Download Repression of the Defense Gene PR-10a by the Single

The Plant Cell, Vol. 13, 2525–2537, November 2001, www.plantcell.org © 2001 American Society of Plant Biologists
Repression of the Defense Gene PR-10a by the
Single-Stranded DNA Binding Protein SEBF
Brian Boyle and Normand Brisson1
Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada H3C 3J7
The potato pathogenesis-related gene PR-10a is transcriptionally activated in response to pathogen infection or elicitor treatment. Characterization of the cis-acting elements of the PR-10a promoter revealed the presence of a silencing
element between residues 52 and 27 that contributes to transcriptional regulation. In this study, we have isolated a
silencing element binding factor (SEBF) from potato tuber nuclei that binds to the coding strand of the silencing element in a sequence-specific manner. The consensus binding site of SEBF, PyTGTCNC, is present in a number of PR
genes and shows striking similarity to the auxin response element. Mutational analysis of the PR-10a promoter revealed an inverse correlation between the in vitro binding of SEBF and the expression of PR-10a. SEBF was purified to
homogeneity from potato tubers, and sequencing of the N terminus of the protein led to the isolation of a cDNA clone.
Sequence analysis revealed that SEBF is homologous with chloroplast RNA binding proteins that possess consensus
sequence–type RNA binding domains characteristic of heterogenous nuclear ribonucleoproteins (hnRNPs ). Overexpression of SEBF in protoplasts repressed the activity of a PR-10a reporter construct in a silencing element–dependent
manner, confirming the role of SEBF as a transcriptional repressor.
INTRODUCTION
A variety of defense-specific events are induced in plants in
response to pathogen infection, including the production of
reactive oxygen species, activation of G-proteins, reinforcement of the cell wall, and induction of signal transduction
cascades leading to the transcriptional activation of defense
genes (Dixon et al., 1994; Blumwald et al., 1998). Although
key components of the signaling cascades are being discovered, few transcription factors have been identified that
integrate these signals at the transcriptional level.
The well-characterized PR genes induced by pathogen invasion provide excellent models to study the transcriptional
regulation of defense genes. PR genes are subdivided into
11 classes based on sequence homology (Van Loon et al.,
1994). The PR-10 gene family is subdivided into two groups
and encodes small, acidic intracellular proteins of 15 to 18
kD (Osmark et al., 1998). Although RNase activity has been
demonstrated for PR-10 proteins, they do not possess any
sequence similarity to classic RNases (Moiseyev et al.,
1994; Swoboda et al., 1996). In potato, three members of
this family have been identified, of which PR-10a is the best
characterized.
Expression studies have identified cis-acting elements involved in PR-10a gene regulation. An elicitor response ele-
1 To
whom correspondence should be addressed. E-mail normand.
[email protected]; fax 514-343-2210.
Article, publication date, and citation information can be found at
www.aspb.org/cgi/doi/10.1105/tpc.010231.
ment (ERE) located between nucleotides 135 and 105 is
essential and sufficient for elicitor-induced gene expression
(Matton et al., 1993; Després et al., 1995; Desveaux et al.,
2000). PBF-2, a single-stranded DNA binding factor, appears to play a role in the activation of PR-10a from the ERE
(Després et al., 1995; Desveaux et al., 2000). Although the
presence of the ERE is sufficient for PR-10a activation, removal of the silencing element (SE), which is located between 52 and 27, leads to further activation, suggesting
that this element participates with the ERE in the regulation
of PR-10a (Matton et al., 1993; Després et al., 1995). Therefore, a thorough understanding of PR-10a regulation requires that we understand the contribution of the SE and its
trans-acting factors.
In this article, we demonstrate, using transient expression
studies, that only nine nucleotides containing the sequence
GACTGTCAC are required for full SE activity. We have identified a nuclear factor (silencing element binding factor
[SEBF]) that shows sequence-specific binding to the sequence PyTGTCNC. Database searches revealed the presence of this sequence in the promoter of numerous PR
genes. SEBF was purified to homogeneity from potato tubers, and sequencing of the N terminus of the protein led to
the isolation of a cDNA encoding SEBF. The deduced amino
acid sequence shows a high degree of sequence similarity
to chloroplast RNA binding proteins. Subcellular partitioning
of leaf cells demonstrated that SEBF is located in both chloroplasts and nuclei, suggesting functions in both cellular
compartments. Finally, overexpression of recombinant SEBF
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in a transient expression system resulted in SE-dependent
transcriptional repression of a PR-10a–uidA reporter fusion,
confirming the role of SEBF as a transcriptional repressor.
RESULTS
Identification of SEBF
Previous studies identified a negative regulatory sequence
(SE) in the promoter of the potato PR-10a gene (Matton et
al., 1993; Després et al., 1995). Therefore, we sought to
identify a nuclear protein able to regulate PR-10a expression through binding to this sequence. Because PR-10a appears to be regulated positively at the ERE by a singlestranded DNA (ssDNA) binding protein (Desveaux et al.,
2000), we searched for proteins that could bind either single- or double-stranded forms of the SE. A crude nuclear
extract of potato tubers was incubated with single- or double-stranded radiolabeled SE and analyzed by electrophoretic mobility shift assay (EMSA). Figure 1 demonstrates
that a nuclear factor (SEBF) bound the coding strand of the
SE (lane 1). SEBF was not able to bind the noncoding strand
(lane 3) or double-stranded DNA (lanes 4 to 6). These results
show that SEBF is a ssDNA binding protein that recognizes
only one strand of the SE.
