Originally published In Press as doi:10.1074/jbc.M909267199 on April 4, 2000
J. Biol. Chem., Vol. 275, Issue 26, 19897-19905, June 30, 2000
Tobacco Transcription Factor TGA2.2 Is the Main Component of
as-1-binding Factor ASF-1 and Is Involved in Salicylic
Acid- and Auxin-inducible Expression of as-1-containing
Target Promoters*
Ricarda
Niggeweg,
Corinna
Thurow,
Carsten
Kegler, and
Christiane
Gatz
From the Albrecht-von-Haller-Institut fuer Pflanzenwissenschaften,
Universitaet Goettingen, Untere Karspuele 2, 37073 Goettingen, Germany
Received for publication, November 15, 1999, and in revised form, March 17, 2000
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ABSTRACT |
In higher plants, activating sequence-1
(as-1) of the cauliflower mosaic virus 35 S promoter
mediates both salicylic acid (SA)- and auxin-inducible transcriptional
activation. Originally found in promoters of several viral and
bacterial plant pathogens, as-1-like elements are also
functional elements of plant promoters activated in the course of a
defense response upon pathogen attack. Nuclear
as-1-binding factor (ASF-1) and
cellular salicylic acid response
protein (SARP) bind specifically to as-1. Four
different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and
TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a
major component of either complex, as shown by supershift analysis and
Western blot analysis of DNA affinity-purified SARP. Minor amounts of a
protein immunologically related to TGA2.1 were detected, whereas TGA1a
was not detectable. Overexpression of either TGA2.2 or a dominant
negative TGA2.2 mutant affected both SA and auxin (2,4D) inducibility
of various target promoters encoding as-1-like elements,
albeit to different extents. This indicates that TGA2.2 is a component
of the enhancosome assembling on these target promoters, both under
elevated SA and 2,4D concentrations. However, the effect of altered
TGA2.2 levels on gene expression was more pronounced upon SA treatment
than upon 2,4D treatment.
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INTRODUCTION |
Plants activate a spectrum of defense genes upon pathogen attack.
Recognition of the pathogen is followed by the activation of different
signal transduction chains, which are functionally connected to a large
number of transcription factors regulating the activity of defense gene
promoters (1). One of these defense responses, known as systemic
acquired resistance (SAR),1
confers immunity against a broad spectrum of pathogens upon local infection with a necrogenic pathogen (2). SAR is associated with the
co-ordinate induction of a set of host genes presumably involved in
plant defense mechanisms such as glucanases, chitinases, and
pathogenesis-related (PR) proteins (3, 4). Salicylic acid (10 µM SA) is both necessary and sufficient to induce this set of genes (3, 5) and to confer SAR. Activating sequence-1 (as-1) is a functionally important element of a subgroup of
SA-inducible defense genes (e.g. Nt103, also
referred to as GNT35 (Ref. 6), and PR-1a (Ref.
7)). Although elevated levels of SA (1 mM) are required to
induce the isolated element (8), its regulation is likely to affect the
expression of several SAR genes. This is corroborated by the finding
that as-1-binding bZIP transcription factors of the TGA
family interact with ankyrin-rich repeat protein NPR1
(nonexpressor of PR-1), which is essential for
SA-inducible expression of PR-1 in Arabidopsis
thaliana (9-11). The isolated element also responds to elevated
levels of auxin (50 µM 2,4D; Ref. 12), indicating its
functional connection to other signal transduction pathways independent
from the SAR.
as-1-like elements are characterized by two
imperfect TGACGTCA motifs spaced by 4 bp (13). Using electrophoretic
mobility shift assays (EMSAs), a nuclear protein complex called ASF-1
was identified (13). ASF-1 is most likely involved in SA induction of
the as-1 element, as oligonucleotides encoding two perfect TGACGTCA motifs are recognized with higher affinity by ASF-1 and confer
higher SA inducibility of transcription (8). An as-1 binding
activity identified in leaf cell extracts has been called salicylic
acid response protein (SARP; Ref. 14). Its main component is a 40-kDa
protein that is immunologically related to bZIP transcription factor
TGA1a. Both SARP activity and ASF-1 activity increase upon SA treatment
of leaves (14, 15). Biochemical evidence supports the hypothesis that,
in the absence of SA, binding of SARP to as-1 is inhibited
by a not yet identified protein (SAI). Knowledge of the molecular
composition of ASF-1 or SARP will be a crucial step toward the
identification of SAI.
TGA1a was the first of the four so far characterized members of the
tobacco as-1-binding family of bZIP factors to be isolated (16). A related tobacco clone called PG13 was subsequently
reported (17). In A. thaliana, cDNA clones coding for
seven TGA-like factors (TGA1 to TGA6 and PERIANTHIA; Refs. 18-23) have
been isolated and grouped into three distinct classes based on sequence
similarities. The bZIP domains of these factors are highly conserved,
conferring similar DNA binding specificities and heterodimerization
properties. Tobacco TGA1a and PG13 are grouped into class I. Using
class II A. thaliana TGA5 cDNA as a probe, we have
isolated cDNAs coding for Nicotiana tabacum TGA2.1 (24)
and N. tabacum TGA2.2 (25). N. tabacum TGA2.2 is
79% identical to A. thaliana TGA2 at the amino acid level.
TGA2.1 is 76% identical to A. thaliana TGA2, but is
characterized by an additional 128-amino acid N-terminal domain.
At present, information on the in vivo function of
individual TGA factors is limited. In Arabidopsis, the
perianthia mutant is characterized by five instead of four
petals, but the target genes of PERIANTHIA are not yet known (23).
Injection of recombinant TGA1a into tobacco leaf cells led to
transcriptional activation from the as-1 element (26),
indicating that increasing the amount of TGA1a can lead to constitutive
promoter activation. TGA1a is predominantly expressed in roots (16).
This observation has been taken as evidence that TGA1a is responsible
for root specific expression from the element. In addition, increased
TGA1a levels lead to increased activation of the as-1
element upon 2,4D treatment, as revealed by transient expression
analysis using protoplasts from tobacco cell line BY-2 (27).
In this study, we elucidate the composition of ASF-1 and SARP and thus
define two members of the TGA family (TGA2.1 and TGA2.2) as candidates
for being involved in regulating the as-1 element in leaves.
Biochemical analysis identified the TGA2.2 homodimer as the main
component of these complexes. Functional analysis of transgenic plants
expressing either TGA2.2 and or a dominant negative TGA2.2 mutant
supports its involvement in gene expression in leaves as expression of
genes containing the as-1 element within their regulatory
sequences was altered both in response to SA and in response to
2,4D.
