Báo cáo khoa học: Salicylic acid and the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element of the PR-1a promoter pot

Salicylic acid and the hypersensitive response initiate distinct signal
transduction pathways in tobacco that converge on the
as-1
-like
element of the
PR-1a
promoter
Rose Gru¨ ner*, Georg Strompen†, Artur J. P. Pfitzner and Ursula M. Pfitzner
Universita
¨
t Hohenheim, Institut fu
¨
r Genetik, FG Allgemeine Virologie, Stuttgart, Germany
Tobacco pathogenesis-related protein 1a (PR-1a) is induced
in plants during the hypersensitive response (HR) after
exposure of plants to salicylic acid (SA) and by develop-
mental cues. Gene activation by these diverse stimuli is
mediated via an as-1-like element in the PR-1a upstream
region. To further analyze the significance of this cis-acting
sequence, an authentic as-1 element from the cauliflower
mosaic virus 35S RNA promoter was inserted into the
PR-1a promoter in place of the as-1-like motif. Reporter
gene analysis in transgenic tobacco plants demonstrated that
as-1 can functionally replace the as-1-like element in the
PR-1a promoter in response to all stimuli. However,
reporter gene induction from the as-1 carrying promoter was
enhanced in response to SA compared to the wild-type
promoter, and the ratio of reporter gene activities in SA
treated leaf tissue to tissue exhibiting the HR increased with
the as-1 promoter construct. Our findings support a model
where PR-1a gene expression relies on at least two distinct

signal transduction pathways initiated by SA and by a yet
unknown signal produced during the HR, that promote
different, albeit related, transcription complexes on the
PR-1a as-1-like element. Analysis of PR-1 proteins in plants
expressing salicylate hydroxylase yielded additional evidence
that an HR dependent pathway leads to high level PR-1 gene
induction in tobacco.
Keywords: GUS reporter gene expression; PR-1 protein
induction; salicylate hydroxylase; transgenic tobacco plants.
Infection of plants with pathogens usually results in a
distinct host response depending on the genetic constitution
of the plant and the pathogen. In an incompatible inter-
action, the pathogen remains localized at the primary
infection sites that often are visible as necrotic local lesions
on the leaves. This local defense reaction is referred to as the
hypersensitive response (HR). Subsequently, the HR trig-
gers a general resistance mechanism rendering uninfected
parts of the plant less sensitive to further attack by
pathogens, a phenomenon called systemic acquired resist-
ance (SAR) [1]. The elicitation of the HR and SAR
reactions is accompanied by the coordinated induction of a
heterogeneous group of proteins in the infected and
uninfected leaves, commonly referred to as pathogenesis-
related (PR) proteins.
PR proteins were first described in tobacco plants infected
with Tobacco mosaic virus (TMV) exhibiting the HR [2,3].
By now, related proteins have been identified across the
plant kingdom in both dicotyledonous and monocotyled-
onous species. In tobacco, seven families of PR proteins are
known [4]. Yet, the biological functions of PR proteins are

not clear. It is intriguing that PR proteins are also expressed
in substantial amounts in healthy plants upon the transition
to flowering [5–7], suggesting that they play a role during
plant development.
Although not defined clearly by their function, the
expression of PR genes has served as a reliable marker for
the induction of SAR [8–11]. Therefore, PR genes are also
referred to as SAR genes [8]. To identify components of the
complex defense signaling pathways, several groups have
studied the regulation of PR gene expression. Initially, PR-1
proteins were found to be inducible to high levels by the
exogenous application of salicylic acid (SA) in healthy
tobacco plants [12]. Consistent with this finding, tissue levels
of SA have been demonstrated to increase significantly
locally and systemically in plants displaying the HR [13–15].
Furthermore, plants that are inhibited in SA accumulation
show defects in SAR and in the expression of PR genes
[16–19]. It was thus hypothesized that SA is an endogenous
regulator of pathogen resistance and of PR gene expression
[20]. Subsequently, some PR genes were found to be
responsive to agents other than SA. From these findings and
from the study of Arabidopsis mutants exhibiting aberrant
SAR expression patterns, it appears that different pathways
encompassing SA or ethylene/jasmonic acid as signal
molecules can lead to the induction of PR genes and SAR
in plants infected by pathogens [21].
Correspondence to U. M. Pfitzner, Universita
¨
t Hohenheim, Institut fu
¨

