Molecular Plant
•
Volume 2
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Number 1
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Pages 152–165
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January 2009
RESEARCH ARTICLE
Heat Shock Factors HsfB1 and HsfB2b Are Involved
in the Regulation of Pdf1.2 Expression and
Pathogen Resistance in Arabidopsis
Mukesh Kumara, Wolfgang Buschb, Hannah Birkec, Birgit Kemmerlingd, Thorsten Nürnbergerd and
Friedrich Schöfflc,1
a
b
c
d
Present address: Heinrich-Pette-Institut, Martinistrabe 52, D-20251 Hamburg, Germany
Max-Planck-Institut für Entwicklungsbiologie, Abteilung Molekularbiologie, Speemannstrabe 37–39, D-72076 Tübingen, Germany
Universität Tübingen, Zentrum für Molekularbiologie der Pflanzen (ZMBP)—Allgemeine Genetik, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
Universität Tübingen, Zentrum für Molekularbiologie der Pflanzen (ZMBP)—Biochemie der Pflanzen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany
ABSTRACT In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we
investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b
plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdf1.2a/b in mutant plants.
The Pdf expression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria
brassicicola. The single mutant hsfB2b and the double mutant hsfB1/B2b were significantly improved in disease resistance
after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is
devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE
binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the
shut-off of the heat shock response. The identification of Pdf genes as targets of Hsf-dependent negative regulation is the
first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.
Key words: Abiotic/environmental stress; transcriptional control and transcription factors; transcriptome analysis; defense responses; disease resistance; Arabidopsis.
INTRODUCTION
Environmental adaptation of plants depends on elaborate systems for stress sensing and signaling, common and stressspecific responses, and probably also on a hierarchical control
of reactions. There is evidence that heat shock factors (Hsfs)
play important roles in stress sensing and signaling of different
environmental stresses and that different stresses, including
also high temperature, induce reactive oxygen species (ROS)
in plants (Dat et al., 1998). ROS, particularly H2O2, are important
components in biotic and abiotic stress response and signaling
mechanisms.
Hsfs recognize consensus binding motifs, so-called heat
stress elements (HSE: 5#-nGAAnnTTCnnGAAn-3# or 5#-nTTCnnGAAnnTTCn-3’) conserved in promoters of heat-inducible
genes of all eukaryotes. The classical target genes are the heat
shock protein (Hsp) genes, which are (including the HSE motifs
in the promoter region) highly conserved in eukaryotes (Wu,
1995). Hsfs display a modular structure with a highly conserved
N-terminal DNA-binding domain (DBD), which is characterized
by a central helix-turn-helix motif, and an adjacent bipartite
oligomerization domain (HR-A/B) composed of hydrophobic
heptad repeats. Hsf trimerization via the formation of a triple
stranded alpha-helical coiled coil is a prerequisite for high-affinity DNA binding and subsequently for transcriptional activation of heat shock genes. Other functional domains of Hsf
include clusters of basic amino acid residues (NLS) essential
for nuclear import, leucine-rich export sequences in the HRC region (NES) and—less conserved—a C-terminal activator domain (CTAD) rich in aromatic, hydrophobic, and acidic amino
acids, the so-called AHA motifs (Nover et al., 1996, Döring
et al., 2000). Sequence comparisons indicate that the combination of a C-terminal activator motif (AHA) with the consensus
1
To whom correspondence should be addressed. E-mail friedrich.
schoeffl@zmbp.uni-tuebingen.de, fax +49-7071-295042, tel. +49-70712978831.
ª The Author 2009. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPP and
IPPE, SIBS, CAS.
doi: 10.1093/mp/ssn095
Received 9 September 2008; accepted 26 November 2008
Kumar et al.
sequence FWxx(F/L) (F/I/L) to an adjacent nuclear export signal
(NES) represents a signature domain for many plant activator
Hsfs (Koskull-Döring et al., 2007).
Based on structural characteristics and phylogenetic comparison, plant Hsfs are subdivided into three classes and several
subgroups. Recent studies in Arabidopsis showed that different class A-Hsfs play important roles during early and late
stages of stress response. Early Hsfs are considered to be constitutively expressed in the cell and may become activated immediately upon stress treatment. Late Hsffunctions are considered for
those Hsfs whose expression is significantly enhanced upon stress
treatment, which suggests that these Hsfs require the action of
early Hsfs for their own expression (Wunderlich et al., 2007).
Most research on Hsfs was focused on the functional characterization of class A-Hsfs of Arabidopsis, which comprises 15
different genes/proteins, some of which are known to function
as transcriptional activators of stress target genes. Recent evidence obtained from the identification of class A-Hsf-knockout
mutants and microarray expression profiling indicates that
some early, like HsfA1a and HsfA1b (Busch et al., 2005), and
the late HsfA2 share an overlapping set of target genes.
Much less is known about the function of class B-Hsfs, which
differ from class A by a structural variation within the oligomerization domain and the lack of an AHA-motif that is required for transcriptional activation function of class A-Hsfs
(Koskull-Döring et al., 2007). There are only five different class
B-Hsf genes present in the Arabidopsis genome, two of which
are considered to have early functions (B3 and B4), three are
considered to act as late Hsfs (HsfB1, HsfB2a, HsfB2b) because
their mRNA expression is significantly increased upon heat
stress and there is evidence that this heat-induced expression
requires the combined action of the early class A-HsfA1a and A1b (Lohmann et al., 2004; Busch et al., 2005).
In contrast to Hsfs of class A, classes B- and C-Hsfs have no
evident function as transcription activators on their own
(Kotak et al., 2004; Czarnecka-Verna et al., 2000, 2004). There
is evidence that tomato HsfB1 interacts with HsfA1a and may
also be involved in CREB-dependent expression of housekeeping genes during recovery (Bharti et al., 2004). Interestingly,
the orthologous Arabidopsis HsfB1 did not show such properties in the same assay systems (Bharti et al., 2004). In Arabidopsis, HsfB1 is the most strongly heat-induced class B-Hsf (Busch
et al., 2005). Surprisingly, transgenic overexpression of Hsf4
(synonymous for HsfB1) had no effect on the expression of
heat shock proteins (Hsp) or the development of thermotolerance (Prändl et al., 1998). Since class B-Hsfs have the capacity to
bind to similar or the same sites in the heat shock gene promoters as class A-Hsfs, it was proposed that they may act as
repressors of target gene expression (Czarnecka-Verna et al.,
2000, 2004).
In the present study, we investigated T-DNA knockout
mutants of HsfB1 and HsfB2b for understanding their functional roles during the later phases of heat shock response.
HsfB1/B2b are the only two out of three class B-Hsfs, which
are clear targets of class Hsf-dependent enhancement of ex-
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pression and for which viable knockout mutants were available. Microarray analysis for the identification of putative
target genes revealed that, in hsfB1/hsfB2b double knockout
plants, the major targets are Pdf1.2 genes, which are involved
in immunity against infection by necrotrophic microorganisms.
The identification of Pdf genes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of
Hsf in the regulation of biotic and abiotic responses.
