Priming of plant innate immunity by rhizobacteria and beta-aminobutyric acid: differences and similarities in regulation.

Abstract

Pseudomonas fluorescens WCS417r bacteria and beta-aminobutyric acid can induce disease resistance in Arabidopsis, which is based on priming of defence. In this study, we examined the differences and similarities of WCS417r- and beta-aminobutyric acid-induced priming. Both WCS417r and beta-aminobutyric acid prime for enhanced deposition of callose-rich papillae after infection by the oomycete Hyaloperonospora arabidopsis. This priming is regulated by convergent pathways, which depend on phosphoinositide- and ABA-dependent signalling components. Conversely, induced resistance by WCS417r and beta-aminobutyric acid against the bacterial pathogen Pseudomonas syringae are controlled by distinct NPR1-dependent signalling pathways. As WCS417r and beta-aminobutyric acid prime jasmonate- and salicylate-inducible genes, respectively, we subsequently investigated the role of transcription factors. A quantitative PCR-based genome-wide screen for putative WCS417r- and beta-aminobutyric acid-responsive transcription factor genes revealed distinct sets of priming-responsive genes. Transcriptional analysis of a selection of these genes showed that they can serve as specific markers for priming. Promoter analysis of WRKY genes identified a putative cis-element that is strongly over-represented in promoters of 21 NPR1-dependent, beta-aminobutyric acid-inducible WRKY genes. Our study shows that priming of defence is regulated by different pathways, depending on the inducing agent and the challenging pathogen. Furthermore, we demonstrated that priming is associated with the enhanced expression of transcription factors.

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Blackwell Publishing LtdOxford, UK NPHNew Phytologist0028-646X1469-8137© The Authors (2009). Journal compilation © New Phytologist (2009)285110.1111/j. 1469-8137.2009.02851.x March 20090 0 419??? 431???Original Article
XX
Priming of plant innate immunity by rhizobacteria
and β-aminobutyric acid: differences and similarities
in regulation
XX
Sjoerd Van der Ent1,2, Marieke Van Hulten1, Maria J. Pozo1,3, Tomasz Czechowski4,5, Michael K. Udvardi4,6,
Corné M. J. Pieterse1,2 and Jurriaan Ton1,7
1Graduate School Experimental Plant Sciences, Plant–Microbe Interactions, Institute of Environmental Biology, Faculty of Science, Utrecht University, PO Box 800.84,
3508 TB Utrecht, The Netherlands; 2Centre for Biosystems Genomics, PO Box 98, 6700 AB Wageningen, The Netherlands; 3Department of Soil Microbiology and
Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Prof. Albareda 1, 18008 Granada, Spain; 4Max-Planck Institute of Molecular Plant Physiology, Am
Mühlenberg 1, 14476 Golm, Germany; 5CNAP Research Laboratories, Department of Biology (Area 7), University of York, Heslington, PO Box 373, York YO10 5YW,
UK; 6Samuel Roberts Noble Foundation, Plant Biology Division, 2510 Sam Noble Pky, Ardmore, OK 73401, USA; 7Rothamsted Research, Department of Biological
Chemistry, West Common, Harpenden, Hertfordshire AL5 2JQ, UK
Summary
• Pseudomonas fluorescens WCS417r bacteria and β-aminobutyric acid can induce
disease resistance in Arabidopsis, which is based on priming of defence.
• In this study, we examined the differences and similarities of WCS417r- and
β-aminobutyric acid-induced priming.
• Both WCS417r and β-aminobutyric acid prime for enhanced deposition of callose-rich
papillae after infection by the oomycete Hyaloperonospora arabidopsis. This
priming is regulated by convergent pathways, which depend on phosphoinositide-
and ABA-dependent signalling components. Conversely, induced resistance by
WCS417r and β-aminobutyric acid against the bacterial pathogen Pseudomonas
syringae are controlled by distinct NPR1-dependent signalling pathways. As
WCS417r and β-aminobutyric acid prime jasmonate- and salicylate-inducible genes,
respectively, we subsequently investigated the role of transcription factors. A
quantitative PCR-based genome-wide screen for putative WCS417r- and β-
aminobutyric acid-responsive transcription factor genes revealed distinct sets of
priming-responsive genes. Transcriptional analysis of a selection of these genes
showed that they can serve as specific markers for priming. Promoter analysis of
WRKY genes identified a putative cis-element that is strongly over-represented
in promoters of 21 NPR1-dependent, β-aminobutyric acid-inducible WRKY genes.
• Our study shows that priming of defence is regulated by different pathways,
depending on the inducing agent and the challenging pathogen. Furthermore, we demon-
strated that priming is associated with the enhanced expression of transcription factors.
Abbreviations: BABA, β-aminobutyric acid; BTH, benzothiadiazole; ET, ethylene;
IR, induced resistance; ISR, induced systemic resistance; JA, jasmonic acid; MeJA,
methyl jasmonate; PR, pathogenesis-related; qRT-PCR, quantitative reverse
transcriptase-polymerase chain reaction; SA, salicylic acid; SAR, systemic acquired
resistance; TAIR, The Arabidopsis Information Resource; TF, transcription factor.
Author for correspondence:
Jurriaan Ton
Tel: +44 1582763133 2893
Email: jurriaan.ton@bbsrc.ac.uk
Received: 2 January 2009
Accepted: 3 March 2009
New Phytologist (2009) 183: 419–431
doi: 10.1111/j.1469-8137.2009.02851.x
Key words: Arabidopsis thaliana, callose,
NPR1, priming, transcription factors,
WRKY.
Introduction
Many plant pathogens have evolved the capacity to circumvent
or suppress host defence mechanisms that are controlled by
the plant’s innate immune system. As an evolutionary
response, plants have evolved additional defence strategies
that allow them to detect and halt pathogen invasion at an
early stage of infection. Resistance (R) gene-mediated resistance

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is a well-known example of a counter-evolved defence system
that enables plants to respond quickly to pathogens that
suppress immune responses (McDowell & Woffenden, 2003;
Jones & Dangl, 2006). In addition, plants have evolved the
ability to enhance their basal resistance after perception of
specific biotic or abiotic stimuli, such as pathogen attack, root
colonization by selective rhizobacteria or selected chemicals.