r
Genetik, FG Allgemeine Virologie, Emil-Wolff-Str. 14,
D-70599 Stuttgart, Germany.
Fax: + 49 711459 2937, Tel.: + 49 711459 2395,
E-mail: pfi
Abbreviations: HR, hypersensitive response; PR, pathogenesis-related;
SA, salicylic acid; SAH, salicylic acid hydroxylase; SAR, systemic
acquired resistance; TMV, Tobacco mosaic virus.
*Present address: Baxter Deutschland GmbH, Edisonstr. 3–4,
D-85716 Mu
¨
nchen-Unterschleißheim, Germany.
Present address:Universita
¨
tTu
¨
bingen, ZMBP, Entwicklungsgenetik,
Auf der Morgenstelle 3, D-72076 Tu
¨
bingen, Germany.
(Received 29 July 2003, revised 9 October 2003,
accepted 22 October 2003)
Eur. J. Biochem. 270, 4876–4886 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03888.x
To dissect the functional architectures of PR gene
promoters responsive to different signal molecules, repor-
ter gene constructs were generated and tested for regulated
gene expression. In addition, in vivo and in vitro studies
were performed to unravel interactions of nuclear proteins
with DNA sequences in the promoter regions of PR
genes. By these means, diverse sequence elements and

their cognate binding proteins have been identified in
accordance with the view that PR genes are regulated
differently. In the )906 bp promoter region of the tobacco
gene encoding the acidic PR-1a protein, a duplicated
TGACG motif has been shown to control reporter gene
expression in transgenic plants in response to SA, to the
HR, and to developmental stimuli [22]. This element is
referred to as as-1-like motif because of its relatedness to
the as-1 element. The as-1 element was originally identi-
fied in the 35S RNA promoter from cauliflower mosaic
virus(CaMV)[23],andhasbeenshowntomediatea
moderate induction of the 35S RNA promoter by SA
[24]. Transcription factors belonging to the TGA family of
basic leucine zipper proteins interact in vitro with as-1
[23,25], and, similarly, with the PR-1a as-1-like motif [22].
Likewise, a related cis-acting element involved in SA
responsive reporter gene expression has been identified in
the homologous Arabidopsis PR-1 promoter by functional
analyses in transgenic plants [26]. The Arabidopsis cis-
acting element comprises the sequence TGACG, which is
directly repeated only 12 bp upstream of the identified
promoter element. TGA transcription factors have been
showntobindtothismotifin vitro [27–29], and an
inducible in vivo footprint has revealed significant changes
in DNA accessibility to the identified element upon SAR
induction [26]. In addition, overexpression of trans-
dominant TGA mutant proteins, which are no longer
able to bind to their target sequences, has resulted in
reduced accumulation of chemically inducible PR-1
mRNAs in transgenic tobacco and Arabidopsis plants

[30,31]. Thus, as-1-like elements and TGA transcription
factors seem to be intimately connected with the expres-
sion of PR-1 genes in both tobacco and Arabidopsis. This
association is even more supported by the finding that
TGA factors from Arabidopsis, tobacco and rice have
been shown to interact in vivo and in vitro with NPR1/
NIM1 [27–29,32,33]. NPR1/NIM1 has been identified as a
key regulator of SAR in Arabidopsis acting downstream
of SA in the SAR signaling pathway [10,11,21], and
analysis of npr1 mutant plants has established that
efficient expression of the Arabidopsis PR-1 gene after
SAR induction relies on a functional NPR1/NIM1 gene
[10,11]. Furthermore, it has been demonstrated recently
that Arabidopsis TGA2 acts as a transcriptional activator
in transgenic plants in response to SA, and that this
activity is abolished in the npr1 mutant [31]. Taken
together, substantial evidence has accumulated suggesting
a prominent role for as-1-type elements and TGA
transcription factors in the regulated expression of some
PR genes.
Here we report that the level of SA inducible gene
expression is controlled by the as-1-like element in the
strong )1533 bp PR-1a promoter from tobacco. To further
analyze the functional significance of as-1-like elements and
their binding factors in the induction of PR genes, we have
modified the PR-1a promoter to contain an authentic as-1
element. As-1 can functionally replace the as-1-like element
in the PR-1a promoter. Substitution by as-1 enhances SA
inducible reporter gene expression from the promoter by a
factor of 3 in transgenic plants, and the ratio of inducible