Binding of SEBF to the SE Correlates with the
Transcriptional Repression of PR-10a
Mutational analysis of the SE was performed to determine
which nucleotides constitute the SEBF binding site and
whether these nucleotides are important in vivo for the regulation of PR-10a expression. Mutated forms of the SE coding strand were synthesized (Figure 2A) and used as probes
in EMSA studies (Figure 2B). The same mutations also were
introduced into the PR-10a promoter fused to the uidA reporter gene encoding -glucuronidase, and the resulting
transcriptional activity was measured in a transient expression system (Figure 2D). As shown in Figure 2B, binding of
SEBF to mutants m3 and m4 was reduced significantly
compared with that of the wild type. In contrast, introduction of these mutations in the promoter of PR-10a led to an
increased activity of the reporter gene in transient studies
(Figure 2D, m3 and m4). Mutations that showed either a
slight increase (Figure 2B, m2) or decrease (Figure 2B, m5)
in binding did not show altered transcriptional activities (Figure 2D, m2 and m5). Binding of SEBF was abolished completely using mutant m1, in which 80% of the sequence has
been changed (Figure 2B, m1). This absence of binding correlated with the highest level of transcriptional activity observed in transient studies (Figure 2D, m1). Therefore, the
transcriptional activity of the PR-10a promoter is inversely
correlated with the in vitro binding of SEBF at the SE. This
finding suggests that SEBF is the trans-acting factor responsible for SE-mediated repression of PR-10a.
Sequence PyTGTCNC Defines the DNA Binding Site
of SEBF
Figure 1. SEBF Binds Single-Stranded SE.
EMSA was performed with 10 g of crude nuclear preparation and
20,000 cpm of 32P-labeled SE coding strand (CS; lane 1) or noncoding strand (NCS; lane 3). The double-stranded probe (DS) was made
by annealing radiolabeled noncoding and nonlabeled (cold) coding
strands. The ratio of CS to NCS is 0.75:1, 1.5:1, and 3:1 for lanes 4,
5, and 6, respectively. No extract was added in lane 2. Arrows indicate the positions of the CS, NCS, and DS probes and of the SEBF
shift in the gel.
The results presented in Figure 2 indicate that the SEBF
binding site is located within the sequence GACTGTCAC.
To further delineate this element, additional mutations were
introduced into this region, and their effect on SEBF binding
was monitored by EMSA. As indicated in Figure 3, mutations affecting the sequence CTGTCAC reduced the binding
of SEBF dramatically (mutants m8, m9, m11, m13, m14,
m15, m16, m17, and m19), whereas mutations outside of
this region did not reduce binding (mutants m6, m7, m12,
and m20). Mutations of the only A in this sequence did not
affect binding (mutants m10 and m18). In fact, any nucleotide could replace the A without affecting SEBF binding
(data not shown). Substitution of the first C by an A (m13) or
a G in the sequence CTGTCAC led to a strong reduction in
SEBF binding, whereas substitution by T led to a small increase in binding (data not shown). Therefore, the consensus SEBF binding site is PyTGTCNC.
Increased binding of SEBF was observed with mutants
m6 and m10 (Figure 3). Transcription factors are known to
make secondary hydrogen bonds with either the phosphate
backbone or nucleotides outside of their binding sites, and
Single-Stranded DNA Binding Repressor
2527
changes of nucleotides involved in these secondary contacts often modify binding affinity (for review, see Pabo and
Sauer, 1992). Therefore, mutants m6 and m10 may favor
secondary contacts, leading to an increase in SEBF binding.
Purification of SEBF
Further characterization of SEBF required the cloning of its
gene. Toward this goal, SEBF was purified from tuber nuclei
using a combination of anion exchange chromatography
and affinity purification. Table 1 shows that a 20,700-fold
purification of SEBF was achieved. Analysis of the chromatographic fractions by SDS-PAGE and Coomassie blue staining revealed two distinct bands of 29 and 28 kD in the most
purified fraction (Figure 4, lane 4). To determine which of
these two bands possessed DNA binding activity, proteins
from the second affinity purification were transferred to nitrocellulose and probed with the wild-type SE oligonucleotide.
The results indicate that the two purified proteins can interact
independently with the SE (Figure 4, lane 5). Both of these
proteins were subjected to N-terminal sequence analysis.
Cloning of SEBF
Figure 2. Mutational Analysis of the SE.
(A) Scheme of the reporter constructs and mutant oligonucleotides
used in this study. Reporter constructs contain the PR-10a promoter
region from 135 to 136 fused to the bacterial gene uidA encoding -glucuronidase. The positions of the ERE, the SE, and the TATA
box are shown. The transcriptional start site is indicated with an arrow. The common sequence between the wild-type (WT) and mutant
oligonucleotides is indicated by dashed lines, and mutated nucleotides are represented by lowercase letters.
(B) EMSA studies using 10 g of crude potato tuber nuclear preparation and 20,000 cpm of the 32P-labeled single-stranded CS oligonucleotides presented in (A).
(C) EMSA studies using 10 ng of purified recombinant mature SEBF
and 20,000 cpm of the 32P-labeled single-stranded CS oligonucleotides presented in (A).
(D) The sequence from 52 to 27 of the PR-10a promoter fused to
the uidA gene was replaced with the sequences presented in (A).
The resulting plasmids were electroporated in potato leaf protoplasts, and the -glucuronidase (GUS) activity was measured. The
histogram represents fold activity to wild type (WT 1). Transfection
efficiencies were corrected by coelectroporating a luciferase re-
A 50–amino acid sequence was obtained from N-terminal
sequencing of the 29-kD protein. N-terminal sequencing of
the 28-kD protein showed more than one amino acid at
each cycle. However, a clear sequence similarity with that of
the 29-kD protein could be observed, suggesting that the
28-kD band contained degraded forms of the 29-kD protein.