This so-called induced resistance (IR) does not necessarily
require direct activation of defence mechanisms, but can
also result from a sensitization of the tissue to express basal
defence mechanisms more rapidly and more strongly after
subsequent pathogen attack. This sensitization of the plant’s
innate immune system is commonly referred to as ‘priming’
(Conrath et al., 2006). As demonstrated recently, the
induction of priming yields broad-spectrum resistance
with minimal reductions in plant growth and seed set (Van
Hulten et al., 2006). Hence, priming is a cost-efficient resistance
strategy that increases the plant’s ability to cope with
environmental stress.
The classic form of IR develops on attempted or limited
infection by a pathogen, and results in a systemic acquired
resistance (SAR) that protects against various types of pathogen
(Ryals et al., 1996). The signalling pathway controlling SAR
depends on the endogenous accumulation of the phytohor-
mone salicylic acid (SA) and on the presence of the defence
regulatory protein NPR1 (Durrant & Dong, 2004). NPR1
functions in both SA-dependent basal resistance and SAR
by controlling the expression of many stress-related genes,
including those that encode pathogenesis-related (PR)
proteins and proteins involved in the secretory pathway
(Dong, 2004; Wang et al., 2005). Colonization of plant roots
by the nonpathogenic rhizobacterial strain Pseudomonas
fluorescens WCS417r also triggers a systemic resistance against
a wide range of pathogens, including the downy mildew-causing
oomycete Hyaloperonospora arabidopsis, formerly known
as (Hyalo)peronospora parasitica (Göker et al., 2004), and the
bacterial speck pathogen Pseudomonas syringae pv. tomato
DC3000 (Pieterse et al., 1996; Ton et al., 2002). In contrast
with pathogen-induced SAR, this so-called rhizobacteria-
induced systemic resistance (ISR) functions independently
of SA, but requires components of response pathways that
depend on jasmonic acid (JA) and ethylene (ET) (Pieterse
et al., 1998; Van Wees et al., 2008). ISR elicited by WCS417r
bacteria (WCS417r-ISR) is not accompanied by direct
activation of PR genes (Pieterse et al., 1996), but leads to
priming of predominantly JA- and ET-responsive genes
(Van Wees et al., 1999; Verhagen et al., 2004). Both WCS417r-
ISR and SAR require NPR1 (Pieterse et al., 1998), suggesting
that this protein is important in regulating and connecting
different hormone-dependent defence pathways. Recently,
the transcription factors (TFs) MYB72 and MYC2 have been
demonstrated to be crucial for WCS417r-ISR (Pozo et al.,
2008; Van der Ent et al., 2008). MYB72 gene expression is
specifically activated in the roots of WCS417r-colonized
plants (Van der Ent et al., 2008), whereas MYC2 is thought
to regulate ISR expression in systemic plant parts (Pozo et al.,
2008).
In addition to biological resistance-inducing agents, many
chemicals have been reported to trigger IR. Most of these agents
trigger the SAR pathway, as they activate a similar set of PR
genes and fail to induce resistance in SAR-impaired
mutants (Lawton et al., 1996; Dong et al., 1999). However,
the non-protein amino acid β-aminobutyric acid (BABA)
has been shown to trigger a partially different IR response.
Like WCS417r-ISR, BABA-IR is not associated with a
major transcriptional activation of defence-related genes
(Zimmerli et al., 2000). Instead, BABA primes the plant
tissue for enhanced activation of SA-responsive genes, such as
PR-1 (Zimmerli et al., 2000, 2001). BABA-IR in Arabidopsis
against P. syringae DC3000 resembles pathogen-induced SAR
in its requirement for SA and NPR1 (Zimmerli et al., 2000).
Yet, BABA-IR against H. arabidopsis is fully expressed in
Arabidopsis genotypes that are impaired in SAR signalling
(Zimmerli et al., 2000). This SA- and NPR1-independent
form of BABA-IR is based on a priming of cell-based defences,
which manifests as an augmented deposition of callose-rich
papillae at the sites of attempted tissue penetration by the
attacking pathogen (Zimmerli et al., 2000; Jakab et al., 2001;
Ton & Mauch-Mani, 2004). Screening for Arabidopsis
mutants that are impaired in BABA-induced sterility (ibs)
resulted in the isolation of two mutants, ibs2 and ibs3,
which are affected in BABA-induced priming of papillae
deposition. The ibs2 mutant carries a mutation in the SAC1b
gene that encodes a polyphosphoinositide phosphatase (Despres
et al., 2003), suggesting involvement of a phosphoinositide-
dependent signalling pathway in BABA-induced priming of
cell wall defence (Ton et al., 2005). The ibs3 mutant, however,
is impaired in the regulation of the zeaxanthin epoxidase gene
ABA1/NPQ2, which links ABA signalling to BABA-induced
priming of cell wall defence (Ton et al., 2005). The latter
finding is supported by previous observations that the ABA
biosynthetic mutant aba1-5 and the ABA response mutant
abi4-1 are impaired in BABA-induced priming of callose
deposition after inoculation with the necrotrophic fungi
Alternaria brassicicola and Plectosphaerella cucumerina (Ton &
Mauch-Mani, 2004). Hence, BABA-induced priming of
pathogen-inducible papillae deposition involves regulation by
a phosphoinositide- and ABA-dependent signalling pathway.
Despite the differences in signalling pathways between
WCS417r-ISR and BABA-IR, both forms of IR are charac-
terized by primed resistance mechanisms (Van Wees et al.,
1999; Zimmerli et al., 2000; Ton & Mauch-Mani, 2004;
Verhagen et al., 2004). Although priming has been known
for years, the current understanding of its underlying mech-
anisms remains rudimentary. It has been hypothesized
that the induction of priming leads to a subtle increase in the
level of signalling components that play a role in basal
resistance (Conrath et al., 2006). After perception of a second,

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pathogen-derived signal, the enhanced signalling capacity
would facilitate a faster and stronger basal defence reaction. As
primed plants display a faster and stronger transcription of
defence-related genes after pathogen challenge (Zimmerli
et al., 2000; Kohler et al., 2002; Verhagen et al., 2004; Ton
et al., 2007), TFs may well play a role in the onset of priming.
We hypothesize that the induction of priming causes a subtle
increase in the expression of defence regulatory TFs, which is
sufficient to prime defence genes, but too weak to activate
them directly. However, primed deposition of papillae is a rel-
atively rapid defence reaction that is unlikely to be controlled
at the transcriptional level, suggesting that transcription-
independent mechanisms are involved as well.