RESULTS
Isolation and Characterization of hsfB1 and hsfB2b
Knockout Mutants
In order to analyze the function of the heat shock-induced Hsf
genes, AtHsfB1 and AtHsfB2b, a loss-of-function strategy was
employed. The T-DNA insertion lines Salk_012292 (insertion in
HsfB1) and Salk_047291 (insertion in HsfB2b) were obtained
from the stock collection of mutants available at the Signal Salk
institute (http://signal.salk.edu/cgi-bin/tdnaexpress) and characterized. Homozygous mutants were identified via PCR screening using gene-specific and T-DNA-specific primers. The
positions of the T-DNA insertions were confirmed and precisely
mapped by DNA sequencing. In HsfB1, the insertion maps to the
second exon 367 nucleotides upstream of the stop codon, in
HsfB2b to a position 277 nucleotides downstream from the first
exon border (Figure 1A). The homozygous mutants hsfB1–/– and
hsfB2b–/– are designated as hsfB1 and hsfB2b, respectively.
The expression of the respective Hsf in hsfB1 and hsfB2b
plants was investigated at the transcript level using qRT–PCR
both at control temperature and after 1 h of heat stress in
comparison to wild-type (WT) plants. It was shown that, at
22C, HsfB1 and HsfB2b are expressed at low levels (15% for
HsfB1 and 4% for HsfB2b compared to actin2 = 100%) in
WT plants (Figure 1B). After heat stress, the transcript levels
of HsfB1 were increased in WT and hsfB2b plants (approximately 12- and 30-fold) but no HsfB1 transcripts were detected
in hsfB1 plants with or without HS. HsfB2b mRNA levels were
up-regulated in WT and hsfB1 plants by factors of three to fivefold after HS but HsfB2b transcripts were not detected in
hsfB2b plants with or without HS. These results indicate that
the T-DNA insertions caused a complete knockout of the expression of the respective Hsf genes in hsfB1 and hsfB2b single
mutant plants.
However, there was no obvious phenotypic effect on morphology or thermotolerance associated with either of the single hsfB1 or hsfB2b mutant lines. Therefore, we isolated
a double knockout mutant hsfB1–/–/hsfB2b–/– (abbreviated
hsfB1/B2b). Following a crossing of the homozygous single
knockout lines, we isolated a double knockout mutant homozygous for both Hsf mutations, in subsequent generations by
PCR screening. The transcript levels of HsfB1 and HsfB2b were
determined in a time course experiment from 0 to 8 h heat
stress in the hsfB1/B2b double mutant compared to WT plants
using qRT–PCR. As previously shown by Lohmann et al. (2004),
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Hsf-Regulated Pdf-Expression–Pathogen Resistance
Figure 1. mRNA Quantification of HsfB1 and HsfB2b in Single and Double Mutants.
(A) T-DNA insertion sites in hsfB1 and hsfB2b lines as determined by sequencing PCR products obtained from genomic DNA of the tagged
lines using either gene-specific 5’ or 3’ primers in combination with T-DNA left border (LB) primers. Triangles indicate the T-DNA elements.
Exons are depicted as grey boxes, introns as open boxes, non-translated regions as hatched boxes. The position and direction of primers for
Hsf genes or T-DNA sequences are marked by horizontal arrows. T-DNA elements and primers are not drawn to scale.
Poly (A)+-RNA was isolated from leaves and analyzed by qRT–PCR for the mRNA levels of HsfB1 and HsfB1b of wild-type (WT) and single
mutant (hsfB1 and hsfB2b) plants that had been subjected to heat stress (37C, 1 h) or control temperature (22C, 1 h) (B), or double knockout (hsfB1/B2b) plants that had been subjected to heat stress (37C) or control temperature (22C) for 0–8 h (C).
PCR levels were normalized with respect to Actin2 mRNA (= 100%). Data points show means (n = 2) and range (B), and means (n = 3) and
S.D. (C), respectively.
HsfB1 and HsfB2b mRNAs are expressed at low levels (20
and0.2%, respectively, compared to actin2) at 22C in WT.
The expression levels are significantly increased after heat
stress (Figure 1C): for HsfB1 to a high level (from 20 to
275% of actin2), for HsfB2b to a lower level (from 0.2 to
3.5% of actin2) after 1–2 h heat stress. No mRNAs were
detected for either of the two Hsfs in the double knockout mutant hsfB1/B2b, neither at control temperature nor after heat
stress. This indicates that HsfB1/B2b expression is completely
eliminated in hsfB1/B2b plants. Despite this clear knockout effect at the level of gene expression, there was no obvious phenotypic difference with respect to growth, fertility and
thermotolerance between hsfB1/B2b, single knockout, or
WT plants.
In order to test whether the combined functions of HsfB1 and
HsfB2b are generally required for late effects in heat shock gene
Kumar et al.
expression, we determined the mRNA expression profiles of a
number of known Hsf target genes (Hsp17.6, Hsp23.6, Hsp70,
Hsp83.1, Hsp101, and GolS) in a time course experiment from
0 to 8 h heat stress (Figure 2). Interestingly, the mRNA profiles,
which differ individually from each other, are almost identical
for WT and hsfB1/B2b double knockout plants. Hence, the deficiency of HsfB1 and HsfB2b has little if any effect on the expression profiles of typical Hsf-A1a/1b target genes (Lohmann
et al., 2004; Panikulangara et al., 2004; Busch et al., 2005).
Expression Profiling: Identification of Putative
HsfB1/HsfB2b Target Genes
In order to understand the functions of HsfB1 and HsfB2b, we
tried to identify genes whose expression is affected in hsfB1/
B2b plants, at either 22C or after heat stress. Considering the
heat-induced expressions of HsfB1 and HsfB2b, it was implicated that the effect should become more obvious after longer
times of heat stress. As suggested by Figure 2, we applied heat
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stress at 37C for 2 h to detect differences in gene expression
between WT and hsfB1/B2b plants.
Whole genome Affymetrix microarray mRNA hybridizations
were performed using RNA samples isolated from leaves that
had been subjected to either 2 h at 22C (control) or heat stress
temperature. By conducting a Welch’s t-test (confidence 95%,
Multiple Testing Correction Benjamini and Hochberg) on the
normalized hybridization data of the two control mRNA samples (not subjected to heat stress), it was examined whether
HsfB1 and HsfB2b exert an influence on basal gene expression.
The datasets of genes expressed at 22C in WT versus hsfB1/B2b
plants were compared. Only 16 genes appeared to be differentially expressed by a factor of more than two (Figure 3; black
spots, left-hand panel), two of them, Pdf1.2a and Pdf1.2b,
were outstanding with respect to the extraordinary differences in expression levels, with a more than 15-fold increase in
hsfB1/B2b plants (Table 1). The expression of Pdf genes is
known to be induced in response to necrotrophic pathogens
Figure 2. mRNA Expression Profiles of Heat Shock Factor Target Genes.
Levels of mRNAs of different Hsp and GolS1 in wild-type (WT) and hsfB1/B2b double mutant plants during heat stress (HS). Poly (A)+-RNA
was isolated from leaves that had been heat-stressed at 37C for the indicated times, converted into cDNA and subjected to real-time PCR.
Relative amounts were calculated and normalized with respect to Actin2 mRNA (= 100%). Data points show means and range (n = 3).
