probe. All experiments were independently performed twice with similar results.

WRKY70 in Disease Resistance 321

NPR1-independent. The results indicate that WRKY70 is likely to

be associatedwith the SA- andNPR1-mediatedSAR response in

plants.

Overexpression or Antisense Suppression of WRKY70

Modulates Plant Disease Resistance

To elucidate the putative function of WRKY70 during plant

defense to pathogens, we generated A. thaliana overexpression

or antisense suppression lines with WRKY70. From [50 in-

dependent overexpression or antisense suppression lines,

homozygous progenies containing a single insert were used for

further experiments. As shown in Figure 2A, five overexpression

lines exhibited substantially elevated levels of WRKY70 tran-

scripts, and eight antisense suppression lines expressed no

detectable WRKY70 transcripts after CF treatment. The five

overexpression lines displayed distinctmorphological traits; they

were smaller in size than control plants, exhibited changes in

morphology with lancet shaped and slightly twisted leaves

(Figure 2B), and were delayed in flowering (data not shown). By

contrast, antisense suppression lines were slightly larger in size

than control plants (Figure 2B) and showed an early flowering

phenotype (data not shown).

To determine the contribution of WRKY70 to disease re-

sistance in A. thaliana, we examined the resistance of the trans-

genic plants to two virulent bacterial pathogens, strain SCC1 of

the necrotroph E. c. carotovora and Pseudomonas syringae pv

tomato strain DC3000. Antisense suppression of WRKY70

appeared to sensitize the plants to SCC1 infection, leading to

more rapid spreading of disease, maceration of nascent and

young leaves, and dehydration of mature leaves (Figure 2C).

Furthermore, the survival of antisense suppression plants was

drastically reduced. By contrast, most WRKY70 overexpression

plants did not exhibit spreading maceration to systemic leaves

and displayed clearly enhanced survival of SCC1 infection. In

agreement with this result, overexpression of WRKY70 led to

enhanced resistance to P. s. tomato DC3000 (Figure 2C). These

data suggest that the constitutive activation of the WRKY70-

controlled defense mechanisms directly contributes to the

enhanced disease resistance.

WRKY70 Does Not Affect Endogenous Levels of

JA, ET, and SA

SA is a central mediator of plant defenses and is required for the

SAR response. One possibility to explain the mode of action

of WRKY70 is that this transcription factor might be involved in

an amplification loop or signaling cascade that modulates SA

biosynthesis. This is supported by the strong morphological

Figure 2. Characterization of WRKY70 Transgenic Plants.

(A) Overexpression and antisense suppression of WRKY70 in transgenic

A. thaliana plants. RNA gel blots showing WRKY70 expression in

untreated or CF-treated leaves. Overexpression, antisense suppression,

and vector control lines are indicated by S, A, and pCP60, respectively.

(B) Morphology of T3 progeny of 3-week-old transgenic A. thaliana lines.

(C) Resistance of WRKY70 transgenic plants to virulent bacterial

pathogens. Survival of transgenic plants inoculated locally with E. c.

carotovora SCC1. Yellow and red arrowheads mark local and systemic

leaves, respectively. Representative plants were photographed 5 and 7 d

after inoculation. The survival of the plants after infection was examined.

Each experiment consisted of 15 individual plants from each line. The

data represented the mean values and standard error from three

independent experiments in the representative lines (Mann-Whitney U

test: P\0.05). Three independent overexpression and two independent

antisense lines were tested in comparison to vector control plants with

similar results. Growth of P. s. tomato DC3000 in planta was monitored

24 and 72 h after inoculation. Data represents the mean bacterial titer of

at least six plants per time point. Error bars represent 95% confidence

limits of log-transformed data (Mann-Whitney U test). At least two

independent homozygous lines from each construct were tested with

similar results. Ecc, E. c. carotovora; Pst, P. s. tomato.

322 The Plant Cell

phenotypes observed in overexpression and antisense plants,

suggesting that the endogenous hormone balance might be

altered. To test this possibility, we determined whether the

endogenous levels of hormones implicated in plant defensewere

affected in WRKY70 transgenic plants. The basal levels of free

SA, ET, and JA were not significantly different between the

different transgenic lines (Figure 3). Interestingly, the content of

conjugated salicylate glucoside was clearly reduced inWRKY70

overexpression plants (Figure 3A). We conclude that over-

expression or antisense suppression of the WRKY70 gene does

not cause major changes in endogenous levels of free SA, ET, or

JA in A. thaliana leaves and argue that the modulation in disease

resistance is not a result of alterations in the balance of these

hormones.