In this study, we compared the involvement of two cellular
responses in WCS417r-ISR and BABA-IR: (1) the formation
of callose-rich papillae and (2) the expression of TF genes. To
study the mechanisms behind priming of papillae deposition,
we focused on the roles of IBS2 and IBS3 in WCS417r-ISR
and BABA-IR against H. arabidopsis WACO9. As WCS417r-
ISR and BABA-IR are both associated with priming for
enhanced transcription of defence-related genes (Zimmerli
et al., 2000; Verhagen et al., 2004), we also examined the
involvement of TFs during the onset of the WCS417r- and
BABA-induced defence priming. These analyses revealed sets
of TF genes that are directly responsive to priming treatment
with WCS417r bacteria or BABA, and identified a putative
cis-element in promoters of NPR1-dependent, BABA-inducible
WRKY genes.
Materials and Methods
Plants and microorganisms
Seeds of Arabidopsis thaliana (L.) Heynh. accession Col-0 and
its mutants ibs2-2, which carries a T-DNA insertion in the
5′-untranslated region of SAC1b (At5g66020; this mutant is
also referred to as s-031243; Ton et al., 2005), ibs3-2, which
harbours an ethyl methanesulfonate (EMS)-induced mutation
in the ABA1/NPQ2 gene (also referred to as npq2-1; Niyogi
et al., 1998; Ton et al., 2005), myb72-1 (Van de Mortel et al.,
2008) and npr1-1 (Cao et al., 1994) were cultivated as described
previously (Pieterse et al., 1996). Pseudomonas fluorescens strain
WCS417r and P. syringae pv. tomato DC3000 (Whalen et al.,
1991) were cultivated as described previously by Pieterse et al.
(1996). Hyaloperonospora arabidopsis strain WACO9 was
cultivated as described by Ton et al. (2002).
Induction treatments and disease assays
ISR was elicited by transplanting 2-wk-old Arabidopsis
seedlings into a sand–potting soil mixture containing 5 × 107
colony-forming units WCS417r bacteria per gram of soil.
Control soil was supplemented with an equal volume of
10 mm MgSO4. BABA-IR was triggered by applying BABA
(Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands)
as a soil drench at the indicated concentrations. Treatment
with methyl jasmonate (MeJA) was performed by dipping
5-wk-old Col-0 plants in an aqueous solution containing
50 µm MeJA (Serva, Brunschwig Chemie, Amsterdam,
The Netherlands) and 0.015% Silwet L-77 (Van Meeuwen
Chemicals B.V., Weesp, The Netherlands), as described
previously (Pieterse et al., 1998). Benzothiadiazole (BTH;
CIBA-GEIGY GmbH, Frankfurt, Germany) was administered
by spraying 5-wk-old plants with a solution containing
19 mg l−1 wettable powder and BTH at the indicated
concentrations. Leaf rosettes were harvested at the indicated
intervals after application and immediately frozen in
liquid nitrogen. IR assays with P. syringae DC3000 and
H. arabidopsis WACO9 were performed as described by
Pieterse et al. (1996) and Ton et al. (2005), respectively.
To visualize colonization by H. arabidopsis WACO9, infected
leaves were stained with lactophenol–trypan blue and
examined microscopically at 5 d after inoculation, as described
by Koch & Slusarenko (1990). Quantification of callose
deposition was performed as described by Ton & Mauch-Mani
(2004).
Northern blot analysis and quantitative reverse
transcriptase-polymerase chain reaction (qRT-PCR)
analysis
Total RNA was extracted from pooled shoot samples, as
described by Van Wees et al. (1999). For northern blot
analysis, 10 µg RNA was denatured using glyoxal and
dimethylsulphoxide (Sambrook et al., 1989), electrophore-
tically separated on a 1.5% agarose gel, and blotted onto
Hybond-N+ membranes (Amersham, ’s-Hertogenbosch, The
Netherlands) by capillary transfer. The electrophoresis and
blotting buffer consisted of 10 and 25 mm sodium phosphate
(pH 7.0), respectively. Northern blots were hybridized
with gene-specific probes for PR-1 and LOX2, as described
previously (Pieterse et al., 1998). To check for equal loading,
RNA gel blots were stripped and hybridized with a gene-
specific probe for 18S rRNA. qRT-PCR analysis was
performed basically as described by Czechowski et al. (2004)
(for further details, see Supporting Information Notes S1).
Statistical analysis of expression data
Cluster analysis (Euclidean distance) and principal component
analysis of the transcriptional patterns of the selected TF genes
were based on the expression values from three independent
biological samples per treatment, using TIGR Multi-experiment
Viewer software (Saeed et al., 2003). Both analyses were
performed with the loge-transformed values of the fold
induction ratio of each gene, which was defined as the
expression value in each replicate sample divided by the mean
expression value of the three corresponding control samples.

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Log-transformed fold induction values of TF-encoding genes
from independent experiments were analysed using a paired
Student’s t-test or a nonparametric Wilcoxon Mann–Whitney test.
Promoter analysis
Promoter analyses were based on the 1000-bp sequences
preceding the 5′-end of each transcription unit, which were
obtained from the Sequence Bulk Download and Analysis
tool of The Arabidopsis Information Resource (TAIR)
(http://www.arabidopsis.org/tools/bulk/sequences/index.jsp).
The promoter sequences of WCS417r- and BABA-inducible
TFs were examined for the over-representation of cis-acting
elements, using the POBO bootstrapping program (Kankainen
& Holm, 2004) and SCOPE for parameter-free de novo
computational motif discovery. POBO and SCOPE analyses
were performed under the program settings recommended
at http://ekhidna.biocenter.helsinki.fi/poxo/pobo/help#p2 and
http://genie.dartmouth.edu/scope/, respectively. Differences
in DNA element frequency between selected groups of
promoters were analysed statistically with SPSS 11.5 software,
using a chi-squared test to compare proportions between: (1)
the total number of promoters in the selected group; (2) the
number of promoters containing the DNA element; and (3)
the total number of DNA elements. The TAG[TA]CT motif
in the promoter regions of the BABA-responsive WRKY
genes was identified with the Statistical Motif Analysis tool of
TAIR (http://www.arabidopsis.org/tools/bulk/motiffinder/
index.jsp). SCOPE analysis was used to verify the analysis and
identify variations to the TAG[TA]CT motif.