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and the defensins exhibit protective functions. Only four of
the differentially regulated genes are down-regulated in
hsfB1/B2b plants by a factor of more than two. Among the
down-regulated genes, the strongest effect was on HsfB1
(18.44-fold), which is consistent with the knockout mutation
in this gene. These data indicate that, at 22C, the expression
levels of .99.93% of all genes represented on the chip are
identical in WT and hsfB1/B2b plants.
Figure 3. Comparison of Expression Characteristics Wild-Type versus hsfB1/B2b Double Mutant Plants.
Expression levels (relative units) of all genes (gray spots) detected by microarray hybridization; black spots indicate genes differentially
expressed by the following criteria: more than two-fold difference, confidence 95%, multiple testing correction Benjamini and Hochberg.
The data points for Pdf1.2a and Pdf1.2b are marked with a full circle; dashed circles mark the expression levels genes (HsfB1 and HsfB2b)
significantly down-regulated mutant plants.
(A) Control condition (room temperature: 22C) comparison of wild-type (WT) and hsfB1/B2b double mutant.
(B) Heat shock (37C) comparison of WT and hsfB1/B2b double mutant.
Table 1. List of Up-Regulated (A) and Down-Regulated (B) Genes in hsfB1/B2b Double Mutant Compared to WT at Control Temperature.
(A)
Affymetrix
identifier
249052_at
WT (C)
WT (HS)
hsfB1/B2b
(HS)
hsfB1/B2b
(C)
hsfB1/B2bC
WTC (FC)
AGI
Annotation
48.86
73.46
1,346.00
1,242.00
25.43
At5g44420
Plant defensin protein, putative (PDF1.2a)
110.70
156.80
2,341.00
1,802.00
16.28
At2g26020
Plant defensin protein, putative (PDF1.2b)
259169_at
26.62
27.75
116.00
145.70
5.47
At3g03520
Phosphoesterase family protein
245228_at
89.70
48.74
118.20
271.00
3.02
At3g29810
Phytochelatin synthetase-like protein
266841_at
64.77
17,191.00
17,090.00
280.30
3.00
At2g26150
Putative heat shock transcription factor, AtHsfA2
263374_at
48.03
7,591.00
7,769.00
141.00
2.94
At2g20560
DNAJ heat shock family protein
256518_at
18.43
1,518.00
1,415.00
44.14
2.40
At1g66080
Expressed protein
260025_at
93.37
8,128.00
8,786.00
207.30
2.22
At1g30070
SGS domain-containing protein
248258_at
164.30
759.50
990.50
352.00
2.14
At5g53400
Nuclear movement family protein
257365_x_at
249867_at
12.72
59.00
98.56
25.98
2.04
At5g23020
2-isopropylmalate synthase-like protein
247771_at
153.00
527.90
606.70
311.70
2.04
At5g58590
Ran binding protein 1 homolog-like
262656_at
112.20
2,449.00
2,263.00
226.80
2.02
At1g14200
Zinc finger (C3HC4-type RING finger) family protein
(B)
Affymetrix
identifier
WT (C)
WT (HS)
hsfB1/B2b
(HS)
hsfB1/B2b
(C)
hsfB1/B2bC
WTC (FC)
246214_at
141.60
2,986.00
10.25
7.68
261662_at
30.64
6.71
6.59
5.50
245449_at
179.90
176.20
126.30
247213_at
17.51
13.60
13.80
AGI
Annotation
18.44
At4g36990
Heat shock transcription factor Hsf4
5.58
At1g18350
MAP kinase kinase 5, putative
76.94
2.34
At4g16870
Retrotransposon-like protein
7.92
2.21
At5g64900
Expressed protein
FC, fold change; AGI, Arabidopsis genome initiative gene model (www.arabidopsis.org); HS, heat stress (37C); C, control (22C).
Kumar et al.
In a second comparative analysis, a t-test was applied for
a dataset comprising the more than two-fold differences in
RNA levels of heat-stressed WT versus heat-stressed hsfB1/
B2b plants (Figure 3; right-hand panel). The scattering of
the expression levels (gray spots) indicates the robustness
of the analysis. The black spots mark the expression levels of
31 genes, which were differentially expressed; 15 genes
showed significantly higher, and 16 genes lower levels in
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WT compared to hsfB1/B2b plants (Table 2). Thus, only a relatively small fraction (0.93%) of 3333 heat stress-affected genes
(data not shown) can be attributed to the functions of HsfB1/
B2B in WT. Interestingly, Pdf1.2a and Pdf1.2b appeared to be
also up-regulated (fold changes of 14–18) in hsfB1/B2b plants;
both are expressed at approximately the same high levels after
heat stress as compared to control temperature. Among the
genes down-regulated in hsfB1/B2b plants are, as expected,
Table 2. List of Down-Regulated (A) and Up-Regulated (B) Genes in hsfB1/B2b Double Mutant Compared to WT After Heat Stress.
(A)
Affymetrix
identifier
WT (C)
WT (HS)
246214_at
141.6
254878_at
16.5
hsfB1/B2b
(HS)
hsfB1/B2b
(C)
2,986
10.25
7.68
980.1
11.9
8.95
8.256
WTHS __
hsfB1/B2bHS (FC)
AGI
Annotation
At4g36990
Heat shock transcription factor Hsf-B1
At4g11660
Heat shock transcription factor-like protein,
Hsf-B2b
2.649
At3g06140
Zinc finger (C3HC4-type RING finger) family
protein
291.2
82.37
256400_at
9.51
40.66
15.35
253916_at
46.93
33.14
13.63
51.46
2.431
At4g27240
Zinc finger (C2H2 type) family protein
247141_at
45.29
28.95
12.47
51.44
2.321
At5g65560
Pentatricopeptide (PPR) repeat-containing
protein
252268_at
10.41
24.49
10.71
12.5
2.288
At3g49650
Kinesin motor protein-related
246314_at
69.22
69.06
31.97
69.19
2.16
At3g56850
ABA-responsive element binding protein 3
2.156
At4g20800
FAD-binding domain-containing protein
2.132
At1g05460
RNA helicase SDE3 (SDE3)
2.118
At2g07684
Hypothetical protein
2.067
At4g37240
Expressed protein
254489_at
261381_at
263507_s_at
246200_at
6.139
14.19
6.432
153.6
14.96
28.85
14.5
100.7
6.938
13.53
6.849
48.71
5.966
11.06
5.708
136.1
263795_at
15.31
33.61
16.41
14.21
2.048
At2g24610
Cyclic nucleotide-regulated ion channel,
putative
264689_at
68.18
30.83
15.11
65.42
2.04
At1g09900
Pentatricopeptide (PPR) repeat-containing
protein
260198_at
6.09
12.08
2.032
At1g67635
Hypothetical protein
247922_at
64.07
23.14
2.03
At5g57500
Expressed protein
(B)
Affymetrix
identifier
249052_at
WT (C)
48.86
WT (HS)
73.46
257365_x_at
110.7
156.8
247529_at
515.7
15.3
247533_at
70.64
4.264
5.945
11.4
64.12
hsfB1/B2b
(HS)
18.33
At5g44420
Plant defensin protein, putative (PDF1.2a)
14.93
At2g26020
Plant defensin protein, putative (PDF1.2b)
45.02
11.7
17.55
41.1
102.7
61.25
150.4
256940_at
246405_at
257129_at
15.97
1,008
9.129
6.455
Annotation
1,242
27.4
5.032
AGI
1,802
47.41
1,908
hsfB1/B2bHS
WTHS (FC)
1,346
254300_at
250379_at
hsfB1/B2b
(C)
2,341
248450_at
259502_at
6.849
45.88
14.69
24.15
54.48
19.82
44.24
16.92
35.97
545.7
57.39
2.943
At5g61520
Monosaccharide transporter STP3
2.743
At5g61570
Protein kinase family protein
25.66
2.614
At5g51290
Ceramide kinase-related
57.52
2.498
At4g22780
Translation factor EF-1 alpha-like protein
2,613
4.682
22.56
865.6
9.101
2.455
At1g15670
Kelch repeat-containing F-box family protein
2.276
At5g11590
Transcription factor TINY
2.256
At3g30720
Expressed protein
2.232
At1g57630
Disease resistance protein (TIR class), putative
2.125
At3g20100
Cytochrome p450 family
257876_at
10.42
11.45
24.13
14.04
2.106
At3g17130
Invertase/pectin methylesterase inhibitor
family protein
256595_x_at
32.25
30.15
61.96
38.15
2.055
At3g28530
Expressed protein
2.033
At3g54580
Proline-rich extensin-like family protein
2.026
At3g55820
Hypothetical protein
251842_at
251756_at
8.311
23.5
9.711
13.95
19.74
28.26
8.103
28.71
FC, fold change; AGI, Arabidopsis genome initiative gene model (www.arabidopsis.org); HS, heat stress (37C); C, control (22C).