reporter gene expression from the as-1 containing promoter
inSAtreatedtissuetonecrotictissueinfectedwithTMVis
increased compared to the wild-type promoter. These
findings emphasize that as-1 related elements and their
binding factors play crucial roles in the regulation of the
tobacco PR-1a gene. Furthermore, our results demonstrate
that the properties of the PR-1a promoter are subject to
change by insertion of a variant as-1 element. The data are
in accord with the conclusion that the tobacco PR-1a
promoter is targeted by at least two distinct signal trans-
duction pathways, elicited by SA and by a yet unknown
signal produced during the HR, respectively, which mediate
PR-1a gene activation through a common DNA sequence,
the as-1-like element.
Experimental procedures
Construction of plasmids containing reporter genes
Recombinant DNA techniques were performed according
to standard procedures [34].
For the construction of the PR-1 reporter gene, a 0.27 kb
StyI/PstI fragment from kW38-1, comprising parts of the 5¢
region and the open reading frame encoded by the W38-1
gene [35], was inserted into StyI/PstI cleaved pT5S/HDP
containing the PR-1a gene. The resulting plasmid was
linearized with EcoRV, which cuts in the PR-1a 3¢ untrans-
lated region 56 bp 3¢ to the stop codon, and a SacI linker was
added to the blunt ends. The W38-1::PR-1a chimeric gene
was excised from the plasmid as StyI/SacI fragment and
inserted into p-1533PR1a[GUS] [7], from which the GUS
reporter gene had been removed as a StyI/SacIfragment.
The )1533as-1m4[GUS] construct was generated by the

addition of the 0.64 kb HindIII/XhoI fragment from
p-1533PR1a[GUS] to the 5¢ end of p-906as-1m4[GUS] [22].
The as-1m4 mutation replaces the as-1-like element occuring
from positions )592 to )577 in the PR-1a promoter.
To obtain construct )906as-1[GUS], site-directed muta-
genesis was employed on p-906PR1a encompassing the
PR-1a sequence from )906 to +28. The DNA was primed
with PR-46 (5¢-TGACGTTAA
CTAACTAT-3¢) containing
an A to C nucleotide substitution with respect to the wild-
type promoter (underlined). By this procedure, a unique
HpaI restriction enzyme site (bold) was introduced in
p-906PR1a at position )597 just upstream of the as-1-like
motif (from )592 to )577). The mutant plasmid was
digested with HpaIandBamHI and ligated to a 0.6 kb
HpaI/BamHI fragment obtained from p-906PR1a by PCR
amplification with a pUC universal primer and PR-47
(5¢-CAAGCTTGTTAACTGACGTAAGGGATGACGGC
CATGTTCAAGTTT-3¢), which contains an authentic as-1
element as found in the CaMV 35S RNA promoter (in bold
letters). The as-1 element was inserted at position )591 in
the PR-1a promoter. The as-1 containing )906 bp PR-1a
promoter region was added as HindIII/BamHI fragment
to p0[GUS] to give plasmid )906as-1[GUS]. To obtain
p-1533as-1[GUS], the 0.64 kb HindIII/XhoIfragmentfrom
Ó FEBS 2003 Analysis of PR-1a gene regulation (Eur. J. Biochem. 270) 4877
p-1533PR1a[GUS] was ligated to HindIII/XhoIcleaved
p-906as-1[GUS].
All plasmids generated by site-directed mutagenesis or
PCR amplification of fragments were verified by DNA