Results
WCS417r- and BABA-induced priming of callose
deposition after H. arabidopsis infection differs in the
requirement for NPR1
The effectiveness of BABA-IR against H. arabidopsis is almost
entirely based on NPR1-independent priming of callose-
containing cell wall papillae (Zimmerli et al., 2000; Ton et al.,
2005). Primed callose deposition in SAR-expressing plants,
however, requires NPR1 (Kohler et al., 2002). Furthermore,
we have recently found that augmented callose deposition
also occurs during the expression of WCS417-ISR against
H. arabidopsis (Van der Ent et al., 2008). To further elucidate
the role of NPR1 in the priming of cell wall defence, we
quantified levels of H. arabidopsis-induced callose in Col-0
and npr1-1 plants after treatment with BABA and WCS417r.
Both priming agents reduced downy mildew disease and
colonization by H. arabidopsis in wild-type plants, although
BABA protected the leaves more effectively than did
WCS417r (Fig. 1a,b, respectively). WCS417r and BABA both
mediated an augmented deposition of callose-rich papillae
at 2 d after inoculation with H. arabidopsis spores (Fig. 1c),
which was proportional to the level of protection (Fig. 1a,b).
Despite these similarities, WCS417r- and BABA-induced
priming of cell wall defence differed in the requirement for
NPR1: although WCS417r-treated npr1 plants failed to
deposit primed levels of callose, BABA-treated npr1 plants
displayed a similar augmentation in callose deposition as
BABA-treated wild-type plants (Fig. 1). In addition, the level
of callose deposition correlated with the level of protection:
WCS417r-colonized npr1 plants failed to express ISR, whereas
BABA-treated npr1 was protected to similar levels as BABA-
treated wild-type plants (Fig. 1c). Hence, WCS417r-induced
priming of cell wall defence depends on NPR1, whereas the
same priming during BABA-IR is regulated independently
of NPR1.
WCS417r- and BABA-induced priming of cell wall
defence requires IBS2 and IBS3
BABA-induced priming of cell wall defence is dependent
on IBS2 and IBS3, which encode a polyphosphoinositide
phosphatase and a zeaxanthin epoxidase, respectively (Ton
et al., 2005). To test whether IBS2 and IBS3 also contributed
to WCS417r-induced priming of cell wall defence, we
quantified levels of ISR and callose deposition in wild-type,
ibs2 and ibs3 plants. In contrast with wild-type plants, ibs2 and
ibs3 failed to express WCS417r-ISR against H. arabidopsis
(Fig. 2a). Furthermore, WCS417r-treated ibs2 and ibs3 plants
did not deposit primed levels of callose at 2 d after pathogen
inoculation (Fig. 2b). This demonstrates that WCS417r-
induced priming of cell wall defence requires IBS2 and IBS3.
Conversely, the ISR-impaired mutants npr1 and myb72
expressed wild-type levels of BABA-IR against H. arabidopsis
WACO9 and deposited primed amounts of callose on BABA
treatment (Fig. 3). Hence, WCS417r-ISR and BABA-IR
against H. arabidopsis differ in their requirement for NPR1
and MYB72, but both pathways converge in their requirement
for IBS2 and IBS3 to mediate primed papillae deposition and
IR against H. arabidopsis.
WCS417r-ISR and BABA-IR against P. syringae DC3000
function independently of IBS2 and IBS3
To test the involvement of IBS2 and IBS3 in WCS417r-ISR
and BABA-IR against the bacterial pathogen P. syringae
DC3000, we quantified levels of bacterial speck disease in
wild-type, ibs2 and ibs3 plants after treatment with WCS417r
or BABA. Since as WCS417r-ISR and BABA-IR against
P. syringae DC3000 both require NPR1 (Pieterse et al., 1998;
Zimmerli et al., 2000), the npr1 mutant was included as a
negative control genotype. As shown in Fig. 4, both WCS417r
and BABA conferred statistically significant levels of disease
protection in wild-type, ibs2 and ibs3 plants, but not in npr1
plants. This demonstrates that WCS417r-ISR and BABA-IR
against P. syringae DC3000 do not require IBS2 and IBS3, but

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depend on NPR1. Furthermore, these results illustrate that
WCS417r-ISR and BABA-IR against P. syringae DC3000 are
regulated differently from WCS417r- and BABA-induced
priming of cell wall defence against H. arabidopsis WACO9.
WCS417r and BABA prime defence gene induction in
response to exogenously applied JA and SA, respectively
The expression of WCS417r-ISR against P. syringae DC3000
is associated with a faster and stronger induction of JA-
inducible genes after pathogen infection (Van Wees et al.,
1999; Hase et al., 2003; Verhagen et al., 2004). Recently,
Pozo et al. (2008) reported that colonization by WCS417r
enhances the plant’s transcriptional response to JA. In
agreement with this, we found that WCS417r-treated plants
display an augmented induction of the JA-responsive marker
gene LOX2 at different time points after treatment of the leaves
with 50 µm MeJA (Fig. 5a). Hence, WCS417r bacteria prime
the responsiveness to JA, suggesting that WCS417r-induced
priming targets signal transduction steps downstream of JA
Fig. 1 Primed expression of cell wall defence against Hyaloperonospora arabidopsis WACO9 during expression of WCS417r-induced systemic
resistance (WCS417r-ISR) and β-aminobutyric acid-induced resistance (BABA-IR) in Arabidopsis wild-type (Col-0) and npr1 plants. (a)
Quantification of WCS417r-ISR and BABA-IR against H. arabidopsis WACO9 at 8 d after inoculation. ISR was triggered by transferring 2-wk-old
seedlings to potting soil containing Pseudomonas fluorescens WCS417r bacteria. BABA was applied to 3-wk-old plants by soil drenching to a
final concentration of 80 µM BABA. One week after transplanting and 1 d after soil-drench treatment with BABA, leaves were inoculated
with H. arabidopsis spores. Disease ratings are expressed as the percentages of leaves in disease classes I (no sporulation), II (trailing necrosis),
III (< 50% of the leaf area covered by sporangia) and IV (heavily covered with sporangia, with additional chlorosis and leaf collapse).
Asterisks indicate statistically significantly different distributions of leaves in disease severity classes compared with the water control
(χ2 test; α = 0.05). (b) Colonization by the pathogen at 8 d after inoculation was visualized by lactophenol–trypan blue staining and
light microscopy. (c) Pathogen-induced papillae deposition at 2 d after inoculation was quantified by the percentage of callose-inducing
spores in the epidermal cell layer of calcofluor–aniline blue stained leaves using epifluorescence microscopy (UV). The data presented are
from a representative experiment that was repeated twice with similar results.