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HsfB1 (fold-change 291) and HsfB2b (fold-change 82) mRNAs.
This clearly proves the Hsf-gene knock character of the mutant
line. All other differentially expressed genes, up- or downregulated in hsfB1/B2b plants, show only relatively little differences in expression (fold change 2–3) compared to WT.
In order to test the reliability of the microarray data, we reexamined the mRNA levels of Pdf1.2 and a number of other
differentially expressed genes using qRT–PCR (Table 3). By
and large, the results of qRT–PCR confirm the gene-chip hybridization data but, as expected, the PCR data of the strongly
heat-induced genes such as AtHsfA2 and AtHsfB1 show
a greater discrepancy in expression levels to the microarray
data. Similar observations have been reported in the analysis
of other hsf mutants (Busch et al., 2005).
Analysis of Pdf1.2 Gene Promoter Region and
Hsf–HSE Binding
Pdf1.2 genes are up-regulated in hsfB1 and hsfB1/B2b plants
and therefore we inspected the promoter region of Pdf1.2a
and Pdf1.2b. Both genes are highly conserved in coding and
in promoter regions. Using the internet tool http://arabidopsis.
med.ohio-state.edu/AtcisDB/, several other putative binding
motifs in the promoter sequence of Pdf1.2a and Pdf1.2b were
identified. Both promoters share a number of common motifs,
such as DPBF1 and -2, RAV1-A, BoxII, GATA box, GCC-box, and
Ibox. The GCC-box mediates jasmonic acid-induced activation of
Pdf1.2 expression in Arabidopsis (Brown et al., 2003).
However, there was no perfect match to the three-boxed HSE
consensus sequences nGAAn/nTTCn/nGAAn, the preferred binding site for Hsfs. One variant HSE, representing only two boxes
(nGAAn/nTTCn), could be detected in Pdf1.2b at position –629
to –619 upstream of the transcription start site, while this was ab-
sent in Pdf1.2a. However, another imperfect HSE (nGAAn/nATCn)
that represents only one complete box consensus sequence was
present in both promoters, at positions –594/–584 of Pdf1.2a and
at positions –678/–668 in Pdf1.2b. Since both genes and promoters appear highly homologous and co-regulated, we concentrated further investigations only on the Pdf1.2a promoter.
Using EMSA, we were unable to identify any difference in
the formation of binding-specific complexes in the Pdf1.2a
promoter between WT and hsf-mutant plants, neither without
nor after heat stress (not shown). This result suggests that no
Hsf can directly bind to the promoter upstream region.
In a second approach, we assayed for any changes in late Hsf
binding capacity in WT and hsfB1/B2b plants by using synthetic
HSEs, which contain the perfect three-box consensus sequence
nGAAn/nTTCn/nGAAn (Lohmann et al., 2004) as a probe. In
a time course experiment covering up to 4 h heat stress prior
to protein extraction, there was a reproducible difference in
the EMSA profiles between WT and hsfB1/B2b plants (Figure
4). The hsfB1/B2b plants show an earlier onset (60 min), a prolonged appearance (240 min), and a stronger intensity of a signal that represents the major late Hsf–HSE binding complex. In
WT, the same signal appears with a maximum after 2 h heat
stress and is already diminished after 240 min.
Effects of Pathogen Infection on Pdf Expression and
Pathogen Resistance in WT and Hsf-Knockout Plants
There is ample evidence that Pdf1.2a and Pdf1.2b are induced
during pathogen attack or jasmonic acid application
Table 3. Comparison of Gene Expression Levels Determined by
Gene Chip Hybridization and qRT–PCR.
Gene
WT (C)
WT (HS)
a
RT–PCR GC
b
RT–PCR
a
hsfB1/B2b (C) hsfB1/B2b (HS)
GC
b
RT–PCRa GCb RT–PCRa
GCb
Pdf1.2a
1.3
0.9
2.3
2.0
39.1
23.7
47.6
Pdf1.2b
1.4
2.1
2.1
4.2
36.2
34.4
61.8
65.1
AtHsfA2
1.8
1.2 1965.4 459.5
7.6
5.3
1922.9
475.7
DnaJ
1.3
0.9
2.9
2.7
293.1
215.8
Atmkk7
1.7
0.6
0.0
0.2
0.0
0.1
0.0
0.2
AtHsfB1
12.9
2.7
238.6
79.6
0.5
0.1
0.4
0.3
0.1
0.3
3.0
26.2
0.0
0.2
0.0
0.3
Stp3
16.2
9.7
0.7
0.4
18.2
10.4
1.2
1.3
F7H2.2
16.9
36.1
0.7
1.6
22.4
49.7
1.1
4.2
AtHsfB2b
267.1 202.8
37.4
a mRNA level determined quantitative PCR. Relative amounts
were calculated and normalized with respect to Act2 mRNA
(= 100%). Data represents mean 6 range (n = 2).
b Expression estimates based on the gene chip (GC) analysis; data
represent normalized and averaged intensity values of the
replicates relative to the signal of Act2 mRNA hybridization
(= 100%).
Figure 4. The Formation of Late Hsf-HSE Binding Complexes in
Wild-Type and hsfB1/hsfB2b Plants.