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EXPERIMENTAL PROCEDURES |
In Vitro Transcription/Translation--
The coding regions of
TGA2.1, TGA2.2 and TGA1a were cloned
into pBluescript SK under the control of the T7 RNA polymerase promoter
(25). RNAs were synthesized using the T7 transcription kit (MBI) with 1 µg of plasmid DNA. TGA proteins were obtained using the rabbit
reticulocyte lysate system (Promega). 0.5 µg of RNA were included in
each reaction. To control the efficient synthesis of the different
factors, products were radiolabeled by [35S]methionine
incorporation and separated on a 10% SDS-PAGE (28). The gel was
incubated in 50% methanol, 10% glacial acetic acid for 30 min and in
7% acetic acid, 7% methanol, 1% glycerol for 5 min before drying it
on a gel dryer. The gel was exposed to a Bioimager screen overnight.
EMSAs--
For EMSAs using as-1 as a probe, a 77-bp
HindIII/EcoRI fragment was cut out from the
pUC18-as-1 vector. This vector was constructed by cloning the following
oligonucleotide into the XbaI site of the multiple
cloning site of pUC18:
HindIII-XbaI-TGACGTAAgggaTGACGCAc-XbaI
EcoRI.
In addition, the 274-bp 5xas-1 PCR fragment described below
(see "Affinity Purification of SARP") was amplified with
nonbiotinylated primers and cloned into the vector pUC57/T (MBI
Fermentas). For radiolabeling, a 250-bp fragment was cut out using the
internal EcoRI site of the PCR fragment and the
XbaI site of the vector.
As G-box probe, a 55-bp XbaI/EcoRI fragment was
cut out of the pUC18-G-box vector. This vector was constructed by
cloning a 28-bp BamHI/BglII fragment containing
the G-box element, which was obtained from the plasmid pECK box II
(kindly provided by Dr. B. Weisshaar, Max Planck Institute for Plant
Breeding, Cologne, Germany), into the BamHI site of
the pUC18 vector:
XbaI-GGATCCCTTATTCCACGTGGCCATCCGGAGATCCCCGGGTACCGAGCTC-EcoRI (the G-box motif is written in bold letters).
The fragments for gel retardation were radiolabeled by filling in the
5'-overhangs with [
-32P]dATP and
[-32P]dCTP with the Klenow fragment and gel-purified
on a 5% polyacrylamide gel. Binding reactions were done as described
previously (29). For supershift analysis, 1 µl of the respective
antiserum or preimmune serum was added. Quantification of complexes was
accomplished by Bioimager analysis (Fujix BAS 1000, Fuji, Tokyo, Japan).
Generation of Antibodies--
Generation of the antibodies
against the N termini of TGA1a, TGA2.1, and TGA2.2 is described by
Niggeweg et al. (25). To generate a general antibody
directed against TGA factors, a synthetic peptide encoding the basic
domain (RRLAQNREAARKSRLRKKA) was coupled to ovalbumin (Pierce) and used
for rabbit immunization. The antibody was purified against the peptide
coupled to CNBr-activated Sepharose according to a procedure
recommended by Amersham Pharmacia Biotech. The antibodies directed
against TGA2.1 and TGA2.2 were affinity-purified using protein extracts
from recombinant Escherichia coli strains expressing the
antigen. Incubation of the serum with the antigen blotted on a membrane
was done as described (30).
Expression of Recombinant TGA Factors in E. coli--
The
cDNAs encoding either TGA2.1 or TGA2.2 were
excised from the respective pSK vectors (25) as
KpnI/BamHI fragments and ligated to the protein
expression vector pETmcs under the control of the T7 RNA polymerase
promoter. The pETmcs vector is a derivative of the vector pET3b with
further restriction enzyme sites (KpnI, SnaBI,
and HindIII) added to the multiple cloning site downstream of the ATG start codon. This cloning strategy added three amino acids
to the N termini of TGA2.1 and TGA2.2. These constructs were
transformed into E. coli BL21(DE)-competent cells. To
express TGA1a, a pET vector containing the cDNA of TGA1a
(7) was used; the vector was kindly provided by Dr. U. Pfitzner
(University of Hohenheim, Hohenheim, Germany). 0.4 ml of an overnight
culture was added to 20 ml of medium and incubated at 37 °C until
A600 = 0.6-0.8 was reached. The expression of
the encoded protein was induced for 3 h with 0.6 mM
isopropyl-1-thio-
-D-galactopyranoside. The pellet of the
cells were resuspended in 10 volumes of denaturation buffer (8 M urea, 0.1 M NaH2PO4,
10 mM Tris-HCl, pH 8).
Western Blot Analysis--
Tobacco and bacterial proteins were
first separated on a 10% SDS-PAGE (28) and then transferred onto a
polyvinylidene difluoride membrane (Millipore) as described (31). The
membrane was blocked in PBS-T (0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4,
4 mM Na2HPO4, 0.05% Tween 20)
containing 5% nonfat dried milk overnight at 4 °C by shaking. It
was washed once for 5 min in PBS-T containing 1% nonfat dried milk and
then incubated with the respective affinity-purified antibodies diluted
in 1% nonfat dried milk/PBS-T for 2 h at room temperature. The
membranes were washed in 1% nonfat dried milk/PBS-T four times for 5 min. They were incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody at a dilution of 1:5000 in PBS-T for
1.5 h at room temperature, washed four times for 5 min in PBS-T
and twice for 5 min in PBS. Detection was carried out with a
chemiluminescence reaction using the ECL detection kit (Amersham Pharmacia Biotech) according to manufacturer's protocols. The membrane
was finally exposed to autoradiography film (Cronex T10, Eastman Kodak
Co.). Before incubation of a processed filter with a second primary
antibody, it was washed once with PBS and then incubated in stripping
buffer (100 mM -mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8) for 60 min at 50 °C by shaking.
Washing was performed twice in PBS-T.
Plant Material and Treatments--
Tobacco plants (N. tabacum cv. Samsun NN) were grown under a 16-h light
(22 °C)/8-h (19 °C) regime in 85% humidity. Fully expanded,
young leaves from tobacco plants were taken either for protein or RNA
extraction. For induction experiments, discs of these leaves were
floated on 1 mM salicylic acid (SA) or 1 mM 3-hydroxybenzoic acid (HBA) in 10 mM phosphate buffer, pH
6.8, or on 50 µM 2,4-dichlorophenylacetic acid (2,4D) or
50 µM 2,6-dichlorophenylacetic acid (2,6D) in 50 mM phosphate buffer, pH 5.7. These treatments were
performed in a climate chamber under a 16-h light (24 °C)/8-h (22 °C) regime.