Fig. 2 Primed expression of cell wall defence
against Hyaloperonospora arabidopsis
WACO9 during WCS417r-induced systemic
resistance (WCS417r-ISR) in leaves of
Arabidopsis wild-type (Col-0), ibs2 and ibs3
plants. (a) Quantification of WCS417r-ISR
against H. arabidopsis. For details, see legend
to Fig. 1a. (b) Quantification of papillae
deposition. For details, see legend to Fig. 1c.

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accumulation. In contrast with WCS417r-ISR, BABA-IR
against P. syringae DC3000 is marked by primed induction of
SA-responsive genes after pathogen infection (Zimmerli et al.,
2000; Van Hulten et al., 2006). To assess whether BABA-
induced priming acts through increased responsiveness to
SA, leaves of water- and BABA-treated plants were sprayed
with increasing concentrations of the SA analogue BTH
and examined for induced transcription of the SA-inducible
marker gene PR-1. At 6 h after treatment with either 50 or
200 mg l−1 BTH, BABA-treated plants showed strongly
augmented induction of PR-1 in comparison with nonprimed
control plants (Fig. 5b). At this time point, BABA-treated
plants displayed a faint induction of PR-1 in comparison
with control-treated plants. In a separate experiment, similar
priming effects were observed at 24 h after treatment with 150
and 300 mg l−1 BTH, whereas the direct effect of BABA on
PR-1 expression was no longer detectable (Fig. 5b). Together,
these results demonstrate that BABA boosts the plant’s
responsiveness to SA, suggesting that signal transduction steps
downstream of SA are targeted by priming.
Fig. 4 WCS417r-induced systemic resistance (WCS417r-ISR)
and β-aminobutyric acid-induced resistance (BABA-IR) against
virulent Pseudomonas syringae DC3000 in leaves of Arabidopsis
wild-type (Col-0), ibs2, ibs3 and npr1. (a) Quantification of
WCS417r-ISR at 4 d after inoculation of the leaves with a suspension
of virulent P. syringae DC3000. Data presented are the means of the
average percentage of diseased leaves per plant (± SD). Asterisks
indicate statistically significant differences compared with noninduced
control plants (Student’s t-test; α = 0.05; n = 20–25). (b)
Quantification of BABA-IR against P. syringae DC3000. Inoculation
and disease scoring were performed as described above.
Fig. 3 Primed expression of cell wall defence
during β-aminobutyric acid-induced resistance
(BABA-IR) against Hyaloperonospora
arabidopsis WACO9 in leaves of Arabidopsis
wild-type (Col-0), npr1 and myb72 plants.
(a) Quantification of BABA-IR against
H. arabidopsis. For details, see legend to
Fig. 1a. (b) Quantification of papillae
deposition. For details, see legend to Fig. 1c.
Fig. 5 Priming for augmented induction of defence genes after
exogenously applied jasmonic acid (JA) and salicylic acid (SA).
(a) WCS417r-induced priming for accelerated induction of the
JA-inducible LOX2 gene. Shoots of 5-wk-old Arabidopsis plants
(Col-0) were dipped in a solution containing 50 µM methyl jasmonate
(MeJA). Leaf rosettes were harvested at the indicated time points
after MeJA treatment. (b) β-Aminobutyric acid (BABA)-induced
priming for enhanced transcription of the SA-inducible PR-1
gene after treatment with the SA analogue benzothiadiazole (BTH).
Five-week-old plants (Col-0) were soil-drenched with 250 µM BABA
and, 1 d later, sprayed with the indicated concentrations of BTH on the
leaves. Leaf rosettes for RNA blot analysis were collected at 6 or 24 h
after BTH treatment.

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WCS417r and BABA influence expression of TF
genes systemically
To examine whether transcriptional priming by WCS417r
and BABA is based on enhanced expression of TFs, we
performed a genome-wide screen for putative WCS417r- and/
or BABA-inducible TF genes. RNA was extracted from leaves
of control-, WCS417r- and BABA-treated plants and analysed
by qRT-PCR using the previously described set of 2248 primer
pairs (Czechowski et al., 2004; McGrath et al., 2005; Libault
et al., 2007). Samples from water- and BABA-treated npr1
plants were included in the analysis to distinguish between
NPR1-dependent and NPR1-independent priming effects by
BABA. The screen identified 90 TF genes with more than
two-fold induction after WCS417r treatment, whereas 31
TF genes were more than two-fold repressed by WCS417r
(Fig. S1A; Table S1; see Supporting Information). Furthermore,
soil drenching of wild-type plants with BABA resulted in more
than two-fold induction of 186 TF genes and more than
two-fold repression of 44 TF genes, whereas BABA treatment
of npr1 plants resulted in more than two-fold induction of 135
TF genes and more than two-fold repression of 141 TF genes
(Fig. S1A; Table S1). Of all putative BABA-inducible genes,
only 32 (10%) were induced in both wild-type and npr1
plants (Fig. S1B), indicating a major role of NPR1 in
BABA-induced expression of TF genes. Furthermore, of all the
247 WCS417r- and/or BABA-inducible TF genes, only 27
genes (10.9%) were induced by both WCS417r and BABA.
This suggests that WCS417r and BABA target largely different
groups of TF genes. Analysis of the 1-kB promoter sequences
of the WCS417r- and BABA-inducible TF genes revealed
a statistically significant over-representation of G-box and
PLGT1-box elements, which are associated with pathogen- and
salt stress-responsive genes (Dröge-Laser et al., 1997; Faktor
et al., 1997; Boter et al., 2004; Park et al., 2004). Promoters of
BABA-inducible TF genes in npr1 displayed a significantly
stronger enrichment in G-box elements than those of BABA-
inducible TF genes in the wild-type (Fig. S2, see Supporting
Information), suggesting a function of G-box elements in
the NPR1-independent induction of TF genes by BABA.
Furthermore, the group of BABA-inducible promoters in
wild-type plants displayed a statistically significant enrichment
of WRKY-binding W-box elements, which was absent in
promoters of WCS417r-inducible genes and in the set of
BABA-inducible genes in npr1 (Fig. S2). This suggests a role
of WRKYs in the NPR1-dependent activation of TF genes
by BABA.
To validate the responsiveness of the putative WCS417r-
and BABA-inducible genes, we quantified the expression levels
of a selection of 37 TF genes in three biological replicate
samples by qRT-PCR (Table S2, see Supporting Information).