Leaves of Arabidopsis wild-type (WT) (Col-0) and double Hsf mutant
(hsfB1/hsfB2b) plants were incubated at 37C for different periods
of time (0, 10, 30, 60, 120, and 240 min). Total protein was extracted
from each sample and subjected to EMSA using radioactive-labeled
synthetic HSE consensus sequence as a probe. The first lane contains
only the free probe without protein. The major late Hsf–HSE binding complex (Lohmann et al., 2004) is marked by an arrowhead; the
unspecific constitutive binding complexes (Lohmann et al., 2004)
are marked by asterisks.
A
3000
ExPdf1.2a/ExActin (%)
Kumar et al.
2500
Control
MeJA
2000
1596
1500
965
1000
579
344
500
0
10
2
WT
ExPdf1.2a/ExActin (%)
B
5000
4000
8
hsfB1
6
hsfB2b
hsfB1/B2b
WT
hsfB1
hsfB2b
hsfB1/B2b
3000
1980
2000
1376
11311130
689
1000
9
0
425
197
0
52
72
96
hours after spray infection
disease index
C
400
WT
hsfB1/B2b
300
200
100
0
0
3
5
7
10
12
DAI
D
Hsf-Regulated Pdf-Expression–Pathogen Resistance
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159
(Penninckx et al., 1998). We tested whether Pdf genes are still
responsive to jasmonic acid induction in double mutants
(hsfB1/B2b) and single Hsf-mutant (hsfB1 or hsfB2b) plants.
Leaves of 5-week-old plants were sprayed with 100 lM MeJ
and samples were collected after 6 h treatment for mRNA
analysis. The results show (Figure 5A) that (1) Pdf1.2a mRNA
is induced by MeJ to higher levels in hsf mutant plants, and
(2) the induced levels in the mutants exceed the induced levels
of WT plants.
We examined whether the infection with the necrotrophic
fungus Alternaria brassicicola leads to enhanced induction of
Pdf1.2a expression and an improved pathogen tolerance in
Hsf-knockout plants. Leaves of 6-week-old plants were inoculated with A. brassicicola in a spray assay for the analysis of
Pdf1.2a mRNA levels using qRT–PCR (Figure 5B) and in a spot
assay (two 5-lL drops of 5 3 105 conidial spores suspended in
water per leaf) for the determination of the disease index (Figure 5C). The results show that Pdf1.2a expression is significantly induced at 72 h post inoculation. This timing in
Arabidopsis WT is in good agreement with published data
(Schenk et al., 2003) showing a significant increase in mRNA
levels 72 h after infection. The mRNA levels appeared to be induced to higher levels in the Hsf knockout plants compared to
WT.
The disease resistance of WT and mutant lines was monitored
on days 3, 5, 7, 10, and 12 after infection with A. brassicicola
(Figure 5C). Infected leaves of double knockout plants (hsfB1/
hsfB2b) provided significantly higher levels of tolerance (lower
disease index) against A. brassicicola infection compared to WT
plants. These differences in pathogen resistance can be seen
from the beginning (3 days after infection (DAI)) throughout
the whole experiment (12 DAI). Comparing the disease index
of all four lines on day 10 after infection, there is a significant
difference between hsfB2b and hsfB1/hafB2b plants, which
were more resistant, compared to WT and single knockout hsfB1
plants (Figure 5D).
350
DISCUSSION
300
disease index
d
250
Considering the complexity of the Hsf gene family, the isolation of knockout mutants for individual Hsf genes is crucial for
the determination of their functional role and biological importance in plants. Although single and double mutations
in HsfB1 and HsfB2b lead to a complete loss of mRNA accumulation, there was no detectable effect on the heat stress response. This finding contrasts the functions of class A-Hsfs in
200
150
100
50
0
WT
hsfB1
hsfB2b
hsfB1/B2b
Figure 5. Effects MeJ and A. brassicicola Infection on Pdf1.2a Expression and Pathogen Resistance in Arabidopsis Wild-Type, Single,
and Double Knockout Hsf Mutants.
Expression of Pdf1.2a was quantified after 6 h treatment with MeJ
(A) or after 72 and 96 h post infection with A. brassicicola (B). Data
were normalized with respect to Actin 2 expression (= 100%),
points show means and S.D. (n = 3).
(C) Leaves of 6-week-old Arabidopsis wild-type (WT) (Col-0), and
hsfB1/B2b plants were drop-inoculated with spores of A. brassicicola. The disease index was calculated 3, 5, 7, 10, and 12 d after inoculation (DAI). Data points show means and S.D. (n = 3–5).
(D) The disease index calculated on day 10 post inoculation of WT
(Col-0), double knockout hsfB1/B2b, and single knockout mutants
hsfB1, hsfB2b plants. Columns represent means and S.D. (n = 3–5).
Significant differences are indicated by asterisks (a = 5).
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which double knockout mutations in hsfA1a/hsfA1b caused
a strong attenuation of the induction of a number of target
genes including Hsp17.6, Hsp70, Hsp83.1, and Hsp101 (Lohmann
et al., 2004). In our present study, the class B-Hsf mutations
hsfB1/B2B had no effect on the expression of these HsfA1a/
HsfA1b target genes or any other known heat shock genes in
Arabidopsis. Hence, HsfB1 and HsfB2b are not directly involved
in the regulation of the onset of the heat shock response. This is
reminiscent to tomato, in which, when the expression of
LbHsfB1 was compromised in a transgenic approach, plants
showed no obvious effect on the heat shock response (Mishra
et al., 2002).
Plant Defensin Genes Are De-Repressed in the hsfB1/B2b
Mutant Plants
In order to understand the biological roles of HsfB1/B2b, we
performed microarray expression analysis to identify possible
target genes and pathways affected in hsfB1/B2b mutant
plants. The total numbers of differentially expressed genes
(control versus heat stress) in WT (3333 = 15%) and hsfB1/
B2b plants (2875 = 13%) are relatively high but only 44 genes
were identified as targets of the HsfB1/B2b-dependent transcriptional regulation. Comparable expression profiling results
have been previously obtained by Busch et al. (2005) in a different genetic background of Arabidopsis (ecotype Wassilewskija), where a total of 112 class A-Hsf target genes has been
identified.
Out of the 44 putative HsfB1/B2B-dependent target genes, 15
were differentially regulated (12 up and three down-regulated)
at control temperature, 29 (15 up- and 14 down-regulated)
upon heat stress in mutant plants compared to WT. The major
effect was the enhanced expression (16 to 25-fold) of Pdf1.2a
and Pdf1.2b, both at heat stress and control temperature conditions. Pdf1.2a and Pdf1.2b encode small polypeptides that
protect plants against pathogen attack (Penninckx et al.,
1996, 1998; Broekaert et al., 1995; Thomma et al., 2002).
The almost selective up-regulation of Pdf1.2a and Pdf1.2b
does not exclude the importance of other differentially
expressed genes with a fold change of two to three. Besides
Pdf1.2a and Pdf1.2b, three other genes de-repressed in
hsfB1/B2b plants are involved in plant defense: (1) Stp3, a green
leaf-specific, low-affinity monosaccharide transporter (Buttner
et al., 2000), (2) a kelch repeat-containing F-box family protein,
which has been shown to be induced after pathogen attack
(www.genevestigator.ethz.ch/), and (3) a TIR class disease resistance gene, which is highly induced by pathogens (www.genevestigator.ethz.ch/). These genes are down-regulated after
heat stress in WT but heat stress has only little effect on the
repression of these genes in hsfB1/B2b plants. Another gene,
encoding a 2-isopropylmalate synthase-like protein (also
known as methylthioalkymalate synthase-like protein), which
might be involved in resistance to insect herbivores (Kroymann
et al., 2003), shows an enhanced mRNA level (approximately
two-fold) in hsfB1/B2b plants at control temperature.