Preparation of Protein Extracts from Tobacco
Leaves--
Preparation of nuclei was done as described previously
(29). Nuclei were not purified using Percoll, but directly used for nuclear protein extraction as described previously (32). Cellular extracts were prepared according to Jupin and Chua (14). Instead of
using Centricon tubes, conventional dialysis was used to desalt the
extract. This alteration did not change the binding characteristics of SARP.
Cell Culture and Preparation of Cellular Extracts from BY-2
Cells--
Bright Yellow-2 (BY-2) tobacco (N. tabacum) cell
suspension cultures were grown in a modified Murashige and Skoog medium
with 3% sucrose, 0.9 g/liter myo-inositol, 0.9 mg/liter thiamine, and 0.22 mg/liter 2,4D at 26 °C in the dark on a rotary shaker. Every week the cells were subcultured by transferring 3 ml of the culture to
27 ml of fresh medium. Cells were harvested 4 days after subculturing by suctioning off the medium, frozen in liquid nitrogen, and stored at
80 °C. After grinding the frozen cells in liquid nitrogen in a
mortar, the fine powder was used to prepare cellular extracts as
described above for cellular extracts from tobacco leaves. Typically, 2 ml of cellular extracts containing about 5 mg of protein/ml were
obtained from 2 g of BY-2 cells.
Affinity Purification of SARP: Preparation of DNA Affinity
Beads--
A 274-bp PCR fragment containing five as-1
elements (5xas-1) was amplified with a 5' biotinylated 27-bp upstream
primer and a 23-bp downstream primer using the 5xas-1 Gus plasmid
(kindly provided by Dr. S. Prat, Barcelona) as template and purified
from agarose gel with the QIAquickTM gel extraction kit (Qiagen). The sequence of the 5xas-1 PCR fragment is: biotin,
5'-CAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCCACTGACGTAAgggaTGACGCAcAATCCCACTAGGATCCTCCACTGACGTAAgggaTGACGCAcAATCCCACTAGGATCCTCCACTGACGTAAgggaTGACGCAcAATCCCACTAGGATCCTCCACTGACGTAAgggaTGACGCAcAATCCCACTAGGATCCGTCATCTCCACTGACGTAAgggaTGACGCAcAATCCCACTATCCTTCGCAAGACCCT-3'. as-1 elements are shown in bold and small letters. The
internal EcoRI site is underlined.
The biotinylated DNA fragment was immobilized on streptavidin-coated,
paramagnetic beads (DynabeadsTM M-280 streptavidin; Dynal) as follows.
After prewashing the Dynabeads in 1× B&W buffer (2 M NaCl,
10 mM Tris-HCl, pH 7.5, 1 mM EDTA) according to
the manufacturer's specifications, 1 mg of beads were resuspended in
0.5× B&W buffer containing 200 ng of the purified 5xas-1
PCR fragment. Coupling occurred during incubation for 30 min at room
temperature with gentle rotation of the tube. Afterwards the beads were
washed in 1× B&W buffer and equilibrated in binding buffer (25 mM Hepes-KOH, pH 7.6, 10% glycerol, 10 mM
MgCl2, 0.2 mM CaCl2, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl
fluoride). For purification of SARP, all incubations were done at room
temperature and with gentle rotation of the tubes. Cellular extract
prepared from BY-2 cells was diluted 1:4 in binding buffer. To deplete
the amount of unspecific DNA binding proteins and GBFs (G-box binding
factors), 20 ng of poly(dI/dC) (Sigma) and 2 ng of G-box
double-stranded oligonucleotide (5'-GATCTCTTATTCCACGTGGCCATCCGGATC-3'; the G-box motif is
written in bold letters) per µg of cellular protein were included as
competitor DNA. The mixture was added to 1 µg of Dynabeads (coupled
with 0.2 ng of 5xas-1 fragment) per µg of cellular
protein, and binding reaction was extended to 20 min. Afterwards, beads with the adsorbed DNA-binding proteins were separated from the supernatant and washed twice for 10 min with 1 volume of binding buffer
(supplemented with 0.5 mg/ml BSA) including the same concentration of
poly(dI/dC) and G-box oligonucleotide as used in the binding reaction.
Finally elution of DNA binding proteins was performed by twice adding
0.125 volume of elution buffer (binding buffer supplemented with 2 M NaCl; if the eluted fraction was used for EMSAs, 0.5 mg/ml BSA was added) to the beads (eluted fractions 1 and 2; elution
time, 15 and 10 min, respectively). The initial cellular extract and
the eluted fraction 1 were analyzed for specific as-1 and
G-box binding activities in EMSAs. Because the eluted fraction 1 is
twice as concentrated compared to the initial extract, we added 1 volume of cellular extract corresponding to 5 µg of protein and 0.5 volume of eluted fraction 1 to the binding reactions. Because of the
very low protein content, 10 µg of BSA was included in binding
reactions with the eluted fraction. Eluted fractions 1 and 2 from
initially 3.4 mg of cellular proteins were collected, dialyzed against
water, freeze-dried, and resuspended in 25 µl of denaturation buffer
(8 M urea, 0.1 M
NaH2PO4, 10 mM Tris-HCl, pH 8). 2.5 µl of the sample were separated on a 10% SDS-PAGE and subjected to
Western blot analysis.
Expression of TGA2.2 and TGA2.2trd in Tobacco--
To introduce
the mutations in the basic domain of TGA2.2, a cDNA
fragment encoding the N terminus and basic domain of TGA2.2 was
amplified by PCR. The lower primer (CCC AAG CTT CAT ATG CCT TTT TCC TTA
GAC GAC TTT TCC GCG GTG GTT CAC G introducing a HindIII site) being complementary to the basic DNA-binding domain contained nucleotide substitutions to change the sequence AGCAGCTCGAA on the
upper DNA strand to
ACCACCGCGGA. These
substitutions caused an exchange of two alanine to proline residues.
The upper primer encoded the sequence GGG GTA CCA ACC ATG GCT GAT ATC
AGT CCT A, introducing a KpnI site and the Kozak sequence.
The PCR fragment was subcloned as a KpnI/HindIII
fragment into the pSK vector (pSK-28mutNterm). The introduced
nucleotide substitutions were controlled by sequencing. The C-terminal
fragment (HincII/BamHI) was amplified using PCR,
to generate a single SunI restriction site just upstream of
the Stop codon, changing the sequence from CGG GAA TAA to CGT ACG TAA.