Using the amino acid sequence obtained from the 29-kD
protein, a polymerase chain reaction (PCR) strategy was
elaborated to clone a cDNA (see Methods). The amino acid
sequence derived from the cDNA clone is presented in Figure 5. The 50 residues determined by N-terminal sequencing (amino acids 60 to 109, indicated by stars) are preceded
by a 59–amino acid sequence, suggesting that SEBF is synthesized as a precursor (pre-SEBF). The TargetP program,
which predicts the cellular location of a protein (Emanuelson
et al., 2000), predicted that SEBF would be located at the
chloroplast with reliability class 1 (Table 2). Ninety-nine
percent of the proteins having reliability class 1 were predicted correctly when using TargetP on a nonredundant
subset of plant proteins (Emanuelson et al., 2000). For
comparison, the predicted locations of the ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco) small subunit and the chlorophyll a/b binding protein are given. TargetP also predicted that the putative transit sequence in
porter gene. Results represent the mean from a minimum of six individual electroporations. Error bars indicate SEM.
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glycine-rich region. Database searches indicated a high degree of sequence similarity to a family of nucleus-encoded
chloroplast RNA binding proteins (Figure 5). The greatest
similarity is to tobacco cp29A (86% sequence identity), a
member of the group I chloroplast RNA binding proteins
(Ohta et al., 1995).
To confirm that the cDNA-encoded protein corresponds
functionally to SEBF, the mature protein was expressed as a
glutathione S-transferase fusion protein in Escherichia coli,
purified, and tested for binding to the SE. Figure 2C shows
that the recombinant mature SEBF binds to SE oligonucleotides with binding specificity similar to native SEBF (Figure 2B).
Cellular Distribution of SEBF
Figure 3. Determination of the Consensus SEBF Binding Site.
(A) Two-by-two mutational analysis of the SEBF binding site.
(B) Single-nucleotide analysis of the SEBF binding site.
Oligonucleotides used for fine mapping of the SEBF binding site are
listed in each panel. The common sequence between wild-type (WT)
and mutant oligonucleotides is indicated by dashed lines, and mutated nucleotides are represented by lowercase letters. EMSA results show the binding of SEBF to oligonucleotides containing the
mutated nucleotides. Studies were performed using 10 g of crude
nuclear preparation and 20,000 cpm of the 32P-labeled singlestranded oligonucleotides listed in each panel.
SEBF would be cleaved after residue 58, suggesting that
the 59 residues encoded in the cDNA but absent from the
purified protein could act as a chloroplastic transit peptide.
Similar results were obtained using the Predotar (http://
www.inra.fr/Internet/Produits/Predotar/) and iPSORT (http://
www.hypothesiscreator.net/iPSORT/) subcellular location
prediction programs (data not shown).
An acidic region present in the N-terminal region of the
mature protein precedes two consensus sequence–type
RNA binding domains (cs-RBDs; identified as RI and RII in
Figure 5). These domains, also known as RNA recognition
motifs, ribonucleoprotein consensus sequences (RNP-cs), and
RNP motifs (Burd and Dreyfuss, 1994), are separated by a
The purification of SEBF from isolated nuclei (Table 1) indicates that SEBF is a nuclear protein. However, the presence
of a putative transit sequence in recombinant pre-SEBF
suggests that the protein could be present in plastids as
well. This led us to investigate the subcellular localization of
SEBF. Antibodies were raised against recombinant mature
SEBF, and subcellular fractions were obtained from potato
leaves. The integrity of each fraction was verified immunologically and by enzymatic assays. Figure 6 shows that as
expected, alcohol dehydrogenase activity was restricted to
the cytosolic fraction. The nuclei were free of the chloroplastic marker chlorophyll, and little contamination was
observed in the cytosolic fraction. No detectable alkaline
pyrophosphatase or nitrite reductase activities, which are
used as plastid markers (MacDonald and ap Rees, 1983;
Smith et al., 1993), were found in the nuclear fraction. The
chloroplastic fraction was clear of nuclear contamination, as
shown by protein gel blot analysis using antibodies against
the nuclear proteins histone H1 and Cdc2 (Figure 6). Protein
gel blot analysis performed on these fractions using an antiSEBF antibody revealed that a protein immunologically related to SEBF was present in chloroplasts and nuclei (Figure
6). A single 29-kD band is present in both cellular compartments and comigrates with the mature form of recombinant
SEBF (in which the 59–amino acid putative transit peptide
has been removed) (Figure 6, SEBF). No proteins of higher
molecular mass were detected in any fraction, suggesting
that if SEBF is synthesized as a precursor, it is processed
efficiently by peptidases before nuclear import.
SEBF Is a Single-Copy Gene
To exclude the possibility that nuclear and chloroplastic
SEBF could be the products of different genes, genomic
DNA was digested with restriction enzymes and used for
DNA gel blot analysis. As shown in Figure 7A, probing the
genomic DNA with an SEBF cDNA fragment resulted in a
single EcoRI band and two HindIII bands. These were the
expected results for a single-copy gene because no EcoRI
Single-Stranded DNA Binding Repressor
2529
Table 1. Purification of SEBF from Potato Tubers
Fraction
Total Protein (mg)
Total Activitya (pg DNA)
Specific Activity (pg DNA/mg)
Purification (fold)
Yield (%)
Crude nuclear
Q-Sephb
Aff.1c
Aff.2e
112
1.15
4.0 103 d
3.5 103 d
3.3 106
3.0 106
2.3 106
2.1 106
2.9 104
2.6 106
5.8 108
6.0 108
1
90
20,000
20,700
100
91
70
64
a Total
activity was determined by measuring labeled probe bound by SEBF in EMSA and calculating total picograms of DNA bound in each fraction.