Interestingly, among the other genes up-regulated in hsfB1/
B2b plants is the class-A transcription factor AtHsfA2 (approximately three-fold up-regulated basal level), which is the strongest heat-inducible class A-Hsf of Arabidopsis (Busch et al.,
2005). It is proposed that AtHsfA2 forms a heteromeric complex with AtHsfA1a and AtHsfA1b to regulate the expression
of target genes during early and late stages of the heat shock
response (Schramm et al., 2006). AtHsfA2 appears to act dominantly as a late, and AtHsfA1a and AtHsfA1b as early Hsfs that
share a number of common target genes including Apx2,
Hsp26.5, Hsp25.3, Hsp22.0, Hsp18.1, Hsp70, Hsp101, and DNAJ
(Wunderlich et al., 2007).
HsfB2b Is a Negative Regulator of Defensin Gene
Expression and Pathogen Resistance
There is ample evidence for the involvement of jasmonic acid
in the regulation of Pdf1.2 expression and the protective role
of defensins in plant pathogen resistance (Brown et al., 2003;
Nandi et al., 2005; Thomma et al., 1998). Jasmonic acid application induces the expression of Pdf1.2 genes. The Arabidopsis
triple mutant, fad3-2 fad7-2 fad8, which is unable to accumulate jasmonic acid, is susceptible to infection by Pythium spp.
(Vijayan et al., 1998). Moreover, the mutants in jasmonate signaling pathway such as coi1 and jar1 showed enhanced susceptibility to fungal pathogens, such as A. brassicicola, Botrytis
cinerea, etc. (Penninckx et al., 1996; Thomma et al., 1998)
and suppressed Pdf1.2 expression (Penninckx et al., 1998).
MeJ treatment led to further enhanced Pdf1.2 mRNA levels
in hsfB1, hsfB2b, and in double knockout plants by a factor of
approximately 100. There is a clear tendency that the mutant
lines accumulate much higher mRNA levels compared to WT.
This indicates that, in Hsf-mutant plants, the de-repression is
not only restricted to the low basal levels of Pdf1.2a expression.
The effects of MeJ on Pdf1.2a expression are largely reproduced in experiments using biotic stress infection with
A. brassicicola for induction. Pdf1.2a and Pdf1.2b are induced
to very high levels when plants are infected with A. brassicicola
compared to other pathogens (genevestigator data).
The results of our patho-assays are in good agreement with
the hypothesis that defensin expression correlates with pathogen resistance. The single mutant hsfB2b and the double mutant hsfB1/B2b showed significantly improved resistance levels
(lower disease index) compared to WT and, surprisingly, to
hsfB1 plants. The discrepancy between high levels of Pdf1.2
mRNA and only WT-level of disease resistance suggests that
other molecular processes of the pathogen response are negatively affected by HsfB2b and thus result in improved pathogen resistance in plants unable to express HsfB2b. HsfB2b acts
as a negative regulator of Pdf1.2 expression and pathogen resistance. The effect of hsfB2b is reminiscent to another recessive mutation, the overexpressor of cationic peroxidase 3
(Ocp3), which confers a constitutive expression of Pdf1.2 that
results in an enhanced resistance of ocp3 plants to B. cinerea
and P. cucumerina (Coego et al., 2005). The protective role of
Kumar et al.
defensin gene expression against fungal infections in plants
has been further demonstrated for transgenic rice (Kanzaki
et al., 2002) or potato (Khan et al., 2006) plants overexpressing
the wasabi defensin gene.
HsfB1 and HsfB2b seem to exert similar effects on Pdf expression, suggesting that they cooperate in repressing the
basal and pathogen-induced levels. The involvement of HsfB1
in Pdf1.2 expression and pathogen response is strongly supported by expression profiling data (www.genevestigator.
ethz.ch/at/). Many pathogens (P. syringae, P. infestans, B. cinerea, A. brassicicola) and a number of signaling mutants like
Cpr5 (Bowling et al., 1997; Clarke et al., 2001), and nahG (Delaney
et al., 1994) affect the expression of both Pdf genes.
Functional Roles of HsfB1 and HsfB2b
The regulation of Pdf1.2 expression through jasmonate signaling seems to require a region located at position –255 to
–277 bp upstream of the Pdf1.2a transcription start site. This region includes the GCC-box, which is a common motif in the promoters of various genes encoding defense-related proteins, and
has been identified as the specific binding site for members of
the ERF subfamily of AP2/ERF transcription factors (Buttner and
Singh, 1997; Ohme-Takagi and Shinshi, 1995; Zhou et al., 1997).
It is possible that general transcription factors have general
binding capability to different cis-regulatory elements and
may activate gene expression through interactions with ERFs.
Co-regulation of gene expression by the GCC-box and other
promoter elements has been observed. For example, the
GCC-box is required but not sufficient for high-level induction
of the tobacco osmotin gene by ethylene (Raghothama et al.,
1997). A G-box is also linked to jasmonate responsiveness in defense-associated genes in various plant species (Kim et al., 1992;
Mason et al., 1993).
Perfect HSEs (three-box sequences), the regulatory elements
for Hsf-dependent gene expression, are present neither in this
region of the Pdf1.2a promoter nor in the introns or the other
non-translated regions of the gene. An imperfect two-box variant HSE is unable to bind Hsf, as indicated by our negative
results in detecting any changes in protein binding to promoter segments (not shown). This suggests that the negative
effect of HsfB1/B2b on Pdf expression in WT must be indirect,
possibly through interaction with other proteins in the chromatin or in the cell. According to the Arabidopsis Small RNA
Project Database (http://asrp.cgrb.oregonstate.edu/; Gustafson
et al., 2005), there are no microRNAs in Arabidopsis thaliana
that target the transcripts of the Pdf1.2.
Using perfect synthetic HSE sequences as a probe, we were
able to detect a positive effect on the formation of the late
Hsf–HSE complex, which appears earlier, persists longer, and
is more intense in hsfB1/B2b plants compared to WT. The formation of this complex requires HsfA2, the major heat stressand high light-induced class A-Hsf in Arabidopsis (Wunderlich
et al., 2007). A higher binding capacity of the HsfA2-dependent
late complex in hsfB1/B2b plants suggests that HsfB1/B2b may
interact with class A-Hsf in regulating the shut-off of the heat
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Hsf-Regulated Pdf-Expression–Pathogen Resistance
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161
shock response. The absence of other HsfB1/B2b in knockout
plants was not associated with the loss of HSE-binding capacity. This further supports the hypothesis that these Hsfs are not
directly involved in binding to the HSE in vivo. It is conceivable
that these class B-Hsf form heteromeric complexes with class
A-Hsf with an increasing probability during the heat shock response. There is evidence that the tomato LpHsfB1 (ortholog
of AtHsfB1) can interact with the class A-Hsf LpHsfA1a but also
with other general factors like HAC1/CBP and thus specifying
a co-activator function (Bharti et al., 2004). In a similar way, the
Arabidopsis HsfB1 may interact with other Hsfs (possibly with
HsfA2) and/or other general transcription factors or chromatin
components for exerting a negative (repressive) role on target
gene expression. Further studies identifying target genes during later stages of the heat shock response or during recovery
and interaction partners of class B-Hsf will shed more light on
the functions and molecular mechanism of Hsf-dependent repression of genes.