An oligonucleotide encoding the Streptavidin-Tag (WSHPQFEK) was
inserted into pSK-28modCterm vector digested with
SunI/BamHI and controlled by sequencing. The
HincII/BamHI fragment was used to exchange the C
terminus within the pSK vector encoding the wild-type allele. A
NdeI/BamHI fragment of this vector was ligated
into the pSK-28mutNterm vector, which was cleaved with NdeI
and BamHI resulting in the derivative TGA2.2trd. To obtain
the corresponding wild-type TGA2.2 allele with the Kozak sequence, the
cDNA sequence containing the nucleotide substitutions was exchanged
by the corresponding EcoRV/PstI fragment of the
wild-type TGA2.2 cDNA. The cDNAs encoding the TGA2.2
derivatives (TGA2.2 and TGA2.2trd) were cloned under the control of the
cauliflower mosaic virus 35 S promoter (modified by three operators;
Ref. 33), by excising them from the pSK vectors as
KpnI/XbaI fragments and inserting them in
pBINHygTX (32), cleaved with KpnI and XbaI. The
constructs were transformed into Agrobacterium tumefaciens GV3101 competent cells containing the pMP90 vector (34). Transformation of tobacco plants was performed using the Agrobacterium leaf
disc technique as described previously (35).
Northern Blot Analysis--
Total RNA was purified from tobacco
leaves using the RNeasy plant kit (Qiagen). RNA concentrations were
determined spectrophotometrically, and 15 µg of RNA for each sample
were electrophoresed on denaturing agarose gels and transferred to
nylon membranes (Schleicher & Schuell; Ref. 36). RNA blots were
prehybridized in 50% formamide, 50 mM sodium phosphate, pH
6.8, 1% SDS, 10% dextran sulfate sodium salt, and 1 M
NaCl for 1 h at 42 °C. Hybridization was performed in the same
solution with 100 µg/ml salmon sperm carrier DNA and 32P-labeled probe for 14-16 h at 42 °C. The probe was
made by the random-priming method (37) with the Megaprime DNA labeling
system (Amersham Pharmacia Biotech). The cDNA probes used for
Northern blot hybridizations were: tobacco Nt103, a
1-kilobase pair full-length cDNA encoding the auxin-inducible gene
Nt103 (38); tobacco IEGT, a 701-bp cDNA
fragment of the immediate-early SA-induced glucosyltransferase (39); a
715-bp fragment of the tobacco ParA gene (40); tobacco PR-1a, a 531-bp fragment of pathogenesis-related protein 1a
(41); tobacco actin, a 800-bp cDNA fragment (kindly provided by Dr. K. Pawlowski, University of Goettingen, Goettingen, Germany); and a
1-kilobase pair pea 18 S rRNA probe (Ref. 42; kindly provided by Dr. S. Koehne, University of Bielefeld, Bielefeld, Germany). Washing was
performed with 2× SSC, 0.1% SDS at 65 °C for two 30-min periods
and with 0.5× SSC, 0.1% SDS at 65 °C for 20 min as required. Quantification of RNA levels was accomplished by Bioimager analysis (Fujix BAS 1000; Fuji). Hybridization signals of Nt103,
IEGT, ParA, and PR-1a were normalized
using either the 18 S rRNA or the actin mRNA obtained by subsequent
hybridization of the same filter.
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RESULTS |
TGA2.2 Is the Main Component of ASF-1 and SARP--
The presence
of multiple TGA factor encoding genes in tobacco raised the following
question: which members of this family constitute
as-1-binding complexes present in nuclear extracts (ASF-1)
and whole cell extracts (SARP)? Supershift analysis with antibodies
designed to specifically recognize individual TGA factors provides a
highly sensitive assay for the analysis of the composition of
DNA-binding complexes (43). Antibodies directed against the N-terminal
domains of TGA1a, TGA2.1, and TGA2.2 were generated and shown to be
highly specific in supershift analysis using in vitro
translated proteins (25).
A 77-bp-long DNA fragment encoding as-1 was used as a probe
for supershift analysis of either ASF-1 or SARP. ASF-1 and SARP had
similar mobilities indicating that both complexes might represent the
same activity (Fig. 1, a and
b). Strong supershifting was only observed upon addition of
the anti-TGA2.2 serum either to nuclear extracts or to whole cell
extracts. The amount of radioactive probe found in the supershifted
complexes corresponded to the amount lost in the ASF-1/as-1
or SARP/as-1 complex. Supershifted complexes were also
obtained with anti-TGA2.1 serum, although to a lesser extent than with
the anti-TGA2.2 serum. Quantification of three independent experiments
revealed that ~80% of ASF or SARP were supershifted with the
anti-TGA2.2 serum, whereas the anti-TGA2.1 serum recognized 10% of
ASF-1 and 20% of SARP. We failed to detect any TGA1a in either
complex.
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Fig. 1.
Supershift analysis of ASF-1 and SARP.
a, Nuclear extracts were prepared from tobacco leaves. For each
lane, 10 µg of nuclear extracts were added to the binding reaction
with a radiolabeled 77-bp-long as-1-encoding fragment. The
ASF-1 complex is characterized by two protein-DNA complexes
representing single and double occupancy of the two TGACG motifs. At
the top of each lane is indicated whether the preimmune sera
(pTGA2.1, pTGA2.2, pTGA1a) or antisera (TGA2.1, TGA2.2, TGA1a)
were added (1 µl). The mobility of the TGA2.2/as-1 complex
is shown using in vitro translated TGA2.2 protein.
b, cellular extracts were prepared from tobacco leaves (14).
For each lane, 20 µg of cellular extracts were added to a 77-bp-long
radiolabeled as-1-encoding DNA fragment. The SARP complex is
characterized by two protein-DNA complexes representing single and
double occupancy of the two TGACG motifs. At the top of each
lane is indicated whether the preimmune serum (pTGA1a) or antisera
(TGA2.1, TGA2.2, TGA1a) were added (1 µl). c,
cellular extracts were prepared from tobacco cell line BY-2 4 days
after subculturing. For each lane, 5 µg of cellular extracts were
added to a 77-bp-long radiolabeled as-1-encoding DNA
fragment. At the top of each lane is indicated, whether the
preimmune serum (pTGA1a) or antisera (TGA2.1, TGA2.2, TGA1a)
were added (1 µl). Relative amounts of ASF-1 or SARP and supershifted
complexes are indicated below the lanes. Numbers
were average values from three independent extracts (a and
b) and from one extract (c).