sequence analysis.
Construction of an expression vector containing
the
nahG
gene from
Pseudomonas putida
P. putida PpG7 containing plasmid NAH7 (DSM 4476)
was obtained from the German Collection of Microorgan-
isms and Cell Cultures, Braunschweig, and grown in
mineral medium with the addition of 0.05% sodium
salicylate according to the instructions of the supplier. In
order to isolate the nahG gene, two primers were designed.
Primer nahG-1 (5¢-TTCCCGGGGCCATCACGAGTA
CAGCATGA-3¢) is identical to the 5¢ end of the nahG gene
including the ATG translation start codon (bold) with an
additional SmaI restriction enzyme site at its 5¢ end. Primer
nahG-2 (5¢-AAGAGCTCGTCAACGTTGTCACCCTT
G-3¢) is complementary to the 3¢ end of the nahG gene
including the translation stop codon (bold) and carries a
SacI restriction enzyme site at its 5¢ end. The primers were
used in standard PCR amplifications on plasmid DNA
isolated from P. putida by the alkaline lysis procedure. A
single reaction product of 1.3 kb was detected on ethidium
bromide stained agarose gels. The PCR product was
subcloned into pUC19 cleaved with SmaIandSacIand
sequenced over its entire length. The nucleotide sequence
determined was found to be identical to the sequence of the
nahG gene described previously [36]. For fusion of the nahG
gene to the CaMV 35S RNA promoter, the 1.3 kb SmaI/
SacI fragment was integrated into pBin19/35S[GUS] [37]

from which the GUS reporter gene had been removed. The
resulting plasmid was designated pBin19/35S[nahG].
Generation and analysis of transgenic plants
Constructs for expression in plants were integrated into the
binary vector pBin19, and Agrobacterium-mediated trans-
formation was employed to introduce gene constructs into
the genome of tobacco (Nicotiana tabacum L. cv. Samsun
NN) as described previously [22]. Primary transformants
were allowed to self-fertilize, and progeny plants were
selected on medium containing 400 mgÆL
)1
kanamycin.
For the induction of reporter gene constructs and of the
endogenous PR-1 proteins, eight leaf disks of 1 cm diameter
were cut from at least two different fully expanded leaves
of each transgenic plant. The leaf disks were floated for
72–96 h in a Petri dish on water or on neutral solutions of
1m
M
or 5 m
M
SA as indicated. From plants which were
tested for gene induction by application of SA as well as
during the HR, leaf disks for chemical treatment were
collected from a single leaf which was cut from the plant just
prior to virus inoculation. Infection of tobacco plants with
TMV has been described previously [22]. Eight leaf disks
were collected from necrotic tissue of transgenic plants
5–7 days after virus inoculation. For the analysis of PR-1
gene expression in nahG transgenic plants, two zones of leaf

material, a narrow zone including the lesion and a more
distant zone, were excised from the leaves around single
lesions with two different cork borers of 7 and 14 mm
diameter, respectively. Leaf disks were extracted with GUS
lysis buffer. Analysis of GUS reporter gene expression and
immunodetection of PR-1 proteins was conducted as
reported [22,37]. For monitoring the expression of the
PR-1 reporter gene, protein extracts were separated by
gradient gel electrophoresis. GUS activity is given in units
(1 unit ¼ 1nmolof4MUÆh
)1
per mg protein).
To verify the correct insertion of the as-1[GUS] con-
structs in transgenic plants exhibiting high GUS reporter
enzyme induction, genomic DNAs were isolated from
transformants according to Fulton et al. [38]. Standard
PCR was performed on the genomic DNAs using PR-19,
identical to the PR-1a promoter, and a GUS primer
complementary to the 5¢ end of the reporter gene. PCR
products were cut with HpaI which cleaves only in the as-1
containing promoter regions, but not in the PR-1a wild-type
promoter. Reaction products were analyzed on ethidium
bromide stained agarose gels.
Results
The )1533 bp
PR-1a
promoter directs high level
induction of a PR-1 reporter protein in transgenic
tobacco plants
Functional analysis of the tobacco PR-1a promoter by