Q-Sepharose Fast-Flow anion exchange chromatography.
c Aff.1, first round of DNA affinity chromatography.
d Estimated from stained gels.
e Aff.2, second round of DNA affinity chromatography.
b Q-Seph,
sites and a single HindIII site are found within the probe. The
presence of two XbaI fragments is attributable to the presence of an XbaI site in the third intron of the gene (data not
shown). Analysis of potato genomic DNA by PCR led to the
identification of three introns in the gene coding for SEBF
(Figure 7B). These three introns are present at the same position in homologs of SEBF in Arabidopsis and tobacco (Ye
et al., 1991; Ohta et al., 1995). These results clearly indicate
that there is only one copy of the SEBF gene in the potato
genome. Because no introns are present at the 5 end of the
gene (Figure 7C), the absence of a transit peptide on the purified protein is not caused by alternative splicing. This was
confirmed by sequencing 5 rapid amplification of cDNA
ends products, all of which showed sequences identical to
that of SEBF (data not shown). These results are supported
by the conservation of the genomic organization of this gene
in tobacco and Arabidopsis. Together, these results indicate
that the nucleus-localized SEBF is encoded by this gene.
with the overexpression of FK506 binding protein, a control
protein (Figure 8, m4). Repression was not seen when preSEBF was overexpressed with mutant m1 (Figure 8, m1), to
which it does not bind, confirming that the repression observed requires a cis element bound by SEBF and is not the
result of tethering of an activator or translational blocking.
These results demonstrate that SEBF participates in SEmediated PR-10a repression and that expression of the preSEBF results in nucleus-localized activity.
SEBF Can Repress Transcription
The correlation between the binding of SEBF to mutated
forms of the SE and their activity as negative regulatory elements in protoplasts suggests that SEBF is the factor mediating SE-dependent repression. This was confirmed by
coexpressing pre-SEBF and various reporter constructs in
potato protoplasts. Although repression was observed using the wild-type promoter construct, reporter activity was
too low and the results were not statistically significant (Figure 8, WT). This result was expected, because promoter activity with the wild-type SE is already fully repressed. To
circumvent this problem, we overexpressed pre-SEBF in
conjunction with a construction carrying the partially derepressed mutant m4 (Figure 2D), which is bound with less affinity than is wild type by recombinant and purified SEBF
(Figures 2B and 2C). We expected that overexpression of
SEBF would compensate for the lower binding affinity to
this mutant. Figure 8 demonstrates that in fact, a 68% decrease of promoter activity was attained when pre-SEBF
was overexpressed, compared with the activity observed
Figure 4. Purification of SEBF.
Coomassie blue staining of proteins from each step of the purification of SEBF (lanes 1 to 4). The crude nuclear extract (Crude; lane 1)
was loaded onto a Q-Sepharose column. SEBF was eluted at 400
mM NaCl (Q-Seph; lane 2) before two rounds of affinity purification
(Aff.1 and Aff.2; lanes 3 and 4). Lane 5 shows the results of a DNA
probed protein gel blot (Dp) experiment performed with affinity 2–purified SEBF and the wild-type coding strand as a radiolabeled probe.
Arrows indicate the two purified bands. Molecular mass markers are
indicated at left.
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Figure 5. Sequence of SEBF and Alignment with a Family of Nucleus-Encoded Chloroplast RNA Binding Proteins.
The amino acid sequence derived from the SEBF cDNA is presented. A member of each group of nucleus-encoded chloroplast RNA binding
proteins was chosen for sequence comparison (cp29A from group I, GenBank accession number Q08935; cp31 from group II, GenBank accession number P19683; cp33 from group III, GenBank accession number P19684). Amino acids obtained through N-terminal sequencing of the
purified protein are indicated by stars. The beginning of the mature form of SEBF is indicated by the arrowhead above the sequence. The putative transit peptide (T), the acidic region (A), and the cs-RBDs (RI and RII) are underlined. The conserved amino acids that define the cs-RBD are
indicated by triangles below the sequence. Black boxes indicate sequence identity, and gray shading indicates conservative substitutions.
DISCUSSION
In this study, we identified a ssDNA binding factor that binds
with sequence specificity to the coding strand of the SE present
in the promoter of the potato PR-10a gene. A strong inverse
correlation was shown between the binding of SEBF in vitro
and the transcriptional activity of the PR-10a promoter. N-terminal sequencing of the purified factor allowed the cloning of a
cDNA that encodes a protein with RNA binding domains similar to those found in mammalian hnRNPs. Recombinant and
purified SEBF displayed similar binding specificity toward the
SE. Overexpression of the recombinant protein in protoplasts
confirmed its implication in the transcriptional repression of the
PR-10a gene. Subcellular partitioning followed by immunoblotting with anti-SEBF antibodies revealed the presence of a 29kD protein in chloroplasts and nuclei, suggesting that SEBF is
present in both cellular compartments.
ssDNA Binding Proteins and Regulation of Expression
by hnRNP
We demonstrated previously that another cis element of the
PR-10a promoter also interacts with a ssDNA binding pro-
Table 2. TargetP Predictions of the Subcellular Localization of SEBF
TargetP Scores
Precursor (Accession Number)
cTPa
mTPb
SPc
Other
TargetP Predicted Location
RCd
SEBF (AF38941)
Rubisco small subunit (X69759)
Chlorophyll a/b binding protein (CAA78379)
0.987
0.874
0.846
0.015
0.044
0.031
0.010
0.096
0.177
0.010
0.099
0.154
Chloroplast
Chloroplast
Chloroplast
1
2
2
a Score
for chloroplastic transit peptide.
for mitochondrial transit peptide.
c Score for signal peptide.
d Reliability class (RC) is the difference (diff.) between the highest score and the second highest score. The classes are defined as follows: RC1,
diff. 0.8; RC2, 0.8 diff. 0.6; RC3, 0.6 diff. 0.4; RC4, 0.4 diff. 0.2; RC5, 0.2 diff.