WRKY70 Overexpression Plants Show Constitutive

Expression of PR Genes

Enhanced disease resistance inA. thaliana is often accompanied

by the accumulation of elevated levels of transcripts of PR genes

associated with the SA-mediated defense pathway (PR1, PR2,

and PR5; Uknes et al., 1992). Because exogenous SA and E. c.

carotovora elicitors triggered expression ofWRKY70, we sought

to determine whether WRKY70 is directly involved in controlling

expression of these PR genes. RNA gel blots demonstrated that

PR2 and PR5 were constitutively expressed in WRKY70 over-

expression plants (Figure 4A). By contrast, no PR1 band of the

normal size could be detected, but a larger size transcript (�1.5

kb) hybridizing to a PR1-specific DNA probe was constitutive

(Figure 4B). Consistent with this observation, reverse transcrip-

tase (RT)-PCR analysis showed the presence of a much stronger

PR1-specific band in the overexpression line S55 than in control

plants under noninduced conditions (Figure 4C). The identity of

this RT-PCR product was confirmed by sequencing (data not

shown). Interestingly, the larger transcript disappeared rapidly

when plants were treated with SA and was replaced by the PR1

transcript, possibly suggesting a precursor–product relation-

ship. Furthermore, overexpression of WRKY70 promoted SA-

induced accumulation of PR1 mRNA, whereas antisense

suppression of WRKY70 resulted in delayed and reduced

accumulation of these transcripts (Figure 4B). A more detailed

examination of SA-inducible PR1 expression at 8 hpt at lower SA

concentrations revealed that the levels of PR1 transcripts were

remarkably reduced in antisense suppression plants, suggesting

reduced sensitivity to SA (Figure 4B). Together, these results

indicate that WRKY70 is one of the coactivators of SAR-

associated PR genes (PR1, PR2, and PR5) and a limiting factor

for full-scale SA-induced expression of PR1.

WRKY70 Acts Downstream of NPR1 in an SA-Dependent

Signal Pathway

NPR1 functions downstream of SA in the SA-dependent

signaling pathway and is required for the activation of some of

the SA-dependent defense responses (Cao et al., 1994). Yu et al.

(2001) suggest that some WRKY proteins act upstream of NPR1

and positively regulate its expression during the activation of

plant defense responses. The reduced expression of WRKY70

in the npr1-1 mutant (Figure 1D) suggested that WRKY70 could

be downstream of NPR1. To determine whether the effect of

WRKY70 on PR gene expression is mediated by NPR1, we first

examined NPR1 expression in both overexpression and anti-

sense suppression plants. RNA gel blots revealed that over-

expression or antisense suppression of WRKY70 did not cause

any change in NPR1 transcript accumulation (Figure 4A).

Furthermore, epistasis analysis was used to investigate the

relative position ofWRKY70 and NPR1 in the signal transduction

pathway leading to PR gene expression. To achieve this,

WRKY70 transgene from the overexpression line S55 was

crossed into NahG and npr1-1 plants, respectively. The expres-

sion of the WRKY70 trangene was not affected in npr1-1 and

NahG backgrounds (Figure 5A). The constitutive expression of

PR2 or the putative PR1 precursor because of WRKY70

overexpression was still evident under noninduced conditions

in theNahG or npr1-1background, albeit at a somewhat reduced

level (Figure 5A). These observations indicate that WRKY70 acts

downstream of SA and NPR1.

WRKY70 Acts as a Repressor of JA-Inducible Genes

Considerable effort has been directed toward elucidating the

regulatory network controlling expression of JA-inducible genes

Figure 3. Effect of WRKY70 Expression on Hormone Levels.

Levels of SA, ET, and JA in transgenic A. thaliana. Shown are free

(shaded columns) and conjugated (open columns) SA (A), ET evolution

(B), and JA content (C). Data presented here were from overexpression

line S55, antisense suppression line A18, and vector control line pCP60.

The values represent the average of three replicates 6SD. Similar results

were obtained in two independent experiments.

WRKY70 in Disease Resistance 323

involved in plant defense responses. It has been well docu-

mented that increased SA levels antagonize JA signaling (Felton

et al., 1999; Gupta et al., 2000). For example, AtVSP induction in

response to E. c. carotovora exoenzyme elicitors in A. thaliana

appears to be strictly dependent on the JA pathway but

negatively regulated by the presence of SA (Norman-Setterblad

et al., 2000). Similarly, our results show a transient induction of

AtCORI expression by E. c. carotovora elicitors but a transient

reduction in its constitutive expression by SA (Figure 1A). It is

tempting to speculate that WRKY70 acts as a repressor

controlling a subset of JA-responsive genes antagonized by

SA. To test this hypothesis, we examined the effect of WRKY70

levels on the constitutive expression of the JA-inducible genes

AtCOR1 and AtVSP. As shown in Figure 4A, RNA gel blots reveal

that antisense suppression ofWRKY70 led to expression of both

AtVSP and AtCOR1, indicating that WRKY70 is a repressor of

these JA-inducible genes. In accordance with these results,

overexpression of WRKY70 suppressed the MeJA-induced

expression of PDF1.2, another marker for JA response (Figure

5B). Interestingly, this suppression was abolished in S55/npr1-1

plants, suggesting that NPR1 is required for this effect.

Differential Regulation of Defense-Related

Genes by WRKY70

Our results (Figure 4A) suggest that WRKY70 might differentially

control subsets of defense-related target genes. To learn more

about the function of this transcription factor in A. thaliana

defense responses, we wanted to identify potential WRKY70

regulon genes at genome-scale level using microarray technol-

ogy. To this aim, we compared global gene expression in

transgenic WRKY70 and vector control plants by using the

Affymetrix AtGenome1 GeneChip containing�8300 probe sets.

Of [4500 probes that were accurately detectable, 331 (see

supplemental data online) exhibited more than twofold differen-

tial expression in overexpression or antisense plants compared

with their expression in control plants. Among them, 42 genes

with [2.5-fold differential expression previously implicated in

plant defense could be assigned to four major groups (Table 1).