several groups had demonstrated that the 0.9 kb upstream
region of the PR-1a gene is sufficient to yield regulated
reporter gene expression in transgenic tobacco plants
[7,39,40]. Yet, gene induction levels from the 0.9 kb PR-1a
promoter are significantly lower (average fold induction of
GUS reporter gene activity < 75) than induction of the
endogenous PR-1a gene (?100-fold). Previously, we have
shown that a longer PR-1a upstream region, the )1533 bp
promoter, is able to direct higher levels of GUS reporter
gene expression in transgenic plants [7]. To assess the
strength of the )1533 bp PR-1a promoter region in direct
comparison to the expression of the endogenous PR-1
genes, we have fused a PR-1 reporter gene to the )1533 bp
promoter and monitored expression of the reporter protein
in transgenic tobacco plants.
The PR-1 reporter gene is a chimera constructed in vitro
by the replacement of a 0.27 kb StyI/PstI fragment in the
PR-1a gene with the respective fragment from the W38-1
pseudogene [35]. The open reading frame of the chimeric
PR-1 gene, designated W38-1::PR-1a, is colinear with the
open reading frame encoded by the PR-1a gene, and differs
by only six amino acids from the PR-1a preprotein in the
N-terminal moiety of the protein. Most importantly, the
W38-1 pseudogene part of the W38-1::PR-1a chimeric gene
contains an amino acid exchange close to the signal peptide
cleavage site (Gly vs. Arg in the PR-1a preprotein at positon
)2 of the signal peptide), thus giving rise to an alternatively
processed mature protein in plants, which can be distin-
guished through its smaller size from the naturally occurring
PR-1a, PR-1b and PR-1c proteins by SDS gel electrophor-

esis (Fig. 1 and data not shown). When put under control of
the CaMV 35S RNA promoter in transgenic tobacco
plants, the chimeric W38-1::PR-1a gene is equally well
expressed as a similarly constructed 35S[PR-1a] transgene
(Fig. 1 and data not shown). Tobacco plants transformed
with the )1533PR1a[W38-1::PR-1a] construct were infected
4878 R. Gru
¨
ner et al.(Eur. J. Biochem. 270) Ó FEBS 2003
with TMV, and protein extracts from independent trans-
formants were monitored prior to and after infection for
expression of the endogenous PR-1 proteins and the W38-
1::PR-1a reporter protein by immunodetection. As shown
in Fig. 1, two bands were observed consistently with
extracts from transformants exhibiting the HR after virus
infection. The upper, more prominent band corresponds to
the endogenous acidic PR-1 proteins, of which the PR-1a
protein constitutes approximately 50% (data not shown).
The lower band corresponds to the recombinant W38-
1::PR-1a protein. Although expressed weaker than the
endogenous PR-1a protein (approximately 5-fold less than
PR-1a), the recombinant W38-1::PR-1a protein is clearly
detected in all extracts containing considerable amounts of
the endogenous PR-1 proteins, thus demonstrating that the
)1533 bp PR-1a upstream region represents a promoter
that encompasses important cis-acting elements for both
regulated as well as high level expression of the PR-1a gene.
The
as-1
-like element controls the level of SA inducible

gene expression from the strong )1533 bp
PR-1a
promoter in transgenic tobacco plants
Previously, we have shown that an as-1-like motif, located
from positions )592 to )577 in the PR-1a upstream region,
contributes significantly to the level of reporter gene expres-
sion from the weaker )906 bp PR-1a promoter [22]. To
analyze the significance of the as-1-like motif for expression
from the strong )1533 bp PR-1a promoter, we have
introduced the as-1m4 mutation, which completely destroys
the as-1-like motif [22], in the )1533 bp PR-1a upstream
region to give construct )1533as-1m4[GUS] (Fig. 2A).
Tobacco plants were transformed in parallel with
)1533PR1a[GUS] or )1533as-1m4[GUS], and transform-
ants were monitored for inducible reporter gene expression
after floating of leaf disks on H
2
O or on a solution of 1 m
M
SA. As seen previously with the )906 bp PR-1a promoter,
insertion of the as-1m4 mutation drastically reduced reporter
gene expression from the strong )1533 bp promoter region
(Fig. 2B). On the other hand, induction of GUS activity by
SA was still preserved with the mutant promoter, albeit at a
lower level (Fig. 2B), just as observed previously for the
)906as-1m4 mutant PR-1a promoter [22]. Thus, the as-1-like
motif represents an important cis-acting element controlling
the level of expression from both the weaker )906 and the
strong )1533 bp PR-1a promoter regions in a similar way.
Replacement of the