b Score
Single-Stranded DNA Binding Repressor
tein. This element, located between 135 and 105, interacts with the ssDNA binding factor PBF-2 (Després et al.,
1995; Desveaux et al., 2000), which has been shown to activate the transcription of a PR-10a–uidA reporter gene
construct in protoplasts (D. Desveaux and N. Brisson, unpublished data). Therefore, our data suggest that a large region of the PR-10a promoter is contacted by ssDNA binding
proteins. A similar situation has been reported for several
genes in animals (reviewed by Rothman-Denes et al., 1998)
and is illustrated by the promoter of the c-myc proto-oncogene, in which several classes of single-stranded cis elements occur in vivo and are bound by various specific
ssDNA binding factors (Michelotti et al., 1996). The formation of ssDNA regions is known to occur as a consequence
of transcription (Giaever and Wang, 1988), which generates
torsional stress on the DNA, resulting in an upstream negative supercoiling that forces strands to separate. This also
can affect neighboring genes, because the effects of negative supercoiling can be perceived as far as 4 kb from a
transcribed gene (Wang and Dröge, 1997). Localized negative supercoiling also has been shown to be produced by
the binding of the non-sequence-specific HMG proteins,
which generate a bend in the DNA (reviewed by Grasser,
1998). DNA supercoiling then may lead to the formation or
stabilization of noncanonical DNA structures that possess
ssDNA regions, providing a way for ssDNA binding proteins
to gain access to their target sequence (reviewed by
Rothman-Denes et al., 1998). On the other hand, the transcriptional repressor YB-1, a cold shock domain protein,
has intrinsic strand-melting activity that allows it to bind to
its high-affinity ssDNA site (MacDonald et al., 1995). Both
phenomena are mechanisms that could generate ssDNA
and allow SEBF binding to the SE.
hnRNPs are among the best-characterized ssDNA binding proteins that play a role in the regulation of gene expression in eukaryotes. For example, hnRNP D and its homologs
have been shown to activate (Tay et al., 1992; Lau et al.,
2000; Tolnay et al., 2000) or repress (Kamada and Miwa,
1992; Smidt et al., 1995; Chen et al., 1998) transcription
from a variety of promoters. Another cs-RBD–containing
protein, human hnRNP A1, has been shown to repress transcription of the thymidine kinase gene (Lau et al., 2000).
Therefore, it is not surprising that SEBF, a plant protein containing cs-RBDs, can act as a transcriptional repressor.
Other plant proteins containing two cs-RBDs, such as Arabidopsis FMV-3b, carnation CEBP-1, and tobacco ACBF,
also were able to bind specific regulatory cis elements (Didier
and Klee, 1992; Maxson and Woodson, 1996; Séguin et al.,
1997) and therefore represent potential transcriptional regulators.
The presence of RNA binding domains in SEBF, and the
multiple functions associated with this domain, suggests
that SEBF also could play a role in RNA processing. Tobacco chloroplast homologs of SEBF have been shown to
bind mRNA and intron-containing pre-tRNAs (Nakamura et
al., 1999). They also have been shown to confer stability
2531
Figure 6. Cellular Localization of SEBF.
Potato leaves were fractionated into cytoplasm, nuclei, and chloroplasts. Ten micrograms of each fraction were separated by 12%
SDS-PAGE. Proteins were transferred to nitrocellulose, and the
presence of SEBF and the nuclear proteins histone H1 and Cdc2
was revealed with specific antibodies. The first lane (10 ng of recombinant mature SEBF) shows the protein that served to immunize the
rabbits. Chlorophyll (CHL), nitrite reductase (NIT), and alkaline pyrophosphatase (PYR) were used as chloroplastic markers (data are
presented as g chlorophyll·mg1 protein, nmol nitrite·mg1 protein·min1, and mol PO4·mg1 protein·min1, respectively), and alcohol dehydrogenase (ADH) was used as a cytoplasmic marker
(data are presented as increased OD·mg1 protein·min1). N.D., not
detectable; , not determined.
and ribonuclease protection to mRNA in the chloroplasts
(Nakamura et al., 2001). In addition, RNA editing in chloroplasts, like mammalian nuclear editing, also involves the
presence of hnRNPs (Lau et al., 1997; Hirose and Sugiura,
2001).
Dual Localization of SEBF
The presence of a protein of similar size and immunologically related to SEBF in chloroplasts suggests that this protein is targeted efficiently to this cellular compartment. This
is supported by the presence of a putative transit peptide
encoded in the cDNA of SEBF and by studies in other species that have shown that SEBF homologs are present in
chloroplasts (Ye et al., 1991; Mieszczak et al., 1992; Ohta et
al., 1995). Our results, however, indicate that SEBF is encoded by a single-copy gene and that the transit peptide is
not eliminated by differential splicing. This suggests that
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Figure 7. Genomic Organization of SEBF.
(A) DNA gel blot analysis of SEBF. Five micrograms of digested genomic DNA was loaded per lane and probed with a randomly labeled XmnI
fragment from the SEBF cDNA shown in (C). Molecular mass markers are indicated at right.
(B) PCR analysis of the genomic DNA. The positions of the oligonucleotides (T to Z; lanes 1 to 6) on the cDNA are presented in (C). The differences in size between the amplification products of lanes 5 and 6, lanes 3 and 4, and lanes 2 and 4 define the sizes of introns 1, 2, and 3, respectively. Molecular mass markers are indicated at right.