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Next, we approached the question of whether a (so far) unknown TGA
factor might be present in SARP. To be able to detect such a protein,
an antibody directed against the conserved basic DNA-binding domain was
generated. As this antibody also recognizes other plant bZIP factors
(e.g. G-box binding factors (GBFs), Ref. 44), cellular extracts were enriched for TGA factors before subjecting them to
Western blot analysis. As this enrichment turned out to be unreliable
using leaf cell extracts, SARP was prepared from tobacco cell line
BY-2. Supershift analysis confirmed that its composition is very
similar to that of SARP from leaf extracts (Fig. 1c). The
extract was loaded onto magnetic beads coated with a multimer of the
as-1 element. To deplete the extract from GBFs, loading of
the beads was done in the presence of an oligonucleotide encoding the
G-box, followed by washing steps with the same oligonucleotide. After
elution of the complex with 2 M NaCl, ~65% of SARP were regained, whereas the amount of GBFs had decreased considerably (Fig.
2, a and b). In
order to determine whether the enrichment procedure had changed the
composition of SARP, supershift analysis of the eluted fraction was
performed with the antibodies against TGA1a, TGA2.1, and TGA2.2. Again,
low amounts of the complex were supershifted with the anti-TGA2.1
serum, whereas the majority of the complex was supershifted with
anti-TGA2.2 serum, indicating that none of the two factors was
preferentially lost (Fig. 2c). Next, enriched SARP was
loaded on a Western blot along with recombinant TGA1a, TGA2.1, and
TGA2.2 (Fig. 3). Using the antibody
directed against the basic DNA-binding domain of TGA factors, one
prominent and four weaker signals were detected. The prominent band was slightly smaller than recombinant TGA2.2, which is due to a three-amino acid N-terminal extension of the recombinant protein. Thus, the dominant component of SARP is TGA2.2. As the prominent band is stronger
than the total of the other bands, we conclude that a large portion of
SARP is a TGA2.2 homodimer. Probing the blot with the TGA2.2 antibody
confirmed that this band is indeed TGA2.2. The ratio of the intensity
of the signal of SARP-derived TGA2.2 and recombinant TGA2.2 was very
similar to the ratio detected with the antibody against the basic
domain, indicating that no additional protein of the same size
co-migrated with SARP derived TGA2.2. A faint band migrating above
TGA2.2 cross-reacted with the TGA2.1 antibody. As this protein runs
considerably faster than recombinant TGA2.1, we call it TGA2.1-related
activity. The low abundant proteins running below TGA2.2 interacted
with none of the specific antibodies. We do not know whether these
proteins constitute further unidentified TGA factors or degradation
products missing the N termini of TGA2.2 and/or TGA2.1.
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Fig. 2.
EMSAs and supershift analysis of
affinity-purified SARP. a, EMSAs using the 250-bp
radiolabeled 5xas-1 fragment as a probe. Loading was done
after addition of 5 µg (1.06 µl) of cellular extract, prepared from
BY-2 cells, and 0.53 µl of SARP, purified using magnetic beads coated
with a multimerized as-1 element (fraction eluted with high
salt). b, EMSAs using the 55-bp radiolabeled G-box as a
probe. Loading of lanes was done as in a. The ratio of TGA
factors to GBFs increased considerably due to depletion of GBFs.
c, supershift analysis of affinity-purified SARP to
demonstrate that the composition of SARP did not change during the
affinity purification. 0.53 µl of the eluted proteins were added to
the binding reaction containing a 77-bp radiolabeled as-1
encoding fragment. At the top of each lane is indicated
which of the three antisera (TGA2.1, TGA2.2, TGA1a) was added
(1 µl).
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Fig. 3.
Western blot analysis of affinity-purified
SARP. Middle panel, purified SARP was
dialyzed against water, freeze-dried, and resuspended in denaturation
buffer. The sample (2.5 µl, eluted fraction) was electrophoretically
separated on a 10% SDS gel along with crude extracts of E. coli (diluted 1:20) expressing TGA2.2 (0.36 µl), TGA1a (2 µl),
and TGA2.1 (1.25 µl), respectively. Detection of bZIP proteins was
done by using an antibody against a synthetic peptide encoding the
conserved DNA-binding domain. Lane 6 is a longer
exposure of lane 7. Right
panel, the filter was stripped and probed with an
affinity-purified TGA2.2 antibody to prove that the major band of the
eluted fraction of lane 6 is TGA2.2.
Left panel, the filter was stripped again and
probed with an affinity-purified TGA2.1 antibody to identify the size
of the TGA2.1-related activity. Lane 5 is a
longer exposure of lane 1. Arrows point to the
five bands detected by the general antibody anti-TGA; the
double arrow points to the TGA2.1-related
activity. Numbers between the middle and the
right panel indicate the number of amino acids of
the recombinant proteins.
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Functional Analysis of TGA2.2 in Planta--
Having defined TGA2.2
as a major component of ASF-1 or SARP, we analyzed its impact on
as-1-encoding potential target genes. Two types of
transgenic plants expressing different TGA2.2 alleles were
generated (Fig. 4a). The first
one encodes the wild-type allele under the control of the cauliflower
mosaic virus 35 S promoter. The second type of transgenic plants
contains the same chimeric gene with only two point mutations in the
conserved DNA-binding domain. These mutations change two alanine
residues into proline residues. As the DNA-binding domain assumes a
helical structure upon binding, we expected that the introduction of
the helix-breaking amino acid proline should abolish DNA binding while
maintaining heterodimerization properties. TGA mutants with defective
DNA-binding domains have been shown previously to specifically suppress
the activity of endogenous TGA factors by forming inactive heterodimers (32, 45). This was confirmed by co-translation of mutated TGA2.2
(TGA2.2trd) with either TGA2.2 or TGA1a and subsequent gel shift
analysis (Fig. 4, b and c). As expected,
TGA2.2trd is deficient in DNA binding and reduces binding of TGA2.2 and
TGA1a.
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Fig. 4.
Construction of a dominant negative TGA2.2
allele. a, schematic presentation of the dominant
negative TGA2.2 allele (TGA2.2trd). The sequence of the basic
DNA-binding domain is shown, as well as point mutations introduced by
PCR. b, SDS-PAGE of in vitro
35S-labeled TGA factors. The cDNA sequences of
tobacco TGA1a, TGA2.2, and TGA2.2trd were transcribed under the control
of the T7 promoter. Mixtures of equal amounts of RNAs were translated
using the TNT reticulocyte lysate (Promega), containing
[35S]methionine. 5 µl of the in vitro
translation reaction were loaded on each lane to estimate the
efficiency of protein synthesis. c, EMSA of in
vitro translated TGA factors. A 77-bp-long 32P-labeled
DNA fragment encoding the as-1 element was used as a
probe.