Biological Role of Limiting Pdf Expression
The identification of Pdf genes as targets of Hsf-dependent
negative regulation is the first evidence for an interconnection
of Hsf in the regulation of biotic and abiotic responses. The
question arises as to why plant defensin expression is downregulated by heat-inducible Hsfs. Regarding plant defense,
a simple explanation may be that the pathogens are not capable of efficient infection at high temperature and thus plants
do not require the expression of defensins for protection. This
is consistent with our findings that A. brassicicola does not
grow under heat stress temperature conditions, that a heat
stress early after infection causes a delay in the development
of disease symptoms, and that jasmonate induction of Pdf1.2
expression is blocked by heat treatment (not shown). Moreover, higher levels of Pdf1.2 expression may be detrimental
to plant growth and development, which might become effective only on a long-term evolutionary scale. In eukaryotic cells,
it has been demonstrated that beta-defensins have little effect
on the epithelial cells at any concentration. In contrast, alphadefensin promotes proliferation of the epithelial cells at low
concentration but has a cytotoxic effect at high concentration
and may have adverse effects on the host (Nishimura et al.,
2004). In a similar way, Arabidopsis plants carrying the iop1
mutation show much higher induced expression levels of
Pdf1.2, but, during their entire lifespan, the iop1 mutants
stayed significantly smaller (Penninckx et al., 2003).
METHODS
Plant Material, Growth Condition, and Heat Treatment
Arabidopsis thaliana (Columbia-0) was used in experiments.
Col-0 T-DNA insertion lines were obtained from the Signal Salk
institute (http://signal.salk.edu/tdnaprimers.2.html).
Seeds were spread on soil or MS media and kept in the dark
at 4–8C for 2 d to achieve a uniform germination. Ten DAG
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Hsf-Regulated Pdf-Expression–Pathogen Resistance
(days after germination), seedlings were singled out in individual pots and grown in soil with a light/dark cycle of 16/8 h
at 22C, 60% relative humidity and a light intensity of 5000
lumen m 2. All experiments were carried out with 5-weekold plants unless specified.
For heat treatment (HS), fully expanded leaves were collected and pooled, incubated at 37C (for 1, 2, 4, or 8 h,
depending on the experiment performed) in pre-warmed
SIB (1 mM potassium phosphate, pH 6, 1% (W/V) sucrose),
then placed in a shaking water bath with 40 oscillations per
minute. As a control (C), leaf pools were incubated under
the same experimental conditions but at room temperature
(22C). After heat treatment, excess SIB was rinsed off with
sterile water; leaf tissue was blotted dry with filter paper
and immediately frozen in liquid nitrogen. Samples were
stored at –70C for future use.
RNA Isolation, Labeling, and Microarray Hybridization
Frozen leaf tissue (–80), which had been collected from 45 individual plants per biological replicate, was crushed by using
mortar and pistil and RNA was extracted with the Plant RNeasy
Mini Kit (Qiagen, Hilden, Germany). A total of 5 lg RNA was
used as starting material for double-stranded cDNA synthesis
using the Superscript Choice System (Invitrogen, Karlsruhe,
Germany) and an oligo dT-T7 primer (Genset, Paris, France).
Biotinylated cRNA was synthesized from cDNA template using
BioArray High Yield Transcript Labeling Kit (Enzo, Farmingdale, NY, USA). RNeasy columns (Qiagen) were employed to
clean the biotinylated cRNA. The fragmentation of cRNA
was performed according to GeneChip protocol (Affymetrix,
Santa Clara, CA, USA).
The fragmented cRNAs were hybridized to Arabidopsis ATH
1-12151 (Affymetrix Gene-Chips), which represents 22 810
probe sets. GeneChip arrays were hybridized according to
the manufacturer’s protocol (Affymetrix). Hybridized GeneChips were scanned with the GeneChip Scanner 3000 (Affymetrix). Samples were quality-controlled by examining of 3’ to 5’
intensity ratios of control genes.
Isolation of mRNA and Preparation of cDNA for
Quantitative Real-Time PCR
This method has been used for the verification of knockout
mutants and the expression profiles of individual Hsf target
genes. Poly (A)+-RNA was isolated from 60–80 mg frozen leaf
material using the chemagic mRNA Direct kit (Chemagen) and
the amounts were quantified using the dye RiboGreen. cDNA
was synthesized from 100 ng of mRNA using the iScript cDNA
synthesis kit (Biorad). The amount of cDNA was quantified using PicoGreen. Quantitative RT–PCR was performed in triplicates using undiluted (1 ng) and 1/8 and 1/64 diluted cDNA
as template. Real-time PCR was performed in a 50-ll reaction
volume. The primers shown in Table 4 were used.
Isolation of mRNA and Preparation of cDNA for
Quantitative Real-Time PCR
This method was used for determining the Pdf1.2-mRNA expression after MeJ treatment or Alternaria spray assay. Poly
(A)+-RNA was isolated from 50–70 mg frozen leaf material using the chemagic mRNA Direct kit (Chemagen). A total volume
of 15 ll was used for cDNA synthesis using the iScriptcDNA
kit (Biorad). Quantitative RT–PCR was performed with three
dilutions (1, 1/8, and 1/64) of cDNA and in duplicate samples.
As a control, undiluted mRNA was used. The reaction volume
Table 4. Primers Used for Real-Time PCR.
Target gene
Primer sequence
Actin2
5#-AAGCTGGGGTTTTATGAATGG-3’
(At3g18780)
5#-TTGTCACACACAAGTGCATCAT-3’
Hsp17.6
5#-GGTGAGTGGCAAAAGACAGA-3’
(At1g59860)
5#-AAACTTCCCCATCCTCCTCT-3’
Hsp23.6
5#-CGATGAGATTAAGGCGGAGA-3’
(At4g25200)
5#-TCGACGTTTTTAGTTGATCTCG-3’
Hsp70
5#-AGGAGCTCGAGTCTCTTTGC-3’
Analysis of Expression Data
(At2g32120)
5#-AGGTGTGTCGTCATCCATTC-3’
Data were imported in Genespring 7.2 (Silicon Genetics, Redwood City, CA, USA) and retransformed in linear values. Data
for every condition were available in two replicates of each
experiment. Raw data were quantile-normalized and expression estimates were calculated by gcRMA (Wu et al., 2004)
implemented in R. Statistical analysis of data was carried
out on gene sets after eliminating all genes that showed less
then two-fold change between conditions, by Welch’s t-test to
compare the different conditions and to find differentially
expressed genes (selected P-value 0.05); the Benjamini and
Hochberg False Discovery Rate Multiple Testing Correction
was used to reduce the detection of false positives. The data
are deposited in a MIAME compliant fashion in the ArrayExpress database (www.ebi.ac.uk/microarray/) under accession
E-MEXP-1725.