Although this list is not comprehensive, it clearly illustrates

different patterns of gene expression.

The first two groups contain genes that are positively

controlled by WRKY70, including 24 genes upregulated in

WRKY70 overexpression plants. Among these are pathogen-

responsive genes (e.g.,WRKY60,PR2, and At1g75040 encoding

a thaumatin-like protein) as well as genes related to oxidative

stress responses (e.g., GST11 and At3g49120 encoding a per-

oxidase). There are also at least three disease resistance genes

encoding LRR proteins (e.g., At2g32680, At4g04220, and

At3g48080) in group I. GST11 and the senescence-related gene

SAG21 have been characterized as upregulated genes during

A. thaliana SAR (Maleck et al., 2000). Notably, the upregulated

genes also include those potentially involved in cell wall

modification, such as XTR3 and CalS1. The second group

includes five genes downregulated in antisense suppression

plants. Their products contain the transcription factor MYB71

and receptor-like protein kinases probably involved in signal

transduction during pathogen infection.

The other two groups consist of genes negatively regulated by

WRKY70. These include the JA-inducible genes AtVSP1 and

AtVSP2 upregulated in antisense plants as well as genes

downregulated in overexpression plants. This latter group

contains additional JA-inducible genes, like OPR1, but also

several genes encoding proteins with putative signaling and

regulatory functions, like the cell wall–associated receptor-like

Figure 4. Differential Expression of SAR Markers and JA-Responsive Genes.

(A) Effect ofWRKY70 on constitutive expression of SA-inducible and JA-responsive genes in transgenic A. thaliana by RNA gel blot analysis. To ensure

equal loading of RNA, one representative filter was subsequently stripped and rehybridized with a probe against rRNA (rDNA).

(B) Effect of WRKY70 on induction of PR1 by SA.

(C) RT-PCR analysis of PR1 constitutive expression in overexpression transgenic line and control plants. The levels of actin were assayed as controls.

324 The Plant Cell

kinase WAK4 and some transcription factors. Moreover, the

group includes several defense-related genes distinct from the

ones that are positively controlled by WRKY70. These data

suggest that the WRKY70 regulon consists of two subsets of

defense-related genes, one activated by this transcription factor

and the other repressed by it.

To elucidate the extent of the WRKY70 control on JA- and

SA-responsive genes, we compared our data with published

microarray data of Glazebrook et al. (2003). The clusters of

upregulated or downregulated genes inWRKY70overexpression

line S55 in comparison to the antisense suppression line A18

were compared with data sets in which coi1, NahG, and sid2

plants were compared with their respective controls after

treatment with P. syringae pv maculicola. The data demonstrate

that WRKY70 clearly controls a substantial portion of SA- or JA-

(COI1-dependent) responsive genes (Table 2). A substantial

portion (61.4%) of the genes found in both data sets, which are

upregulated in the WRKY70 overexpression plants, is also

upregulated in the coi1 mutant. Accordingly, 52.4% of the

WRKY70 downregulated genes are also downregulated in the

coi1 plants. On the other hand, more than half of the genes

downregulated in WRKY70 overexpression plants are upregu-

lated in NahG plants, and 40.9% of the genes upregulated in

WRKY70 overexpression plants are downregulated in NahG

plants. Similar results can be found for sid2mutants, demonstra-

ting an inverse trend for plants deficient in SA signaling, and for

the coi1mutant deficient in the reception of jasmonate (Table 2).

DISCUSSION

Induced disease resistance in plants relies on the ability of

the host to recognize the potential pathogen and trigger an

appropriate response. Plants often employ distinct recognition

mechanisms and signaling pathways for different pathogen

elicitors. These pathways are not necessarily linear, but the

signals mediated by the major endogenous signal molecules

SA, JA, or JA/ET appear to form a network of synergistic and

antagonistic interactions (Glazebrook, 2001; Kunkel and Brooks,

2002; Spoel et al., 2003). Some pathogens, like the Gram-

negative bacterial necrotroph E. c. carotovora, appear to be

capable of triggering both SA- and JA- or JA/ET-mediated

defense responses (Kariola et al., 2003), and E. c. carotovora

hence provides an excellent model for studies of the interaction

between different signal pathways. Furthermore, induced re-

sistance to this pathogen can be obtained either by SA-mediated

or JA/ET-mediated defenses (Palva et al., 1994; Norman-

Setterblad et al., 2000; Kariola et al., 2003). Here, we show that

E. c. carotovora elicitors indeed cause alterations in the endo-

genous SA/JA balance and trigger accumulation of mRNAs for

both the SAR marker PR1 and the JA-responsive gene AtCOR1

(Figure 1B). Previous studies indicate that themutual antagonism

between SA- and JA-dependent pathways involves a common

regulatory element acting downstream of SA in the SA-mediated

response (Vidal et al., 1997; Norman-Setterblad et al., 2000). To

elucidate the identity of such nodes of interaction, we screened

a subtractive library ofA. thaliana for early E. carotovora–induced

genes (Brader et al., 2001). In this study, we provide strong

evidence that one of the genes identified, WRKY70, regulates

defense-related gene expression via integrating SA- and JA-

signaling events (Figure 6).