as-1
-like element by
as-1
enhances
SA inducible and developmentally controlled reporter
gene expression from the
PR-1a
promoter in transgenic
tobacco plants
To address the question whether the PR-1a as-1-like motif is
a genuine as-1-type element, possibly targeted by the same
transcription factor(s) as as-1, we have introduced an
authentic as-1 element in place of the as-1-like motif in the
)906 and )1533 bp PR-1a promoter regions. The resulting
reporter gene constructs were designated )906as-1[GUS]
and )1533as-1[GUS], respectively (Fig. 3A). Tobacco plants
were transformed in parallel with )906as-1[GUS], )1533as-
1[GUS], or the respective wild-type promoter constructs, and
transformants were monitored for reporter gene expression
after treatment of leaf disks with H
2
Oorasolutionof1 m
M
SA. As shown in Fig. 3B, inducibility of GUS activity was
maintained with anyplant containing either of the as-1[GUS]
transgenes. Surprisingly, plant extracts from transformants
with the as-1[GUS] constructs yielded on average threefold
Fig. 2. Effect of mutation of the as-1-like element on SA inducible
reporter gene expression from the -1533 bp PR-1a promoter in trans-
genic tobacco plants. (A) Sequences of the wild-type and the as-1m4

containing promoter regions in the GUS reporter gene constructs. (B)
Functional promoter analysis in transgenic plants. Tobacco plants
were transformed with constructs )1533PR1a[GUS] or )1533as-
1m4[GUS]. Leaf disks were cut from grown-up independent trans-
formants (10 plants for each construct), and floated for 3 days on
wateroron1m
M
SA. Protein extracts from leaf disks were measured
for GUS activities. Average GUS activities in water and in SA treated
leaf disks and average reporter gene induction were calculated for each
construct. Average reporter gene induction is the ratio of average GUS
activities in SA to water treated leaf disks.
Fig. 1. Expression of a PR-1 reporter protein under control of the
-1533 bp PR-1a promoter in transgenic tobacco plants. Independent
transformants with the )1533PR1a[W38-1::PR-1a] chimeric gene
(lanes 3–6) were inoculated with Tobacco mosaic virus (TMV). Pro-
teins were isolated from plants prior to (–) and 5 days after infection
(+). Extracts were analyzed for expression of the endogenous and the
recombinant PR-1 proteins by immunodetection after electrophoretic
separation of protein extracts. Lanes 1 and 2 contain equal amounts of
protein isolated from transgenic plants expressing the PR-1a or the
W38-1::PR-1a chimeric gene under control of the CaMV 35S RNA
promoter.
Ó FEBS 2003 Analysis of PR-1a gene regulation (Eur. J. Biochem. 270) 4879
higher GUS activities than extracts from plants containing
the respective wild-type promoter constructs (Fig. 3B). To
ensure that an as-1 element in the context of the PR-1a
promoter is indeed able to mediate higher average reporter
gene induction in transformed plants, we have isolated
genomic DNAs from four plants each exhibiting the highest

GUS activities from the transformations with the )906 and
)1533as-1[GUS] constructs. A 1.0-kb fragment was ampli-
fied from the isolated DNAs by PCR, and the PCR products
were cleaved with HpaI (Fig. 3A). In all cases, the highest
GUS activities measured in transformed plants correlated
with the presence of a HpaI restriction endonuclease site
occurring at position )597 only in the )906as-1 and )1533
as-1 sequences, but not in the wild-type promoter regions
(data not shown).
Likewise, GUS reporter gene expression was monitored
in transgenic plants during normal development, as it has
been shown that the PR-1a promoter responds not only to
environmental, but also to developmental signals [7]. Again,
higher levels of GUS activity were observed consistently
with transformants containing the as-1[GUS] transgenes
and not with the wild-type promoter constructs (data not
shown). Taken together, these results demonstrate that as-1
can functionally replace the as-1-like motif in the tobacco
PR-1a promoter in planta.
Replacement of the
as-1
-like element by
as-1
reduces
the ratio of reporter gene activities in necrotic tissue
exhibiting the HR to SA treated tissue
To analyze the effect of an as-1 containing PR-1a promoter
on gene induction during the HR, the selfed progeny from
two independent primary transformants with the )1533as-
1[GUS] construct, 313–6 and 313–7, were selected on MS