Hsp83.1
5#-GCTGCTAGGATTCACAGGATG-3’
(At5g52640)
5#-TCCTCCATCTTGCTCTCTTCA-3’
Hsp101
5#-TAACGGGCCAAAGAGAAGTG-3’
(At1g74310)
5#-CACACGTTGGAGGTCAAGACT-3’
AtHsfB1
5#-GGACCGGGATGAAAAGAATTA-3’
(At36990)
5#-CACGCTGGTTTGAACAGTCTT-3’
AtHsfB2b
5#-TGGAGGAGAATAACTCCGGTAA-3’
(At11660)
5#-ATGCAATGGGGATTCAGTAACA-3’
GolS1
5#-AGCTTAGCCACAATATAATCATCG-3’
(At1g56600)
5#-ATCCTCCAAAACCCATAAAAATTA-3’
Pdf1.2a
5#-CCAAGTGGGACATGGTCAG-3’
(At5g44420)
5#-ACTTGTGTGCTGGGAAGACA-3’
Pdf1.2b
5#-GGTACTTGGTCAGGAGTTTGC-3’
(At2g26020)
5#-ACTTGTGAGCTGGGAAGACA-3’
Kumar et al.
was 30 ll. mRNA expression level of Pdf1.2a was analyzed with
respect to Actin2. The following primers were used:
Actin2-F3 (5#-AAGCTGGGGTTTTATGAATGG-3’),
Actin2-R3 (5#-TTGTCACACACAAGTGCATCAT-3’),
Pdf1.2a_for (5#-TGCTTTCGACGCACCGGC-3’),
Pdf1.2a_rev (5#-TGTAAAATACACACGATTTAGCACC-3’)
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Combined heat stress and MeJ treatment was carried out as
follows: plants were first sprayed with MeJ and then kept in
a growth chamber (set at 37C, light/dark cycle of 16/8 h, relative humidity 60%, light intensity 5000 lumen m 2). As a control, water-sprayed plants were kept in a growth chamber at
22C (light/dark cycle of 16/8 h, relative humidity 60%, light
intensity 5000 lumen m 2).
Pathogen Treatment: Spray Assay
Pathogen Treatment: Spot Assay
Plants were grown in soil with a light/dark cycle of 8/16 h at
22C, 100% relative humidity and a light intensity of 5000 lumen m 2. Pathogen (Alternaria brassicicola) treatment was carried out by apoplectic puncturing 6-week-old plants. Care was
taken to choose leaves with approximately equal size and having the same developmental stage in WT as well as in mutants.
Two leaves per plant were chosen for fungal inoculation.
Plants were inoculated at a concentration of 5 3 105 conidial
spores per milliliter. Each leaf was inoculated with two 5-lL
drops of a suspension in water. Inoculated plants were incubated at 100% RH and 22C for control conditions and 35C
for heat stress treatment, respectively. The spread of fungal
colonization on each leaf was analyzed 3, 5, 7, 10, and
12 DAI. As a control, one plant per line was treated with water.
Detection of Pathogen Colonization on Inoculated Plants
and Disease Index
Pathogen-inoculated leaves were bonitated according to the
disease symptoms shown in Table 5.
The disease index (DI) for each DAI was calculated from the
results of bonitation by the following formula:
X
DI =
Bonitation scores=Number of inoculated leaves 100:
Methyl Jasmonate Treatment
Five-week-old plants were sprayed with methyl jasmonate
(MeJ) solution (100 lM). Mock inoculations were only sprayed
with water.
Table 5. Pathogen Inoculated Leaves Bonitated According to
Disease Symptoms.
Plants were grown in soil with a light/dark cycle of 8/16 h at
22C, 100% relative humidity and light intensity 5000 lumen
m 2. For pathogen treatment (Alternaria brassicicola), 6week-old plants were sprayed with a concentration of
5 3 105 conidial spores per milliliter. Leaf material was collected immediately after spray infection and after 72 and
96 h post inoculation, respectively.
Isolation of Single and Double Hsf Mutants
Individual T-DNA insertion lines for AtHsfB1 and AtHsfB2b
were obtained from the Salk Institute. Suitable primers for
testing the T-DNA insertion (as described by the Salk website:
http://signal.salk.edu/tdnaprimers.2.html) were generated
and used in PCR reactions with genomic DNA as the template.
Using 5’ and 3’ gene-specific primers, WT plants were identified and eliminated from segregating population. Mutants
were identified by specific PCR fragments obtained using
a T-DNA and a gene-specific primer.
To generate a double Hsf mutant, 8–9-week-old single homozygous mutant plants were selected for pollination. For $
(hsfB2b mutant plants), closed flower buds were selected. Flowers were opened with a pin set and anthers were removed.
Flowers were then left opened for 2 d before pollination with
pollen from hsfB1. After 2 d, healthy and non-pollinated stigmas were chosen for fertilization. For # (hsfB1 mutant plants),
fully mature anthers with ripened pollens were chosen.
The resulting F1 heterozygous population was selfed to get
homozygous hsfB1/B2b double mutant plants. WT plants were
identified using gene-specific primers for hsfB1 and hsfB2b.
Accordingly, the double mutation was identified with TDNA left border primer and the gene-specific primer (Table 6).
Electrophoretic Mobility Shift Assay (EMSA)
The Hsf binding to DNA has been tested essentially as described by Lohmann et al. (2004), and the same synthetic
HSE sequences (5#-CCAGAAGCTTCCAGAAGCC) were used as
a probe. Unspecific binding complexes are discriminated
Score
Symptoms
1
No symptom
2
Little brownish at point of inoculation
3
Dark-brown spot at point of inoculation
4
Necrotic spots spreading around
the point of inoculation
5
Maceration
6
Sporulation
AtHsfB2b (At11660)
7
Leaf detachment
T-DNA (Lba1)
Table 6. Double Mutations Identified with T-DNA Left Border
Primer and the Gene-Specific Primers.
AtHsfB1 (At36990)
5#-AAAAGTTCGCCGGAGATGACG-3’
5#-GTCGCAACCTTCGCACTCACT-3’
5#-CACAGAGGTCAATTCCGACGC-3’
5#-CTTCTTCCTCTGCAGCACCCA-3’
5#-ATGGTTCACGTAGTGGGCCATC-3’
164
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Kumar et al.
d
Hsf-Regulated Pdf-Expression–Pathogen Resistance
according to Lohmann et al. (2004) by the ability to be also
formed with mutated HSE, while specific binding complexes
are formed only with perfect consensus HSE sequences but
not with mutated HSE.
FUNDING
The research was funded by grants of the Deutsche Forschungsgemeinschaft (SFB446, project A2).
ACKNOWLEDGMENTS
We thank Dr Jasmin Doll and Markus Wunderlich (ZMBP-Allgemeine
Genetik, Universität Tübingen) for advice and fruitful discussions. No
conflict of interest declared.
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