Our data indicate that increased levels of SApromoteWRKY70

expression. Both the basal and elicitor-induced expression of

WRKY70 appear to involve SA-dependent defense signaling

(Figure 1). This is supported by the lack of expression in NahG

transgenic plants, decreased expression in npr1 mutants, and

induction of the gene by exogenous SA in the absence of any

pathogen elicitor. Moreover, we could show that the mpk4

mutant with constitutively high SA levels (Petersen et al., 2000)

accumulates elevated levels of WRKY70 transcripts (data not

shown). Our results are consistent with those of Brodersen et al.

(2002) showing that WRKY70 is clearly upregulated in the SA-

dependent lesion mutant acd11 (Brodersen et al., 2002). The SA

responsiveness appears common in related WRKY genes and

may indicate their involvement in different steps of the SAR

response in plants. For example, two WRKY70 homologs in

N. tabacum are similarly induced by SA and pathogen infection

(Chen and Chen, 2000). Dong et al. (2003) showed that 49 of

the 72 A. thaliana WRKY genes were differentially regulated in

response to exogenous SA or infection by a bacterial pathogen.

Although WRKY70 expression in leaves was apparently below

the detection limit in that study, a more recent analysis

demonstrated the SA and pathogen inducibility of this gene

(Kalde et al., 2003).

The ankyrin repeat protein NPR1 is essential for activation of

PR gene expression in response to SA, and NPR1 itself is

positively regulated by some SA-inducible WRKY proteins (Yu

Figure 5. Epistasis Analysis.

RNA samples were prepared from mature leaves of 4-week-old soil-

grown plants. For each sample, 20 mg of total RNA was loaded. The

ethidium bromide–stained RNA is shown for equal loading.

(A) WRKY70,PR1, andPR2geneexpression inS55/NahGandS55/npr1-1

plants. RNAgel blots show the accumulation ofPR2mRNAand aputative

precursor of PR1 mRNA (PR1pp). The WRKY70 transgene from the

overexpression lineS55wascrossed into theNahGornpr1-1background.

F3progenieswerecomparedwithNahG,S55, andnpr1-1plants aswell as

with vector control (pCP60).

(B) Effect of the npr1mutation on theWRKY70-mediated suppression of

JA-responsive gene PDF1.2. Leaves were harvested 24 h after spraying

100 mM MeJA for RNA extraction.

WRKY70 in Disease Resistance 325

et al., 2001). Our data show that WRKY70 is not a positive

regulator of NPR1 expression and indicate that WRKY70 is not

upstream of NPR1. First, npr1 mutants exhibit reduced expres-

sion ofWRKY70 (Figures 1C and 1D). Second, overexpression or

antisense suppression of WRKY70 does not change the

constitutive expression of NPR1 (Figure 4A). Third, epistasis

tests reveal that the constitutive expression of PR2 or the

putative PR1 precursor is still activated when a WRKY70 trans-

gene is crossed into the NahG or npr1-1 background (Figure 5).

Together, these results strongly indicate that WRKY70 acts

downstream of NPR1 in an SA-dependent signal pathway.

Notably, the presence of the npr1-1mutation does not affect the

WRKY70 transgene expression level but reducesPR2 transcripts

(Figure 5A). The precise mechanism remains to be determined.

Table 1. Defense-Related Genes Differentially Expressed in WRKY70 Transgenic Plants

Probe Gene code A/C S/C Function/Similarity

W box

(TTGAC[C/T])

W-like box

(TTGACA)

Group I

17068_at At5g60900 N 2.5 S-receptor kinase homolog 2 precursor 3 1

16360_at At4g21380 N 2.6 Receptor-like Ser/Thr protein kinase ARK3 3 1

15616_s_at At1g21250 N 2.7 Wall-associated kinase 1 1 1

12364_at At3g57240 1.8 2.8 Glycosyl hydrolase family 17 (b-1,3-glucanase BG3) 1 1

16620_s_at At5g57560 �2.2 2.9 Xyloglucan endotransglycosylase TCH4 3 1

14638_at At3g49120 N 3.1 Peroxidase 0 4

16054_s_at At1g02920 1.8 3.5 Glutathione S-transferase GST11 8 0

13432_at At2g25000 1.8 3.6 WRKY60 3 6

19181_s_at At4g02380 N 3.7 Senescence-associated SAG21 4 2

13659_at At4g23150 �1.9 3.8 Ser/Thr kinase-like protein 1 0

16365_at At2g32680 1.5 4.4 Similar to Leu-rich repeat disease resistance protein Cf-2.2 2 0

20446_s_at At1g05570 N 4.5 Callose synthase CALS1 (1,3-b-glucan synthase) 3 0

12497_at At2g31880 N 4.7 Putative Leu-rich repeat transmembrane protein kinase 6 0

16366_at At4g04220 N 5.0 Similar to Leu-rich repeat disease resistance protein Hcr2-2A 6 2

14636_s_at At1g75040 N 5.2 Thaumatin-like protein 3 1

18968_at At5g57550 1.8 5.9 Xyloglucan endotransglycosylase XTR3 4 1

20238_g_at At3g13790 N 6.1 Glycosyl hydrolase family 32, identical to b-fructofuranosidase 1 3 4