This selection was based on the genome-wide screen and
contained five different categories of TF genes: (1) responsive
to BABA in wild-type only (10 genes); (2) responsive to BABA
in wild-type and npr1 (six genes); (3) responsive to WCS417r
and BABA in wild-type only (four genes); (4) responsive
to both BABA and WCSC417r in wild-type and responsive to
BABA in npr1 (nine genes); and (5) only responsive to
WCS417r in wild-type (eight genes). Although the MYC2/
JIN1 gene showed less than two-fold induction by WCS417r
in the genome-wide screen, this gene was included as well,
as MYC2/JIN1 has been identified recently as critical for
WCS417r-ISR (Pozo et al., 2008). Finally, the selection of 37
TF genes was supplemented with seven phytohormone-
responsive genes (PR-1, PR-5, RAB18, PDF1.2, LOX2, VSP2
and EBF2) and four constitutively expressed genes (GAPDH,
UBQ10, At1g13320 and At1g62930) (Czechowski et al., 2005).
To test the specificity of the transcriptional response, the
ISR-noninducing rhizobacterial strain P. fluorescens WCS374r
(Van Wees et al., 1997) and the inactive BABA isomer α-
aminobutyric acid (Jakab et al., 2001) were included as
negative control treatments. In a third experiment, a comparison
was made between the BABA responses of wild-type and
npr1 plants. Hierarchical cluster analysis (Fig. 6a) and principal
component analysis (Fig. 6b) of the transcriptional profiles
clearly distinguished the WCS417r-induced profiles from
the control- and WCS374r-induced profiles. Similarly, the
transcriptional profiles of BABA-treated plants formed a
separate cluster from those of control- and α-aminobutyric
acid-treated plants (Fig. 6a,b), whereas the profiles of BABA-
treated wild-type plants clustered from those of BABA-
treated npr1 plants and corresponding control treatments
(Fig. 6a,b). These results not only validate the outcome of the
genome-wide screen, but also illustrate that the transcriptional
behaviour of the selected TF genes can mark the onset of
WCS417-ISR and BABA-IR.
Identification of a novel promoter element in
BABA-inducible WRKY TF genes
Various WRKYs have been shown to regulate SA-inducible
defence genes (Eulgem & Somssich, 2007). Moreover, some
WRKYs are directly regulated by NPR1 (Wang et al., 2006).
In this context, WRKYs may play a significant role in the
NPR1-dependent priming of SA-inducible defences by
BABA. The genome-wide screen for putative BABA-inducible
TF genes identified 21 WRKY genes that were more than
two-fold induced by BABA (Table S1). To verify these results,
we quantified all 71 WRKY genes for BABA responsiveness in
samples from three independent experiments. In wild-type
plants, 22 of the 71 WRKY genes were, on average, more than
two-fold induced by BABA at a statistical significance level of
α < 0.05 (paired Student’s t-test or Wilcoxon Mann–Whitney
test). By contrast, only three WRKY genes, WRKY30, WRKY33
and WRKY54, met these criteria in the npr1 mutant. However,
all BABA-responsive WRKY genes, except WRKY54, showed
fold induction values that were more than two-fold
reduced by the npr1 mutation (Fig. 7a; Table S3, see Supporting

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Information). Hence, 21 of the 22 BABA-inducible WRKY
genes require NPR1 for full responsiveness to BABA. To
investigate the regulation of these BABA-inducible WRKY
genes, we analysed their promoter regions for over-
representation of DNA elements. Using the Statistical Motif
Analysis tool of TAIR, we found a significant over-
representation of two nearly identical motifs, TAGTCT
and TAGACT (binomial distribution; P = 7.28e−05 and
Fig. 6 Systemic expression of selected transcription factor (TF) genes in Arabidopsis after treatment of the roots with WCS417r or β-aminobutyric
acid (BABA). Root colonization with the induced systemic resistance (ISR)-noninducing Pseudomonas fluorescens strain WCS374r (Experiment
1) and application of the inactive BABA isomer α-aminobutyric acid (Experiment 2) were included as negative control treatments. The npr1 mutant
was included to examine the contribution of NPR1 in BABA-responsive TF gene expression (Experiment 3). (a) Cluster analysis of WCS417r- and
BABA-responsive transcription profiles. The colour intensity of induced (red) or repressed (green) genes is proportional to the fold induction values
of each gene. Fold induction was defined as the expression value in each replicate sample divided by the mean expression value of the three
corresponding control samples (water or MgSO4). Loge-transformed fold inductions were subjected to average linkage clustering (Euclidean
distance). (b) Principal component analysis of WCS417r- and BABA-responsive gene expression. The analysis was based on the loge-transformed
fold induction values, as described above. Grey dots represent genes; coloured squares represent treatments.

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P = 9.02e−05, respectively). Subsequent comparison between
promoters of BABA-inducible WRKY genes, promoters of
BABA-noninducible WRKY genes and a set of random
Arabidopsis promoters revealed clear differences in occurrence.
Although TAGTCT and TAGACT were both significantly
over-represented in NPR1-dependent BABA-inducible WRKY
promoters (data not shown), the most contrasting differences
were found for the combined TAG[TA]CT motif (Fig. 7b).
The fact that this motif is strongly over-represented in
NPR1-dependent, BABA-inducible WRKY promoters
(χ2= 15.93; P < 0.001), and not in BABA-nonresponsive
WRKY promoters (χ2= 0.025; P = 0.9), points to the
existence of a TAG[TA]CT-binding factor to regulate NPR1-
dependent activation of WRKY genes by BABA.
Discussion
WCS417r-ISR and BABA-IR are associated with priming
of various pathogen-inducible defence mechanisms (Conrath
et al., 2006). As both forms of IR are effective against
overlapping spectra of pathogens (Zimmerli et al., 2000; Ton
et al., 2002; Ton & Mauch-Mani, 2004), it is plausible to
assume that WCS417r bacteria and BABA prime at least
partially similar defence reactions. In this study, we investigated
the similarities and differences between WCS417r- and
BABA-induced priming. A clear difference between the two
priming responses is that they target distinct classes of defence-
related genes. WCS417r bacteria prime the induction of
JA-dependent genes, whereas BABA primes the induction of
SA-dependent genes (Verhagen et al., 2004; Ton et al., 2005;
Pozo et al., 2008) (Fig. 5). However, both WCS417r and
BABA mediate the augmented deposition of callose-containing
papillae after infection by the oomycete pathogen H. arabidopsis
WACO9 (Figs 1–3). In both cases, priming is impaired in ibs2
and ibs3 mutants (Fig. 2), indicating that WCS417r-ISR and
BABA-IR against H. arabidopsis share similar ABA- and
phosphoinositide-dependent signalling components (Fig. 8).