medium in the presence of kanamycin. Transformant 313–6
had displayed an intermediate reporter gene expression in
the initial SA induction experiment shown in Fig. 3B,
whereas transformant 313–7 had exhibited high responsive-
ness to SA. Resistant seedlings were transferred to soil. In
parallel, seedlings containing wild-type PR-1a promoter
constructs were selected. At the six-leaf stage, leaf disks were
cut from the plants and floated on H
2
O or a solution of
1m
M
SA. Immediately afterwards the same plants were
infected with TMV. GUS reporter gene expression was
determined in protein extracts isolated from leaf disks after
4 days of chemical treatment or 7 days after virus inocu-
lation. As shown in Fig. 4A, reporter gene induction was
considerably stronger in response to TMV infection in
comparison to chemical treatment in transformants con-
taining the )906 or the )1533 bp PR-1a wild-type promoter
constructs (9.2- and 14.3-fold higher GUS activities,
respectively, after TMV infection). In contrast, transform-
ants containing the )1533as-1[GUS] transgene consistently
exhibited higher levels of SA inducible GUS expression
compared to reporter gene induction measured in TMV
infected plants exhibiting the HR. TMV infection of
transformants 313–6 and 313–7 produced only 2.7- to 4.8-
fold stronger GUS activities than chemical treatment
(Fig. 4A). To account for possible errors in GUS activities
between individual plant extracts due to experimental

variations, GUS enzyme extracts were also analyzed
for the expression of the endogenous PR-1 proteins by
immunodetection. In all cases, induction of the endogenous
PR-1 proteins was significantly stronger in virus infected
Fig. 3. Effect of replacement of the as-1-like element by as-1 on SA inducible reporter gene expression from the PR-1a promoter in transgenic tobacco
plants. (A) Sequences of the wild-type and the as-1 containing promoter regions in the GUS reporter gene constructs. A HpaI restriction
endonuclease site, which was generated by a single nucleotide exchange for DNA manipulation, is underlined in the as-1 containing promoter
region. (B) Functional promoter analysis in transgenic plants. Tobacco plants were transformed with constructs )906as-1[GUS], )1533as-1[GUS],
or the respective wild-type promoter constructs. Leaf disks were cut from grown-up independent transformants (10 plants for each construct), and
floatedfor3 daysonwateroron1m
M
SA. Protein extracts from leaf disks were measured for GUS activities. Average GUS activities in water and
in SA treated leaf disks and average reporter gene induction were calculated for each construct. Average reporter gene induction is the ratio of
average GUS activities in SA to water treated leaf disks.
4880 R. Gru
¨
ner et al.(Eur. J. Biochem. 270) Ó FEBS 2003
tissue than in leaf tissue subjected to SA (Fig. 4B). These
results demonstrate that GUS reporter gene induction in
plant lines with the )906 and )1533 bp PR-1a wild-type
promoter constructs reflects induction of the endogenous
PR-1 proteins by the different stimuli. Therefore, as-1
enhances gene expression from the PR-1a promoter in
response to SA, but it does not seem to markedly affect PR-
1a gene induction during the HR.
PR-1 proteins are induced to high levels in necrotic tissue
from tobacco plants expressing the
nahG
gene from
P. putida