19195_at At2g44380 N 6.5 Putative CHP-rich zinc finger protein 1 2

13187_i_at At1g45145 N 8.1 Thioredoxin 7 1

12925_s_at At4g29940 1.7 9.0 Pathogenesis-related homeodomain protein PRHA 0 2

20625_at At3g48080 N 9.2 Similar to disease resistance protein EDS1 0 3

13212_s_at At3g57260 N 12.6 Acidic b-1,3-glucanase 2 (BGL2), PR2 2 0

20345_at At4g01700 N 13.1 Glycosyl hydrolase family 19, similar to type II chitinase 2 0

17840_at At2g43570 1.8 18.4 Glycosyl hydrolase family 19, similar to chitinase class IV 3 1

Group II

19267_s_at At4g02330 �2.9 2.2 Similar to pectinesterase 5 1

16597_s_at At3g24310 �3.6 N Myb family transcription factor MYB71 0 0

19025_at At1g77280 �4.2 �2.3 Similar to receptor-like protein kinase 2 1

16875_at At4g23240 �4.2 1.7 Ser/Thr kinase-like protein 4 3

16840_at At1g30640 �4.3 N Strong similarity to protein kinase 2 2

Group III

20227_s_at At1g52030 2.5 1.7 Myrosinase binding protein MBP1.2 1 3

15125_f_at At5g24780 4.7 �1.8 Vegetative storage protein VSP1 3 2

15141_s_at At5g24770 5.2 �1.9 Vegetative storage protein VSP2 2 2

Group IV

18359_at At3g57710 N �2.6 Wall-associated kinase WAK4 2 0

15581_s_at At2g28190 N �2.8 Copper/zinc superoxide dismutase CSD2 2 1

18253_s_at At1g76680 N �2.9 12-Oxophytodienoate reductase OPR1 2 0

18853_at At4g21760 N �2.9 Glycosyl hydrolase family 1; b-glucosidase 5 1

12251_at At2g34930 N �3.0 Similar to Leu-rich repeat disease resistance protein Cf-2.1 4 0

18035_at At1g53160 N �3.1 Putative transcription factor 0 0

18743_f_at At5g07690 N �3.4 Myb family transcription factor 2 1

18156_at At4g18640 N �3.4 Putative Leu-rich repeat transmembrane protein kinase 1 2

14606_at At2g32990 N �3.6 Glycosyl hydrolase family 9, similar to endo-b-1,4-glucanase 2 1

17517_at At5g36910 N �3.7 Thionin THI2.2 2 1

The table indicates $2.5-fold upregulated or downregulated gene expression in antisense suppression line A18 (A/C) or in overexpression line S55

(S/C) compared to vector control pCP60. W box or W-like box distribution is within 1.5-kb promoter sequences. N,\1.5-fold difference.

326 The Plant Cell

One possible interpretation is that WRKY70 and an SA and

NPR1–dependent uncharacterized factor activate synergistically

expression of the downstream genes, such as PR2. This acti-

vation might act in concert to trigger, even involve, feedback

regulation through WRKY70-controlled factors.

Furthermore, we show that the SA-induced expression of

WRKY70 actually includes two different responses: an NPR1-

independent early response and an NPR1-dependent late

response (Figure 1D). The observed SA-induction pattern is

consistent with that of two other WRKY genes, WRKY18 and

WRKY53 (Yu et al., 2001), and supports the idea that NPR1 is

involved in an amplification loop of SA-induced expression of

WRKY70. The NPR1-independent SA-induced expression of

WRKY70 may coincide with the SA-mediated NPR1-indepen-

dent pathway leading to PR gene expression and resistance

involving additional signal such as reactive oxygen species, cell

wall fragments, or nitric oxide in the cpr6 mutant (Clarke et al.,

1998, 2000).

Evidence presented here shows that WRKY70 controls plant

disease resistance. By overexpressing WRKY70, we were able

to generate enhanced resistance to two virulent pathogens

with different virulence strategy, E. c. carotovora SCC1 and

P. s. tomato DC3000. By contrast, antisense suppression of

WRKY70 led to enhanced susceptibility to E. c. carotovora

SCC1. The spectrum of resistance established in overexpres-

sion plants can be attributed partially to constitutive activation

of a subset of defense-related genes. Several of these genes

have been shown to be responsive to SA and associated with

SAR, including PR genes like PR2 and PR5. Thus, a set of

SAR marker genes likely to be involved in pathogen defense is

upregulated in the overexpression plants and may account for

the enhanced resistance phenotype. Some of these genes are

also upregulated in mutants with increased SA levels exhibit-

ing constitutive SAR (Clarke et al., 1998, 2000; Petersen et al.,

2000). Interestingly, overexpressing WRKY70 caused consti-

tutive expression of PR2 and PR5 but did not lead to

accumulation of PR1 mRNA (Figures 4A and 4B). Several

lines of evidence have hinted to the absence of coordinate

regulation between the SAR genes PR1 and PR2/PR5 (Rogers

and Ausubel, 1997; Nawrath and Metraux, 1999; Clarke et al.,

2000). Our findings provide the direct genetic evidence for

separate regulation of subsets of SA-mediated PR genes and

support the notion that SA might activate several proteins that

act in synergy to induce the expression of PR1.