Mutants ibs2 and ibs3 are not affected in expression of
Fig. 7 Identification of a putative cis-element in promoter regions of NPR1-dependent, β-aminobutyric acid (BABA)-inducible WRKY genes.
(a) Fold inductions of 71 Arabidopsis WRKY transcription factor (TF) genes in Arabidopsis wild-type (Col-0) and npr1 plants after treatment of
the roots with BABA. Asterisks indicate statistically significant levels of induction based on three independent experiments (paired Student’s t-test
or Wilcoxon Mann–Whitney test; P < 0.05). (b) Occurrences of the TAG[TA]CT motif in promoter regions of NPR1-dependent BABA-inducible
WRKY genes (blue broken line), BABA nonresponsive WRKY genes (yellow broken line), and random Arabidopsis promoters (red full line).
Different letters indicate statistically significant differences in occurrence of the TAG[TA]CT motif (χ2 test; α = 0.05).

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WCS417r-mediated ISR and BABA-IR against the bacterial
pathogen P. syringae DC3000 (Fig. 4). Hence, primed defence
against P. syringae requires different signalling mechanisms
than primed defence against the oomycete H. arabidopsis. This
dissimilarity could be related to differences in infection
strategy by the two pathogens. For instance, P. syringae bacteria
enter leaves through natural openings (Melotto et al., 2006),
and are therefore less restricted by cell wall barriers, whereas
H. arabidopsis hyphae actively penetrate the epidermal cell
layer (Slusarenko & Schlaich, 2003).
As WCS417r and BABA prime the transcriptional induc-
tion of JA- and SA-inducible genes, respectively (Fig. 5), we
examined whether this enhanced transcriptional capacity is
related to a direct increase in TFs. To this end, we screened the
level of expression of nearly all TF genes in the Arabidopsis
genome after priming treatment with WCS417r or BABA.
In contrast with earlier findings reported by Verhagen et al.
(2004), we found consistent effects by WCS417r on TF gene
expression in leaves (Figs 6, 7, S1). This discrepancy can be
explained by the different methodologies used to quantify gene
expression. Verhagen et al. (2004) used microarrays to quantify
gene expression, whereas the transcriptional profiling in this
study was based on qRT-PCR. This technique is substantially
more sensitive than hybridization-based techniques and,
consequently, more reliable for the detection of low-abundant
mRNAs that are characteristic for the expression of TF genes
(Czechowski et al., 2004).
Many of the priming-related TF genes in this study have
previously been reported to be responsive to pathogen attack,
during which they promote defence gene expression directly
(McGrath et al., 2005; Wang et al., 2006). However, the
reported fold induction values on pathogen infection are
typically an order of magnitude higher than those observed in
our study on priming treatment (Tables S1–S3). This difference
supports our model, which predicts that priming causes a
subtle increase in the expression of defence regulatory TFs that
is sufficient to prime defence genes, but too weak to strongly
activate them directly. Conversely, a relatively strong induction
of TFs during pathogen infection is consistent with direct
activation of defence genes. It is, nevertheless, plausible that
pathogen-induced TF expression also contributes to a longer
lasting priming of defence genes after the invading pathogen
has been constrained. As most defence regulatory TFs act as
amplifiers in defence signalling cascades, even a modest induction
during priming can be sufficient to enhance the defence
signalling capacity, thereby giving the primed plant a ‘head
start’ during the early stages of pathogen infection.
Our screen for priming-related TF genes revealed that
WCS417r and BABA influence largely different sets of TF
genes (Figs 6, 7, S1). In combination with our previous findings
that WCS417r and BABA prime for different sets of
defence-related genes (Verhagen et al., 2004; Ton et al., 2005),
we propose that WCS417r-induced TF genes contribute to
the priming of JA-inducible genes, whereas BABA-induced TF
genes contribute to the priming of SA-inducible genes (Fig. 8).
In support of this, WCS417r bacteria induce TF genes that
have been related to the regulation of JA- and ET-dependent
defence reactions, such as AP2/EREBP genes (Table S1).
Amongst these, the ERF1 gene encodes a key regulator in the
integration of JA- and ET-dependent signalling pathways
(Lorenzo et al., 2003). In addition to ERF1, JA-induced
expression of defence genes is co-regulated by the MYC2 TF
(Lorenzo et al., 2004). Interestingly, also, the MYC2 gene is
weakly, yet consistently, induced by WCS417r (Fig. 6a; Table
S2). With respect to the BABA-inducible group of TF genes,
the majority of genes are substantially less responsive to BABA
in the npr1 mutant, indicating that NPR1 plays an important
role in BABA-induced expression of TF genes (Figs 6, 7).
These NPR1-dependent, BABA-inducible TF genes include
21 members of the WRKY family. Some of these, such as
WRKY38, WRKY53, WRKY58 and WRKY70, play a role in
the fine-tuning of SA-inducible defences and are directly
Fig. 8 Differences and similarities in the regulation of WCS417r- and
β-aminobutyric acid (BABA)-induced priming of defence. Root
colonization by the rhizobacterial strain Pseudomonas fluorescens
WCS417r triggers induced systemic resistance (ISR) against the
bacterial pathogen Pseudomonas syringae DC3000 and oomycete
pathogen Hyaloperonospora arabidopsis WACO9. This ISR depends
on MYB72- and NPR1-dependent signalling pathways. WCS417r
bacteria enhance the systemic expression of regulatory transcription
factors (TFs) in the jasmonic acid (JA) response, such as MYC2 and
ERF/EREBP, which leads to primed expression of JA-dependent
defences after infection by P. syringae DC3000. In addition, WCS417r
bacteria trigger IBS2- and IBS3-dependent priming of cell wall
defence, which leads to an augmented deposition of callose-rich
papillae after infection by H. arabidopsis. Root treatment with BABA
also induces resistance against P. syringae and H. arabidopsis.
BABA-induced resistance (IR) against P. syringae DC3000 depends
on salicylic acid (SA)- and NPR1-dependent signalling pathways.
Downstream of NPR1, the BABA-induced resistance pathway leads to
the enhanced expression of regulatory TFs in the SA response, such as
WRKYs, which mediate the primed activation of SA-dependent
defences on infection by P. syringae DC3000 and H. arabidopsis.