(C) Deduced genomic organization of SEBF. The cDNA is represented as a line, and the coding region is represented as a box. The N-terminal
transit sequence is presented in black. Introns are indicated as triangles emerging from the cDNA. The oligonucleotides used in the PCR reactions are designated by letters (T, U, V, W, X, Y, Z). The positions and sizes of the introns are deduced from the PCR analysis (B). Restriction
sites are indicated by arrows: H, HindIII; E, EcoRI; Xn, XmnI.
both the nuclear SEBF and the chloroplast protein are derived from the same precursor. Because only the mature
form of SEBF is detected in the nucleus, this also suggests
that the precursor can be processed outside the chloroplast. Similar results were found in potato for the enzyme
starch phosphorylase, which is synthesized as a precursor
and targeted to the amyloplast in young tubers but accumulates in a processed form in the cytoplasm of older tubers
(Brisson et al., 1989). In agreement with these data, Dahlin
and Cline (1991) reported that the import of proteins into
plastids is regulated developmentally, with the plastids losing their ability to import precursors in older tissues. This
differential import of proteins could be controlled by the
phosphorylation of transit peptides, which has been shown
to reduce chloroplast import rates (Waegemann and Soll,
1996). It will be interesting in the future to determine
whether the subcellular localization of SEBF changes during
development. Nonetheless, the dual localization of SEBF in
chloroplasts and the nucleus makes this protein an ideal
candidate for regulating metabolism in both subcellular
compartments during the defense response. It is well established that in addition to the activation of nuclear defense
genes, infection leads to major changes in the primary metabolism of the plant, including reductions in the rate of photosynthesis and synthesis of Rubisco (Kombrink and Hahlbrock,
1990; Somssich and Hahlbrock, 1998). SEBF, therefore,
could play a role in coordinating defense-induced changes
in both compartments.
Single-Stranded DNA Binding Repressor
SEBF Binding Site Is Present in Other Defense Genes
and Is Similar to the Auxin Response
Element (AuxRE)
DNA database searches revealed that the SEBF binding site
is present in the promoter of a number of genes known to
be induced by pathogens (Table 3). Many more sites will be
added to this list when an exhaustive study of SEBF binding
to naturally occurring sites found in the promoter of PR
genes is performed. The SEBF binding site also was found
in numerous wound- or stress-inducible genes. This suggests an important and general role for SEBF in plant defense
responses. It will be interesting to determine whether the
SEBF binding site actually contributes to the regulation of
these genes and if SEBF homologs in other species are involved in this regulation.
Comparison of the SEBF binding site (PyTGTCNC) with
other regulatory elements revealed a strong similarity to the
auxin response element (AuxRE; TGTCTC) present in composite AuxRE. In the absence of auxin, this element represses the action of a constitutive enhancer element
(Ulmasov et al., 1995) and appears to be occupied, regardless of auxin status, by a yet unidentified factor that can interact with auxin response factors (Ulmasov et al., 1999).
Auxin is known to negatively regulate several defense genes
(Grosset et al., 1990; Jouanneau et al., 1991). Recently, an
Arabidopsis pleiotropic mutant was isolated that shows both
increased susceptibility to pathogens and auxin insensitivity,
demonstrating complex interactions between the pathways
2533
leading to these phenotypes (Mayda et al., 2000). These results, and the strong similarity between the SEBF binding
site and the AuxRE, raise the possibility that SEBF also could
be involved in the hormonal control of gene expression.
Conclusion
It is difficult at this stage to formulate a general model explaining the role of SEBF in the regulation of PR-10a expression. Promoter fusion studies conducted in leaf protoplasts
indicate that deletion of the SE is required for optimal EREmediated expression (Matton et al., 1993; this study). This
demonstrates the importance of repression for the proper
regulation of PR-10a expression in this system in which
SEBF could play a major role. Although SE-mediated repression also is observed in transgenic tubers, an enhancer
element positioned between 155 and 135 suppresses
the action of the silencer after elicitor treatment (Després et
al., 1995). This could be the result of a specific interplay between tuber-specific enhancer binding factors and SEBF,
thus allowing maximal gene expression through the action
of the ERE. In this context, post-translational modifications
of SEBF could be required for specific protein–protein interactions to occur. Yeast two-hybrid studies are being conducted to identify proteins that interact with SEBF. In
addition, our future experiments will aim at determining
whether SEBF activity and localization are regulated by
phosphorylation or other post-translational modifications
Figure 8. Overexpression of SEBF Represses PR-10a Expression.
The coding sequence of pre-SEBF and the coding sequence of a control protein were each inserted into plasmid pBI223D (effector plasmids).
These plasmids were coelectroporated in potato leaf protoplasts with the reporter plasmids described in Figure 2D (reporter plasmids). The histogram represents the effect of pre-SEBF overexpression on reporter activity compared with the overexpression of the control protein (control 100). Fold activity to wild-type SE (WT 1) is presented for easier reference to Figure 2D. Transfection efficiencies were corrected by coelectroporating a luciferase reporter gene. Results represent means from a minimum of three individual electroporations. GUS, -glucuronidase. Error bars indicate SEM.
2534
The Plant Cell
Table 3. Occurrence of the SEBF Binding Site in the Promoter of
PR Genes
Gene
Organism
Accession
Number
Sequence
PR-10a
PR-10b
PR-10 (ypr10b)
PR-10 (ypr10a)
PR-10
PR-10
PR-10 (MLP)a
PR-10 (MLP)a
Chitinase
Chitinase
Chitinase
Glucanase
Glucanase
PR-1
PR-1
Osmotin (PR-5)
Peroxidase
Alternative oxidase
Antifungal protein
Potato
Potato
Birch
Birch
Apple
Parsley
Opium poppy
Peach
Potato
Bermuda grass
Arabidopsis
Rice
Tomato
Tobacco
Barley
Tobacco
Wheat
Voodoo lily
Gastrodia elata
M29041
M29042
AJ289770
AJ289771
AF020542
U48862
L06467
AF239177
AF153195
AF105426
Y14590
X58877
AF077340
X66942
Z48728
S68111
X85230
Z15117
AF334813
CTGTCAC
CTGTCAC
CTGTCTC
CTGTCAC
CTGTCAC
TTGTCTC
CTGTCAC
CTGTCAC
TTGTCAC
TTGTCTC
TTGTCTC
CTGTCAC
CTGTCAC
CTGTCAC
TTGTCAC
TTGTCAC
CTGTCAC
CTGTCAC
CTGTCTC
a Major
latex protein.