The observed repression of WRKY70 expression by JA and

enhancement of its basal expression in coi1 mutant plants

indicate that the endogenous JA levels play an important role in

controlling the expression of theWRKY70 gene. It is possible that

the regulation is mediated by a JA-responsive factor acting as

a repressor of WRKY70. RNA gel blot analysis and expression

profiling indicated that WRKY70 itself appears to act as

a repressor of JA-responsive genes. The differential expression

of genes like AtVSP andOPR1 in transgenic plants suggests that

expression of such JA-responsive genes might be strictly

controlled by WRKY70. The differences between groups III and

IV (Table 1) might indicate that other JA-responsive factors are

required for upregulation of the group IV genes.

The results presented here argue for a model (Figure 6) in

which recognition of a pathogen or a pathogen-derived elicitor

would change the endogenous levels of SA and JA and alter

the balance between these two hormones. We show that an

increase in SA levels activates the WRKY70 gene expression,

and conversely, an increase in JA levels represses its

expression. Hence, the level of WRKY70 transcripts and

presumably WRKY70 protein would reflect the cellular SA/JA

balance. The cellular WRKY70 levels would in turn affect

the expression of downstream target genes. This effect of

WRKY70 in transgenic lines is not the result of an alteration in

the levels of free SA or JA but appears to be controlled more

Table 2. Comparison of Gene Clusters Differentially Expressed in

WRKY70 Transgenic Lines, and Mutants or Transgenic Plants

Impaired in Jasmonate or Salicylate Signaling

Line coi1" coi1# NahG" NahG# sid2" sid2#

S55/A18 " 61.4% 9.1% 15.9% 40.9% 4.5% 27.3%

S55/A18 # 14.3% 52.4% 52.4% 9.5% 28.6% 0

S55/A18 indicates the percentage of genes upregulated (") and down-

regulated (#) in the WRKY70 overexpression line S55 in relation to the

antisense supression line A18, which are also upregulated or down-

regulated in coi1, NahG, and sid2 plants in relation to their respective

controls. The source of the data sets was Glazebrook et al. (2003). The

leaves of mutant or transgenic plants were compared to the wild type

(Columbia 0) 30 h after treatment with 104 cfu P. syringae pv maculicola

strain ES4326 per cm�2 leaf. " and #, $1.5-fold different from control.

Figure 6. Working Model Showing WRKY70-Mediated Cross Talk

between SA- and JA-Dependent Defense Signaling.

Recognition of a particular pathogen or pathogen-derived elicitor may

trigger the synthesis of SA or JA (or both) and lead to subsequent

activation of the corresponding signal pathways. The balance between

the two pathways determines the level of WRKY70 expression. As

a consequence, WRKY70 level determines which type of response is

favored. High WRKY70 levels activate expression of SAR-related genes

while repressing JA-responsive gene expression. Conversely, low

WRKY70 levels favor JA-responses over SAR. Thus, WRKY70 acts

directly or indirectly by integrating signals from both pathways, the out-

come being dependent on the initial signal strength.

WRKY70 in Disease Resistance 327

directly by WRKY70. We show that high WRKY70 transcript

levels promote activation of a subset of SA-responsive PR

genes, whereas low levels favor expression of JA-responsive

genes. In conclusion, the evidence presented here suggests

that WRKY70 is a pivotal integrator of SA and JA signals in the

regulation of the plant defense responses. This model would

at least partly explain the mutual antagonism between SA- and

JA-mediated defense signaling and identify WRKY70 as the

postulated node of interaction between these pathways. This

antagonistic interaction, executed by the common regulatory

component WRKY70 for both pathways, possibly signifies the

evolution of a mechanism designated to prioritize different

resistance responses.

How does the WRKY70 transcription factor act as a positive

regulator of a subset of genes, including several known SA-

responsive genes (e.g., PR1)? Several lines of evidence

suggest that SA-responsive WRKY proteins might activate

expression of downstream genes by binding to the cognate W

or W-like boxes (Maleck et al., 2000; Yu et al., 2001). The

presence of W or W-like boxes in the promoters of the genes

upregulated or downregulated by WRKY70 (Table 1) suggests

that such genes might be under direct control of this

transcription factor (Figure 6). However, we cannot exclude

the possibility that WRKY70 indirectly regulates these genes

through yet uncharacterized regulatory factors, which are the

primary targets of WRKY70. For example, WRKY60 with

several W boxes in its promoter is upregulated by over-

expression of WRKY70 and might act as direct regulator for

a subset of WRKY70-controlled genes.

The close inverse correlation of WRKY70 and JA-responsive

gene expression and bioinformatics analysis of available data

sets (Table 2) strongly suggest that WRKY70 can act as

a negative regulator of a subset of JA-responsive genes. How

does WRKY70 control the SA-mediated suppression of JA-

responsive gene expression? One possibility is that WRKY70, as

a DNA binding protein, is inactivated or removed from promoters

of the JA-responsive gene upon JA induction. It has recently

been proposed that COI mediates the removal of transcription

factors tagged by JA-dependent phosphorylation (Turner et al.,

2002). Thus, WRKY70 could be released from its target genes

(e.g., AtVSP) by such a mechanism. However, not all WRKY70-

repressed genes (e.g., PDF1.2) containW orW-like boxes (Spoel

et al., 2003). One possible explanation for WRKY70-mediated

downregulation of such genes is that WRKY70 activates another

negative regulator (Figure 6). Recently, it has been proposed that

SA-mediated suppression of JA-responsive gene expression is

controlled by hypothetical cytosolic NPR1 (Spoel et al., 2003).