In addition, BABA triggers IBS2- and IBS3-dependent priming for
augmented deposition of papillae after infection by H. arabidopsis.
This BABA-induced priming of cell wall defence functions
independently of NPR1.

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targeted by NPR1 (Eulgem, 2005; Wang etal., 2005). Therefore,
we conclude that NPR1 plays an important role in the onset
of BABA-induced priming of SA-inducible defences (Fig. 8).
It is tempting to speculate that the relatively small number
of TFs that are induced by both WCS417r and BABA regulate
defence mechanisms that are primed by both treatments, such
as the expression of callose-rich papillae. Priming of this cell
wall defence is dependent on IBS2 and IBS3, regardless of the
nature of the inducing priming agent (Fig. 2). Yet, this priming
is regulated differently at the level of NPR1 and MYB72.
Our bioassays indicated that NPR1 is necessary for WCS417r-
induced priming of callose, whereas it is dispensable for
BABA-induced priming of callose (Figs 1, 3). Similarly,
MYB72 is important for WCS417r-induced callose priming,
whereas it has no contribution to BABA-induced callose
priming (Fig. 3). As MYB72 is thought to operate at a rela-
tively early stage in the WCS417r-induced signalling pathway
(Van der Ent et al., 2008), we propose that MYB72 and NPR1
act upstream of IBS2 and IBS3 in the WCS417r pathway of
cell wall defence (Fig. 8).
The role of NPR1 in the priming signalling network is
complex. Our data suggest that NPR1 not only controls
WCS417r-induced priming of cell wall defence, but also
regulates WCS417r-ISR against P. syringae DC3000, which is
based on priming of JA-dependent defences (Verhagen et al.,
2004; Pozo et al., 2008). In addition, NPR1 is necessary for
BABA-IR against different pathogens (Zimmerli et al., 2000,
2001), and plays a major role in BABA-induced expression
of TF genes (Figs 6, 7, S1). Finally, NPR1 contributes to
SAR-related priming of pathogen-induced expression of the
PHENYL AMMONIA LYASE gene (Kohler et al., 2002). Hence, the
function of NPR1 in priming depends on the signalling
pathway that is activated upstream (Fig. 8). In this context, it
would be interesting to investigate the role of NPR1-interacting
signalling proteins, such as TGA TFs (Dong, 2004). Another
potentially interesting component is the SNI1 protein, which
functions as a negative regulator of NPR1-dependent gene
expression. Interestingly, SNI1 has been reported to influence
chromatin structure around the SA-inducible PR-1 promoter
(Mosher et al., 2006). Therefore, it is tempting to speculate that
priming antagonizes SNI1-dependent chromatin remodel-
ling, which would enhance the TF-binding capacity to the
gene promoters and successively facilitate a faster and stronger
transcriptional induction of NPR1-dependent defence genes
after pathogen attack.
The direct effects of WCS417r and BABA on TF gene
expression may point to earlier signalling steps in the priming
of defence genes. These transcriptional activations are controlled
by other TFs, whose activity is not necessarily regulated at the
transcriptional level. Such ‘early-acting’ TFs in the pathway
may function as key regulators in the onset of priming. In a
first step in the identification of such factors, we analysed the
promoter regions of WCS417r- and BABA-inducible TFs for
the over-representation of common cis-acting elements. The
promoter regions of both WCS417r- and BABA-inducible TF
genes were significantly enriched in G- and PLGT1-boxes
(Fig. S2). Both elements have been related to transcriptional
responses to pathogen infection, salt stress, JA and ABA
(Dröge-Laser et al., 1997; Faktor et al., 1997; Boter et al.,
2004; Park et al., 2004). Conversely, we found a statistically
significant over-representation of W-boxes in BABA-inducible
genes in the wild-type, but not in the npr1 mutant (Fig. S2).
This result points to the involvement of WRKYs in the
NPR1-dependent induction of TF genes by BABA. In support
of this, we identified 21 WRKY genes that were directly
induced by BABA in wild-type plants, but not, or significantly
less, in the npr1 mutant (Fig. 7a). Promoter analysis of the 21
NPR1-dependent WRKY genes revealed a significant over-
representation of an unknown promoter element (Fig. 7b).
We hypothesize that this TAG[TA]CT element functions as
an important cis-acting element in the NPR1-dependent
activation of TF genes by BABA. Future studies will focus on
the identification of TF proteins that bind to the TAG[TA]CT
element, aiming at the discovery of a novel key regulator in
priming of SA-inducible defence mechanisms.
Acknowledgements
We thank L.C. van Loon for useful suggestions on earlier
versions of the manuscript. We also acknowledge the helpful
suggestions from two anonymous reviewers. This research was
co-financed by the Centre for BioSystems Genomics (CBSG),
which is part of the Netherlands Genomics Initiative/Netherlands
Organization of Scientific Research (NWO), and by grants
865.04.002 and 863.04.019 of the Earth and Life Sciences
Foundation (ALW), which is subsidized by NWO. Research
activities by Jurriaan Ton are supported by a Biotechnology
and Biological Sciences Research Council (BBSRC) Institute
Career Path Fellowship (no. BB/E023959/1).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1 Genome-wide screen for putative WCS417r- and
β-aminobutyric acid (BABA)-responsive transcription factor
(TF) genes in Arabidopsis wild-type (Col-0) and npr1 plants.
Fig. S2 Occurrences of cis-acting elements in the promoter
regions of WCS417r- and β-aminobutyric acid (BABA)-
inducible transcription factor (TF) genes in Arabidopsis
wild-type (Col-0) and npr1 plants.
Notes S1 Experimental details of quantitative reverse
transcriptase-polymerase chain reaction (qRT-PCR) analysis
Table S1 Genome-wide screen for putative WCS417r- and
β-aminobutyric acid (BABA)-responsive transcription factor
(TF) genes
Table S2 Fold induction values of a selection of 37
transcription factor (TF) genes in three biological replicate
samples on treatment of the roots with WCS417r or β-
aminobutyric acid (BABA) in leaves of Arabidopsis wild-type
(Col-0) or npr1 plants
Table S3 Fold induction values of 71 WRKY genes in three
independent experiments on treatment of the roots with
β-aminobutyric acid (BABA) in leaves of Arabidopsis
wild-type (Col-0) or npr1 plants
Please note: Wiley-Blackwell are not responsible for the content
or functionality of any supporting materials supplied by the
authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
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