and whether this factor controls other genes involved in defense responses.
p-135 (Matton et al., 1993) using PCR oligonucleotide 1 (5-GCCAAGCTTTAGATAAAATGACACAAATGTCAAAAATGG-3) and oligonucleotide 2 (5-CCACCCGGGGATCCAGCTTTGAAC-3) to replace
the XbaI site at position 135 with a HindIII site. After digestion with
HindIII and BamHI, the fragment was inserted into pBI201. Mutant
m1 was described previously (pLP9; Desveaux et al., 2000). Mutants
m3, m4, and m5 were created by PCR using mutated oligonucleotides (Figure 2A) and PCR oligonucleotide 2. The PCR fragments
were cleaved with XbaI and BamHI and inserted into the wild-type
plasmid described above. For mutant m2, a HindIII-NcoI PCR fragment was synthesized using oligonucleotide 1 and oligonucleotide 3
(5-CAGTCCATGGTTAAATCAAC-3). Then, a NcoI-BamHI PCR fragment was synthesized using oligonucleotide 2 and oligonucleotide 4
(5-TAACCATGGACTGTCACTTG-3). Ligation of these fragments
into pBI201 cleaved with HindIII and BamHI created mutant m2. Amplifications were performed in a Whatman/Biometra TGradient thermal cycler. All constructs were sequenced to ensure that no
mutations were inserted by PCR. The transient expression system
was described previously (Matton et al., 1993). All values were corrected for the efficiency of electroporation using luciferase activity
resulting from the coelectroporation of 1 g of plasmid pWB216
(Barnes, 1990).
The coding sequence for the precursor silencing element binding
factor (pre-SEBF) was inserted into pBI223D, which contains a double 35S enhancer of Cauliflower mosaic virus. For transrepression
studies, 5 g of expression vector was added to the assays. The
control vector expressed a human fusion protein containing FK506
binding protein (Pelletier et al., 1998) under the same promoter.
Preparation of Extracts
Potato tubers (Solanum tuberosum cv Kennebec) were obtained
from the Quebec Ministry of Agriculture Les Buissons Research Station (Pointe-aux-Outardes, Canada). Tubers were stored in the dark
at 4C and brought to room temperature 24 hr before use. Leaf mesophyll protoplasts were isolated from 4- to 5-week-old potato plants
grown in growth chambers as described by Magnien et al. (1980).
Potato leaves were isolated from plants grown in an environmental
growth chamber (Conviron, Winnipeg, Canada) under long-day photoperiod conditions.
Crude nuclear extracts were prepared as follows: 200 g of potato tubers or 75 g of leaves was homogenized in a blender at maximum
speed for 1 min in 250 mL of cold NEBH (10 mM Pipes-KOH, pH 6.0,
1 M 2-methyl-2,4-pentanediol, 0.15 mM spermine, 0.5 mM spermidine, 10 mM MgCl2, 14.3 mM -mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride). After decantation at 4C for 5 min, the
homogenate was filtered through five layers of Miracloth under vacuum. The filtrate was centrifuged at 4000g for 5 min in a Sorvall
(Newtown, CT) GSA rotor to pellet the nuclei. The supernatant served
as the cytosolic fraction. Nuclei were washed three times in Nonidet
P-40 buffer (10 mM Mes-NaOH, pH 6.0, 260 mM sucrose, 10 mM
NaCl, 1 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 14.3 mM
-mercaptoethanol, 0.1% BSA, and 1% Nonidet P-40) to remove organelle and cytoskeleton contamination. The final pellet was resuspended in 5 mL of 200 mM NaCl SEBF binding buffer (SBB; 10 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 14.3 mM -mercaptoethanol, and
variable NaCl concentrations). Nuclei were ruptured by sonication,
and the lysate was centrifuged at 19,000g for 45 min. The supernatant was either used immediately for SEBF purification or stored at
70C in 10% glycerol. In such conditions, SEBF binding activity
was stable for months. Chloroplasts were prepared by the method of
Gegenheimer (1990).
-Glucuronidase Reporter Constructs and Analysis
Determination of Enzymatic Activities in Extracts
The wild-type construct was created by polymerase chain reaction
(PCR) amplification of the PR-10a promoter fragment of plasmid
Chlorophyll content was measured according to Arnon (1949). Alkaline pyrophosphatase, nitrite reductase, and alcohol dehydrogenase
METHODS
All standard molecular biology procedures were performed according to Sambrook et al. (1989).
Plant Material
Single-Stranded DNA Binding Repressor
were measured according to Gross and ap Rees (1986), Hucklesby
et al. (1972), and Smith and ap Rees (1979), respectively.
Electrophoretic Mobility Shift Assays
Protein extracts (in 200 mM NaCl SBB) were mixed with 20,000 cpm
of 32P end-labeled probe. The binding reaction (40 L) contained 100
ng of poly(dI-dC) to eliminate nonspecific binding. The reaction was
left on ice for 15 min. Electrophoretic separation of the complexes
was achieved by loading the mixture on a 5.7% polyacrylamide gel
prepared in Tris-glycine buffer (25 mM Tris and 195 mM glycine) and
applying 200 V for 2.5 hr at 4C. The gel was exposed overnight to
Kodak X-Omat AR film at 70C.