Therefore, we speculate that the fate of the WRKY70-controlled

negative regulator of JA-responsive genes is likely dependent

upon cytosolic NPR-mediated protein modification. In the ab-

sence of the functional NPR1 protein, the putative negative

regulator in the cytosol would be nonfunctional. In support of this

assumption, the epistasis test indeed shows that the WRKY70-

mediated suppression of JA-induced PDF1.2 expression is

released in the npr1-1 background (Figure 5B).

In conclusion, WRKY70 could form a master switch for

regulatory cascades that control distinct subsets of defense-

related genes, integrating signals from two defense pathways.

Further studies with WRKY70 transgenic plants in combination

with pathway-specific mutants will help to refine the model.

METHODS

Plant Materials and Growth Conditions

Seeds of A. thaliana wild type and mutants were kept at 48C at least

2 d before placement in a growth environment to aid uniform germina-

tion. Seedlingswere transferred to pots (737 cm) after 1-weekgermination

on MS plates (Murashige and Skoog, 1962) or soil. Plants were grown

on a 1:1 mixture of vermiculite:peat (Finnpeat B2, Kekkila Oyj, Tuusula,

Finland) in a 228C growth room under a 12 h photoperiod and a light

intensity of 40 mmol �m�2 � sec�1. The A. thaliana mutants ein2-1 and

npr1-1 were obtained from the Arabidopsis Biological Resource Center

(accession numbers CS3071 and CS3726). The coi1-1 mutant and the

transgenic line expressing the NahG gene were kindly provided by

J. Turner (University of East Anglia, Norwich, UK) and J. Ryals (Ciba

Geigy, Research Triangle Park, NC), respectively. All plants were derived

from the Columbia 0 ecotype.

Cloning of WRKY70, Vector Construction, and

Plant Transformation

A 956-bp full-length fragment for WRKY70 was cloned from a cDNA

library of A. thaliana plants treated with CF by PCR by using the follow-

ing primer pairs: 59-TAAGATACCACTCACCAAAAACTTCCTCAA-39 and

59-CTCATGGTCTTAGTCCTATAGTAGTGGT-39. The full-length PCR

fragment was cloned into a pCR 2.1 vector (Invitrogen, Carlsbad, CA)

and sequenced to verify the sequence. The plasmid harboring the 956-bp

full-length fragment for WRKY70 was digested with SpeI and NotI and

thensubcloned into thecorrespondingsitesXbaIandNotIof thebinaryvec-

tor pCP60, which is derived from pBIN19 containing the 35S promoter of

Cauliflower mosaic virus, multiple cloning sites, and NOS, resulting in the

fusion construct S-pCP60-WRKY70, with the 35S promoter directing

expression in the sense orientation of the full-length WRKY70. A 332-bp

cDNA clone from a substracted cDNA library (CFminus control; Brader et

al., 2001) specific for WRKY70 was cloned into the SmaI-EcoRI sites of

pCP60, resulting in the fusion construct A-pCP60-WRKY70, with the 35S

promoter directing expression in the antisense orientation. The fidelity

of all constructs was confirmed by restriction and sequence analysis.

A. thaliana transformation was performed as described previously

(Clough and Bent, 1998). Transgenic progeny lines with single insertion

lociwere selectedonMSplatescontainingkanamycinandcarried tohomo-

zygosity. The empty vector pCP60 was used to generate transgenic

control plants in a similar manner.

RNA Gel Blot Analysis

Isolation of total RNA, labeling of DNA probes with digoxigenin (DIG), and

RNA gel blot analysis were performed as described previously (Kariola

et al., 2003). Twenty micrograms of total RNA was transferred to a nylon

membrane (Boehringer Mannheim, Basel, Switzerland) and hybridized

with DIG-labeled DNA or RNA probes, as indicated. DIG RNA labeling,

hybridization, and detectionwere performed according tomanufacturer’s

instructions (Roche, Basel, Switzerland). A 332-bp cDNA fragment

cloned to pBluescript II SK1 (Stratagene, La Jolla, CA) was used as a

template for WRKY70-specific DNA and RNA probes. The other DNA

probe templates were amplified by PCR from PR1 (gene identifier [GI]:

3810599), PR2 (GI: 166636), PR5 (GI: 2435405), NPR1 (GI: 1773294),

AtCOR1 (GI: 2460202), and AtVSP (GI: 14994268). Equal loading was

confirmed by hybridization of the filters with an rDNA probe or running

agarose-formaldehyde gels with ethidium bromide.