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Transformants were phenotypically normal, independent of the level of
transgene expression. Fig. 5a
depicts the mRNA levels of the two highest expressing plants of
each construct, which were chosen for further analysis. Plants
transformed with the chimeric TGA2.2 gene expressed 2-fold
less transgenic mRNA levels than the two highest expressing
TGA2.2trd plants, indicating counterselection against high levels of
TGA2.2. This notion is supported by the finding that only two strong
TGA2.2 overexpressors were found in 28 primary transformants analyzed,
whereas five strong TGA2.2trd overexpressors were found in 7 primary
transformants. Western blot analysis with the TGA2.2 antibody confirmed
the synthesis of transgenic TGA2.2 proteins (Fig. 5b).
Western blot analysis using an antibody directed against glutamine
synthetase (kindly provided by Dr. Tischner) indicated, that the
quality of the different extracts was comparable (Fig. 5c).
Subsequently, these extracts were subjected to supershift analysis.
Transgenic plants overexpressing TGA2.2 revealed a ~2.5-fold increase
in SARP activity, whereas transgenic plants encoding TGA2.2trd showed a
~2.3-fold decrease in SARP activity (Fig. 5d). Supershift
analysis did not indicate a dramatic change in the composition of SARP.
The anti-TGA2.1 serum recognized 20% of SARP in wild-type and
TGA2.2trd plants. In plants overexpressing TGA2.2, only 10% of SARP
was recognized by the anti-TGA2.1 serum, indicating that the relative
levels of TGA2.2 and TGA2.1-related protein had shifted toward higher levels of TGA2.2.
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Fig. 5.
Expression of TGA2.2 alleles in transgenic
plants. a, Northern blot analysis of transgenic plants
expressing either TGA2.2 or TGA2.2trd. 15 µg of total RNA were loaded
in each line. Two independent transformants with highest expression
levels are shown. Hybridization was done with a TGA2.2 probe.
Arrows point to the endogenous (upper
arrow) and the transgenic (lower
arrow) transcript. b, Western blot analysis of
transgenic plants expressing either TGA2.2 or TGA2.2trd, using an
antibody directed against the N terminus of TGA2.2. 100 µg of
cellular extracts (14) were loaded. The arrow marks the
specific band. c, Western blot analysis of transgenic plants
expressing either TGA2.2 or TGA2.2trd, using an antibody directed
against glutamine synthetase to control the quality of the extract. 40 µg of cellular extracts (14) were loaded. d, EMSA and
supershift analysis of transgenic plants expressing either TGA2.2 or
TGA2.2trd. Cellular extracts were prepared from tobacco leaves of
either untransformed plants or plants overexpressing TGA2.2 or
TGA2.2trd. For each lane, 20 µg of cellular extracts were added.
Addition of 1 µl of the different antisera is indicated at the
top (TGA2.1, TGA2.2, TGA1a). Radiolabeled probe was
the 77-bp as-1 encoding DNA fragment. The panel
below the lanes indicates the percentage of supershifted
SARP (see also Fig. 1).
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Next, we tested whether altered levels of SARP correlate with altered
expression of potential target genes. SA-responsive genes are grouped
into "immediate early" and "late" genes according to the
kinetics of the induction (8). Nt103 (a glutathione S-transferase; Ref. 46), IEGT (a gene with
homology to a glucosyltransferase; Ref. 39) and ParA (a gene
coding for a nuclear localized gene product with remote similarity to
glutathione S-transferases; Ref. 47) belong to the immediate
early genes, which are transiently induced after 2-3 h of SA
treatment. The respective promoters encode as-1-like
elements, which are recognized by ASF-1 (48, 49). Fig.
6 demonstrates that the kinetics of
induction in response to SA and 2,4D is identical to the kinetics of an
isolated as-1 element fused to a TATA-box and a
-glucuronidase (Gus) reporter gene (32). SA caused a
rapid and transient increase in transcription of Gus,
Nt103, IEGT, and ParA, whereas the
kinetics of 2,4D induction was slower for all four genes. Therefore,
these genes were considered to be potential target genes for TGA2.2.
PR-1a, which encodes a functionally important
as-1 element (7), was chosen as a late gene.
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Fig. 6.
Northern blot analysis of transgenic plants
expressing -glucuronidase
(Gus) under the control of the as-1 element.
Leaves were floated on 1 mM SA or 50 µM 2,4D
before extraction of RNA at the indicated time points. 15 µg of total
RNA were loaded. The blot was subsequently hybridized with probes for
Nt103, IEGT, ParA, and PR-1a.
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The expression profiles of these genes were analyzed in the transgenic
plants described above. Leaf disks of untransformed and transgenic
plants (two independent individuals of each construct) were floated
either on 1 mM SA or 1 mM HBA (a structural
analogue of SA). RNA was prepared and subjected to Northern blot
analysis with probes for Nt103, IEGT,
ParA, and PR-1a. HBA treatment served to show
that induction of these genes is specific for SA and that the
specificity was not changed by the transgene. The most striking effects
were observed with Nt103. Elevated levels of SARP correlated with a 2.9- and 4.7-fold increase in SA responsiveness, whereas reduction of SARP correlated with a 8.5- and 5.6-fold decrease of the
response (Fig. 7). The IEGT
promoter was not as strongly affected by the transgenes as the
Nt103 promoter. In contrast, overexpression of TGA2.2 had no
effect on the expression of ParA or PR-1a,
although expression of TGA2.2trd decreased SA inducibility of these
promoters ~5-fold.
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Fig. 7.
Expression levels of SA-inducible target
genes in transgenic plants overexpressing either TGA2.2 or
TGA2.2trd. Leaves were floated on 1 mM SA or 1 mM HBA for 2 and 3 h before extraction of RNA. 15 µg
of total RNA were loaded. Blots were hybridized with probes for
Nt103, IEGT, ParA, and 18 S rRNA as
indicated. For analysis of PR-1a, leaves were floated on 1 mM SA for 12 and 24 h. Northern blots are only shown
for one of the two independent transformants. Transcript levels were
quantified by Bioimager, and values were corrected for small variations
in loading by standardization to the 18 S rRNA values.
Numbers indicate the amount of increase or decrease of
expression levels of two independent transgenic plants, with the
control plants having a value of 1.0.
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Next we examined whether changing SARP levels also correlated with
effects on 2,4D inducibility of the immediate early genes Nt103, IEGT, and ParA (Fig.