Purification of SEBF from Nuclear Extracts
Crude nuclear extracts were loaded onto a 3 mL Q-Sepharose fast
protein liquid chromatography column (Amersham Pharmacia Biotech) equilibrated in 200 mM NaCl SBB. The column was washed
with 10 volumes of 200 mM NaCl SBB followed by 15 volumes of 300
mM NaCl SBB. SEBF was eluted in 4 mL of 400 mM NaCl SBB. The
eluate then was submitted to two rounds of affinity purification. The
affinity beads were prepared by coupling a biotinylated wild-type SE
oligonucleotide to streptavidin-coated paramagnetic beads (Sigma)
according to Desveaux et al. (2000). The beads were washed twice
with 400 mM NaCl SBB before use. To 1 mL of eluate was added 20
L of 500 mM EDTA, pH 8.0, 10 g of poly(dI-dC), and 5 L of Nonidet P-40. The mixture was added to the affinity beads and left on ice
for 15 min with sporadic mixing. After separation of the beads from
the solution using a magnet, the beads were washed in 1 mL of 200
mM NaCl SBB followed by a 1-mL wash with 1 M NaCl SBB. SEBF
was eluted twice from the beads with 0.1 mL of 2 M NaCl SBB and
incubated at 37C for 5 min. The eluate containing SEBF was desalted using Ultrafree 4 centrifugal filter 10 K BioMax (Millipore, Bedford, MA). Proteins from the equivalent of 1 kg of tubers were pooled
and acetone precipitated. The pellet was resuspended in Laemmli
solubilizing buffer (Laemmli, 1970) and loaded onto a 12% SDSpolyacrylamide gel. The proteins were transferred to a polyvinylidene
difluoride membrane (Bio-Rad) and stained with Coomassie Brilliant
Blue R 250. Bands were cut and sent for N-terminal analysis at the
Eastern Quebec Proteomics Core Facility (Ste-Foy, Canada). A 50–
amino acid sequence was obtained from N-terminal analysis of the
29-kD band (see Figure 5). The last 10 amino acids of this sequence
(residues 41 to 50) eluted with minor contaminating amino acids.
DNA-probed protein gel blot analysis was performed according to
Vinson et al. (1988).
2535
diluted, and the procedure was repeated until it was possible to isolate a single plaque. A positive clone was excised using the ExAssist
system (Stratagene) according to the manufacturer’s instructions.
The clone was sequenced on both strands with Thermosequenase
(Amersham Pharmacia Biotech) in a cycle sequencing reaction that
was then processed on a Li-Cor automated sequencer.
DNA Gel Blot Analysis and Genomic Analysis
Procedures for the extraction of genomic DNA and DNA gel blot
analysis were as described by Ausubel et al. (2001). The following
primers were designed according to the predicted positions of the introns in the tobacco and Arabidopsis SEBF homologs (Ye et al.,
1991; Ohta et al., 1995) and used for PCR analysis of the genomic
DNA: T, 5-GCCGTTCTGTCTTCACAATTCTTTTGCTTC-3; U, 5CAACATCTCCAGCACGCTCAAAAAGCTCAG-3; V, 5-CCTCTGCTTCTTCCTGTAAGCTTGTCATAG-3; W, 5-CAGTTGAAGCCGCCTGTCAACAATTTAATG-3; X, 5-TTGACGGGAGGGCACTGAGGGTGAATTCTG-3; Y, 5-GCTTTCAATTGCATCGTTGACCTCCTTAG-3; and Z,
5-GCTTCAGCAGGACTTACACGGATGGCCCTG-3.
Antibody Production and Protein Gel Blot Analysis
The cDNA encoding SEBF was fused to the glutathione S-transferase
(GST) gene in plasmid pGEX-4T1 (Amersham Pharmacia Biotech).
The fusion protein (GST-SEBF) was expressed in BL21 cells. After lysis of the cells, the fusion protein was purified on a glutathione column (Smith and Johnson, 1988). The GST tag was cleaved with
thrombin (Amersham Pharmacia Biotech) and removed from the extract by a second pass on the glutathione column. Rabbit immunization was performed according to Harlow and Lane (1988). Briefly, 50
g of recombinant SEBF was used to immunize rabbits, followed by a
second injection of 150 g 30 days later to boost the immunological
reaction. The rabbits were killed 14 days after the second injection.
For protein gel blot studies, the SEBF antiserum was used at a dilution 1:5000, and the antibody–antigen interaction was revealed with
the enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Antibodies
to histone H1 (sc-8030) and Cdc2 (sc-53) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:1000.
Accession Number
The GenBank accession number for SEBF is AF389431.
Cloning of SEBF
ACKNOWLEDGMENTS
Degenerate oligonucleotides (5-GTKACWYTITCIGATTTYGAYCA-3
and 5-ARRTTWCCIACRAARATYTT-3) deduced from residues 1 to
8 and 41 to 46, respectively, of the N-terminal sequence of the purified protein were used to generate a 141-bp genomic PCR fragment
of SEBF. A specific primer (5-GTTTGAGTGATGAAGGTGC-3) was
derived from this fragment and used to probe a potato cDNA bank
(Matton and Brisson, 1989) using the method of Israel (1993).
Twenty-three pools of 18,000 phage were used in PCR reactions using the gene-specific primer and the M13 universal primer. Two
pools gave a PCR fragment of 1000 bp. These pools were further
We thank Rajagopal Subramanian for helpful discussions and the
members of the laboratory, particularly Darrell Desveaux, for reviewing the manuscript. We also thank Louise Cournoyer for preparing in
vitro grown plants and Barbara Otrysko for the gift of certified potato
tubers. We also are grateful to Franz Lang, Gertraud Burger, and
their group for sequencing SEBF. This research was supported by
research grants from the Natural Sciences and Engineering Research Council of Canada and from the Fonds pour la Formation de
Chercheurs et l’Aide à la Recherche, Québec.
2536
The Plant Cell
Received June 6, 2001; accepted August 30, 2001.
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Repression of the Defense Gene PR-10a by the Single-Stranded DNA Binding Protein SEBF
Brian Boyle and Normand Brisson
Plant Cell 2001;13;2525-2537
DOI 10.1105/tpc.010231
This information is current as of August 3, 2017
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