328 The Plant Cell

RT-PCR Analysis

RT-PCR analysis was performed with 1 mg of total RNA treated with

DNaseI (Amersham Biosciences, Piscataway, NJ) according to the

manufacturer’s instructions. With SuperScript II RNase H� and Oli-

go(dT)16 RT (Invitrogen), the reverse transcription was performed

according to the manufacturer’s instructions. Subsequent PCR was

performed for 30 cycles under the following conditions: 948C for 45 sec,

588C for 1 min, and 728C for 1 min, followed by a final extension at 728C

for 5 min. Samples were visualized on 1.2% agarose gels. To confirm the

RT-PCR specificity for the PR1 gene, the generated RT-PCR product

was inserted into a pCR 2.1 vector (Invitrogen), and positive clones

were subsequently sequenced. The following primer pairs were used:

59-CTCTTGTAGGTGCTCTTGTTCTTCC-39 and 59-CTTCATTAGTATG-

GCTTCTCGTTCAC-39 for PR1, and 59-GACATGGAAAAGATATGGCAT-

CACAC-39 and 59-AGATCCTTCCTGATATCGACATCAC-39 for actin.

Preparation of E. carotovora Elicitors and Application of Chemicals

For E. carotovora elicitor (CF) preparation, a bacterial culture filtrate was

prepared as described previously (Vidal et al., 1997). CF, SA (5 mM in

water), and MeJA (100 mM in 0.1% [v/v] ethanol) were applied at the

indicated concentrations as 5 mL droplets on four fully expanded leaves

for each soil plant (five drops per leaf). MeJA-treated plants were

immediately placed to a tray with a transparent lid. Control plants were

treated identically with water containing 0.1% (v/v) ethanol. Leaves

were collected at time points indicated.

Pathogen Infections

E. c. carotovora SCC1 inoculation was done by applying 10 mL drops of

suspension (106 cells/mL in 0.9% NaCl) on local leaves (one leaf per

plant). After inoculation, plants were incubated for the indicated period of

time under high humidity conditions. Leaf maceration and plant survival

ratio were examined. Inoculation with P. s. tomato DC3000 was per-

formed by infiltration of mature leaves (one leaf per plant; �5 mL

suspension of 106 cells/mL in 10 mM MgCl2). Inoculated leaves were

harvested 24 or 72 h after infiltration and homogenized in 10 mM MgCl2.

Diluted leaf extracts were used to titer the bacteria. Colony-forming units

(cfu) of 6–8 individual plants were determined from each time point. At

least two independent lines for each construct were tested. All experi-

ments were repeated three times.

Quantification of JA and SA

JA and SAwere extracted and quantified with (6)-9,10-Dihydro-jasmonic

acid and 13C1-SA as internal standards using the protocol of Baldwin et al.

(1997). Conjugated SA was determined after hydrolyzing with hydro-

chloric acid (final concentration 2 M) at 808C for 1 h and proceeding after

that as with free SA and JA.

Expression Profiling

Leaves from a pool of 12 different 4-week old plants were harvested.

Fifteen micrograms of total RNA from pCP60 vector control, antisense

suppression line A18, and overexpression line S55 were submitted to the

Finnish DNA Microarray Center, where synthesis of biotin-labeled cRNA,

microarray hybridization to AtGenome1 GeneChips (Affymetrix, Santa

Clara, CA), and scanning procedures were performed (http://www.btk.

utu.fi/Genomics). Experiments and hybridizations were performed twice

and averaged, and Gene Spring software (Silicon Genetics, Redwood

City, CA) was used for normalization and further analyses. Expression

data were normalized globally by taking the 50th percentile of all

measurements as a positive control for each sample, with the bottom

10th percentile as test for correct background subtraction. The

measurement for each probe set in each sample was divided by the

corresponding averaged values of pCP60 (set to 1) when this expression

value was [0.05. Normalized averaged expression values \0.05 were

adjusted to 0.05. Probe sets with a detection P value\0.06 in at least two

independent samples were defined as accurately detectable (57% of all

probe sets) and corresponded to expressed genes. Genes with a more

than twofold change in expression level in any transgenic plant compared

with the two others (7% of the expressed genes; see supplemental data

online) were clustered by K-means analysis.

Sequence data from this article have been deposited with the EMBL/

GenBank data libraries under accession numbers AL163972, AF421157,

CS3071, and CS3726.

ACKNOWLEDGMENTS

We thank the following for materials: J. Turner (coi1-1), J. Ryals (NahG),

A. Hegedus (pCP60 plasmid), M. Romantschuk (virulent Pst DC3000),

and the Arabidopsis Biological Resource Center (npr1-1 and ein2-1). We

thank Turku Center for Biotechnology for Affymetrix service. This work

was supported by the Academy of Finland (Grants 38033, 42180, 49905,

44252, and 44883; Finnish Center of Excellence Programme 2000–

2005), Biocentrum Helsinki, and Helsinki Graduate School in Bio-

technology and Molecular Biology.

Received September 4, 2003; accepted November 21, 2003.

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WRKY70 in Disease Resistance 331

DOI 10.1105/tpc.016980; originally published online January 23, 2004; 2004;16;319-331Plant Cell

Jing Li, Günter Brader and E. Tapio PalvaSalicylate-Mediated Signals in Plant Defense

The WRKY70 Transcription Factor: A Node of Convergence for Jasmonate-Mediated and

 This information is current as of September 18, 2014

 

Supplemental Data http://www.plantcell.org/content/suppl/2004/02/06/tpc.016980.DC1.html

References http://www.plantcell.org/content/16/2/319.full.html#ref-list-1

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