8). 2,4D induction of Nt103
was 1.7- and 2.0-fold higher in TGA2.2-overexpressing plants and 3.5- and 4.0-fold lower in TGA2.2trd-expressing plants. The response was specific for 2,4D and did not occur with 2,6D. 2,4D inducibility of
IEGT was only slightly affected (2-fold) in TGA2.2trd
plants, but not in TGA2.2-overexpressing plants. Expression of
ParA was not at all altered in the transgenic
plants.
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Fig. 8.
Expression levels of 2,4D-inducible target
genes in transgenic plants overexpressing either TGA2.2 or
TGA2.2trd. Leaves were floated on 50 µM 2,4D or 2,6D
for 7, 12, and 24 h before extraction of RNA. 15 µg of total RNA
were loaded. Blots were hybridized with Nt103,
IEGT, ParA, and actin, as indicated. Northern
blots are only shown for one of the two independent transformants.
Transcript levels were quantified by Bioimager, and values were
corrected for small variations in loading by standardization to the 18 S rRNA or actin values. Numbers indicate the amount of
increase or decrease of expression levels of two independent transgenic
plants, with the control plant having a value of 1.0.
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Expression Pattern and Binding Activity of TGA2.2--
Expression
levels of pre-existing regulatory proteins often increase under
inductive conditions. Therefore, we analyzed whether the expression
level of TGA2.2 changes upon treatment of plants with either SA or
2,4D. SA treatment for up to 36 h neither changed TGA2.2 mRNA
levels nor protein levels. The same results were obtained upon 2,4D
treatment (data not shown).
| |
DISCUSSION |
To further elucidate the regulation of the as-1
element, we analyzed the composition of a nuclear (ASF-1) and a
cellular (SARP) as-1 binding activity previously identified
in leaf extracts (13, 14). The major component of both complexes is a
TGA2.2 homodimer. A TGA2.1-related gene product was also detected,
albeit to significantly lower amounts. Overexpression of TGA2.2 or a
dominant negative TGA2.2 mutant altered SARP levels and had
differential effects on both SA and 2,4D inducibility of various target
genes, albeit to different extents.
TGA2.2 Is the Main Component of ASF-1 and SARP--
Two different
protocols for the enrichment of as-1-binding proteins from
leaf extracts have been reported. Originally, ASF-1 was communicated as
a nuclear DNA binding activity (13). Later, Jupin and Chua (14)
described a cellular factor binding to as-1, which they
called SARP. SARP and ASF-1 were not directly compared with respect to
electrophoretic mobility or binding specificity. The composition of
these complexes is particularly interesting with regard to the
elucidation of the regulatory mechanism leading to SA-inducible gene
expression. According to a model suggested by Jupin and Chua (14), SARP
associates with a hypothetical protein (SAI) in the absence of SA. This
SAI protein is believed to interfere with binding of SARP to
as-1. Dissociation of SARP from SAI, which depends on
phosphorylation of either of the two partners, leads to a 3-fold
increase of SARP activity in leaves. Thus, the knowledge of the
composition of SARP identifies one of the two interacting partners,
yielding a promising strategy for a two-hybrid screen to identify SAI.
Eighty percent of ASF-1 and SARP were supershifted with the anti-TGA2.2
serum. The composition of ASF-1 did not change upon SA or 2,4D
treatment of leaf disks (data not shown). To decide whether TGA2.2 is
represented in the complex as a homodimer or a heterodimer, SARP was
analyzed by Western blot analysis after affinity purification against
as-1-encoding oligonucleotides attached to a solid matrix.
An antibody against the conserved basic domain of TGA-factors
identified TGA2.2 as the dominant protein of SARP, indicating that a
large fraction of TGA2.2 in SARP must be represented as a homodimer.
This is consistent with results reported by Jupin and Chua (14);
Southwestern blot analysis of SARP had indicated that a 40-kDa protein,
which is smaller than TGA1a but immunologically related, is the main component of SARP. The calculated molecular mass of TGA2.2 (36.2 kDa)
is close to the size reported by Jupin and Chua (14). As TGA1a, PG13,
and TGA2.1 have molecular masses of 43.3, 43.3, and 50.0 kDa
respectively, it seems very likely that Jupin and Chua (14) and our
group have indeed analyzed the same activity. Unfortunately, the 3-fold
increase of SARP binding activity upon SA treatment was not
reproducible in our hands (data not shown). As we obtain considerable
amounts of SARP in extracts of untreated leaves, the most likely
explanation is that SAI is so labile that its activity is readily lost
during the extraction. This is consistent with the finding that we
could also not reproduce the formamide induced increase in SARP
activity, which was interpreted to lead to the dissociation of the
SARP·SAI complex (14). TGA2.2 may still be a good bait to isolate SAI
in a yeast two-hybrid system.
A TGA2.1-related Activity Is Present in Minor Amounts in ASF-1 and
SARP--
Only minor amounts of ASF-1 (10%) or SARP (20%) were
recognized by the anti-TGA2.1 serum. As the respective protein is
significantly smaller than the recombinant TGA2.1 protein, it is
presently unclear whether it represents a different immunologically
related protein, a processed form of TGA2.1, or a degradation product
of TGA2.1. If this activity would be represented in SARP as a
homodimer, it should represent ~20% of all TGA factors in the
Western blot analysis. As the band is considerably weaker, we assume
that the TGA2.1-related activity is represented as a heterodimer,
either with TGA2.2 or with one of the residual factors present in the complex. No TGA1a was detected in either ASF-1 or SARP. Similar studies
have been done before in A. thaliana (44). 50% of A. thaliana ASF-1 are supershifted by the anti-TGA2 serum, with 50% of the complex being unaffected. No detectable traces of TGA1 and TGA3
were found. We emphasize that this type of analysis describes only the
relative abundance of TGA factors binding to the as-1 element in vitro. We cannot exclude that additional TGA
factors with reduced or different DNA binding affinities might be
present in planta.
Plants with Increased or Decreased SARP Levels Were Generated by
Overexpression of TGA2.2 and a Dominant Negative TGA2.2
Mutant--
Expression of TGA2.2 or a dominant negative TGA2.2 mutant
led to increased and decreased SARP levels, respectively. The
activities of four different SA-inducible promoters containing
as-1-like elements were analyzed in these plants. Three of
these promoters respond with identical kinetics both to SA and 2,4D as
the isolated as-1 element (Fig. 6), suggesting that they are
subject to the same control mechanism. The analysis of transgenic
plants expressing either TGA2.2 or a dominant negative TGA2.2 mutant
served to investigate the following questions. 1) Is TGA2.2 limiting
for the expression of endogenous plant genes encoding the
as-1 element? 2) Does overexpression of TGA2.2 lead to
constitutive expression of target genes, e.g. by
outcompeting the postulated inhibitory factor SAI? 3) Do SA- and
2,4D-activated signal transduction chains activate TGA2.2?
Analysis of SA-inducible Gene Expression in Transgenic Plants
Expressing TGA2.2 or a Dominant Negative TGA2.2 Mutant--
Out of the
four potential target promoters analyzed, the Nt103 and the
IEGT promoter were clearly affected by both TGA2.2 alleles.
Overexpression of TGA2.2 led to a 3-4-fold higher SA inducibility.
This indicates that TGA2.2 can be activated by SA and that it helps to
activate the promoter either as a homodimer or a heterodimer. Thus, the
answer to question 1 asked above is that TGA2.2 can be limiting for the
expression of endogenous plant genes. No significant increase in
uninduced or mock-induced samples was observed, answering question 2 asked above. In transgenic plants expressing the dominant negative
TGA2.2 derivative, gene expression was significantly reduced
(5-10-fold). This indicates that factors heterodimerizing with TGA2.2,
i.e. endogenous TGA2.2 and/or other members of the TGA
family, are indeed involved in gene expression of these defense genes.
Alternatively, other regulatory components might bind to the
nonfunctional TGA dimer becoming thus limiting for the events happening
at the promoter. At this point, we cannot decide, whether the observed
effects are due to direct binding of TGA2.2 to the Nt103 and
the IEGT promoter, or due to binding of TGA2.2 to a promoter
driving a second trans factor. We consider the latter
possibility to be less likely, because Nt103 expression does
not require de novo protein biosynthesis and TGA2.2 would
thus have to induce this hypothetical trans factor in the
absence of SA.
Increased TGA2.2 levels did not modify the expression of
ParA or PR-1a in response to SA. In contrast,
expression of TGA2.2trd led to decreased transcription from these
promoters, supporting the importance of this family of transcription
factors for SA-inducible transcription. As these promoters display
stronger activities than the promoters driving Nt103 and
IEGT, other factors might become limiting to gain a further
increase upon overexpression of TGA2.2. Alternatively, a different TGA
factor might activate these promoters. In the case of the
PR-1a promoter, an indirect effect must be discussed, as
activation depends on de novo protein synthesis. In this
case, reduced levels of functional TGA proteins might not only affect
the promoter through the PR-1a-encoded as-1 element, but might also influence the expression of a primary response
component, which must be synthesized a priori to obtain maximal promoter activity.
Analysis of 2,4D-inducible Gene Expression in Transgenic Plants
Expressing TGA2.2 or a Dominant Negative TGA2.2
Mutant--
as-1-like elements have also been called
multiple stimuli-responsive elements, as they are responsive to other
chemical stimuli like 2,4D, several heavy metals, and methyljasmonate
(50). Upon 2,4D treatment, Nt103 levels were induced more
strongly in TGA2.2-overexpressing plants as compared with wild-type
plants. This implies that TGA2.2 can also be activated by 2,4D and
answers question 3 asked above. In TGA2.2trd-expressing plants,
Nt103 and IEGT were to a lesser extent
2,4D-inducible than in wild-type plants. The observed effects of the
transgene were less pronounced than upon SA treatment. Several
explanations for the lower sensitivity of these promoters to changing
TGA levels after 2,4D treatment in comparison to SA treatment can be
envisioned. 1) These promoters might be occupied by a different
combination of non-TGA-related trans-factors upon 2,4D
treatment than upon SA treatment. This might enhance the affinity of
TGA factors to these promoters, making them less responsive to altered
levels of functional TGA factors. Indeed, promoter activities are
higher upon 2,4D treatment than upon SA treatment, supporting the
notion that both responses are modulated by different trans-acting factors interacting with TGA factors. 2) A
different heterodimer between TGA2.2 and another TGA factor might be
responsible for 2,4D induction as compared with SA induction. As
outlined above, SARP has been characterized as an in vitro
as-1 binding activity, which selects for the most stable complex.
It might well be that a different combination of TGA factors assembles on different promoters upon different stimuli in vivo. In
this case, we would have to postulate that TGA2.2trd heterodimerizes less efficiently with this factor as compared with the situation upon
SA induction. 3) Other limiting factors allow only a 2-fold (Nt103) or no (IEGT) increase in gene expression.
Any of these explanations might also be relevant for the failure of the
transgenes to alter 2,4D-induced expression from the ParA
promoter. TGA factors may even be dispensable for ParA.
In conclusion, the composition of SARP as well as the analysis of
transgenic plants expressing TGA2.2 or TGA2.2trd suggests that TGA2.2
is involved in activating target promoters encoding an as-1
element. However, TGA2.2 does not encode a potential activation domain,
as shown by its heterologous expression in yeast (25). Thus, we favor
the hypothesis that TGA2.2 acts as in combination with different
members of the TGA family, one candidate being TGA2.1.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Achim Gau (University of
Hannover) for generating the antibody against the basic domain, Dr.
Radunz (University of Bielefeld) for immunization of rabbits, Dr.
Ursula Pfitzner (University of Hohenheim) for the TGA1a expression
vector, Ingo Lenk for providing the ParA probe, and
Alexandra Frey for excellent technical assistance.
| |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinshaft
Grant GA-330/3-2 and a personal fellowship from the Fonds der
Chemischen Industrie (to R. N.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-551-39-7843;
Fax: 49-551-39-7820; E-mail: cgatz@gwdg.de.
Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M909267199
1
The abbreviations are SAR, systemic acquired
resistance; PR, pathogenesis-related; SA, salicylic acid;
as-1, activating sequence-1; bZIP, basic/leucine zipper;
EMSA, electrophoretic mobility shift assay; ASF-1,
as-1-binding factor; SARP, salicylic acid response protein;
BY, Bright Yellow; PAGE, polyacrylamide gel electrophoresis; HBA,
3-hydroxybenzoic acid; 2,4D, 2,4-dichlorophenylacetic acid; 2,6D,
2,6-dichlorophenylacetic acid; GBF, G-box binding factor; IEGT, immediate early glycosyltransferase gene;
ParA, protoplast auxin-regulated gene A, Gus,
-glucuronidase gene; PBS, phosphate-buffered saline; PBS-T,
phosphate-buffered saline with Tween 20; bp, base pair(s); PCR,
polymerase chain reaction.
| |
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