It is envisaged that PR-1 gene induction in local and
systemic tissues of infected plants is mediated by SA
produced during the course of the HR [20,21,41]. However,
our finding showing differential inducibility of an as-1
carrying PR-1a promoter in comparison to the wild-type
promoter in response to SA vs. the HR, would favor an
alternative explanation. In view of the results presented, it
seems plausible that two distinct signal transduction path-
ways could be targeted by SA and by signals released during
the HR, respectively, resulting in the independent activation
of different transcription complexes. These complexes, in
turn, could interact differentially with the wild-type and the
as-1 mutant promoter regions, thus leading to differential
PR-1a gene activation in response to the different stimuli.
To challenge this model, we have made use of tobacco
plants expressing the P. putida nahG gene which codes for
salicylate hydroxylase (SAH). Previously, it has been
reported that expression of the bacterial gene in tobacco
leads to drastically reduced levels of endogenous SA during
the plant defense response [16,19].
The nahG gene from P. putida was cloned in a plant
expression vector under control of the CaMV 35S RNA
promoter and several independent transgenic lines (N. t. cv.
Samsun NN) were generated via Agrobacterium-mediated
transformation. Primary transformants were analyzed for
the activity of SAH by infection with TMV. In wild-type
plants, virus infection induces a local necrotic reaction
which remains restricted to the primary infection site. On
the contrary, six plants out of 9 regenerants transformed
with the nahG expression vector exhibited enlarged local

lesions as depicted in Fig. 5. Typically, lesions had a light-
brown center surrounded by two distinct necrotic regions
each bounded by a thinner dark-brown margin at 14 days
post-infection (Fig. 5C). In contrast, the center of local
lesions on wild-type plants was surrounded by only a single
necrotic region bounded by a dark-brown margin.
This phenotype has been described previously for TMV
infected tobacco plants that were severely depleted from
endogenous SA by the expression of the nahG gene
[16,17,19]. To further demonstrate the enzymatic activity
of SAH, progeny plants from primary transformants, that
were no longer able to restrict lesion growth, were monit-
ored for endogenous SA levels and for their ability to induce
PR-1 proteins after treatment with SA. The amount of total
SA (free SA plus glucosylated SA) reached levels of 7199 ng
per gram fresh weight in TMV infected control leaves (N. t.
cv. Samsun NN), which represents a 90-fold induction over
SA levels in noninfected leaves (79.5 ng total SA per gram
fresh weight). On the contrary, total SA levels in inoculated
tissue from nahG transformants of line 201–10 (58 ng per
gram fresh weight) remained even below the levels detected
in noninfected control leaves. Consistent with this result,
Fig. 4. Comparison of gene expression from the wild-type and the
-1533as-1 PR-1a promoter regions in response to SA and during the HR
in transgenic tobacco plants. (A) Quantitative GUS assay of extracts
from SA treated and TMV infected plants. Leaf disks were cut from
progeny plants of transgenic lines containing the )906PR1a, the
)1533PR1a, or the )1533as-1 (two plants each from lines 313–6 and
313–7) promoter constructs and floated on water or on 1 m
M

SA.
Immediately after cutting the leaf disks, the same plants were ino-
culated with TMV. GUS activities were measured in protein extracts
isolated from leaf disks floated on water or SA after 4 days and from
TMV infected plants after 7 days. For a more facile comparison of
reporter gene expression between individual plant extracts, GUS
activities determined in extracts from TMV infected plants were
assigned 100% activity, and GUS activities determined in floated leaf
disks were expressed as percentage of the activities in virus infected
plants. The ratio of reporter gene activities in TMV infected tissue to
SA treated tissue is given for each plant. 100% GUS activity corres-
ponds to 240.0 units for )906PR1a[GUS], 539.9 units for
)1533PR1a[GUS], 261.6 units for 313–6/A,B, and 921.7 units for
313–7/A,B, respectively. (B) Immunodetection of PR-1 proteins in
extracts from SA treated and TMV infected plants. After measuring
GUS activities, plant extracts shown in A were analyzed for the
accumulation of the endogenous PR-1 proteins. Equal amounts of
protein were loaded on the gels from water (0) or SA treated (S) leaf
disks or from TMV infected plants (T).
Ó FEBS 2003 Analysis of PR-1a gene regulation (Eur. J. Biochem. 270) 4881
plants from lines 201–10 and 201–4 were not able to
accumulate PR-1 proteins to appreciable amounts after
floating of leaf disks on H
2
O or on solutions of 1 m
M
or
5m
M
SA, thus demonstrating the activity of SAH in these

plant lines (Fig. 6A).
Subsequently, plants were infected with TMV. After 7
and 14 days, leaf material was collected from a narrow
zone including the lesion and from a more distant zone
around single lesions of each plant. Whereas samples from
the distant zone of control plants never contained necrotic
leaf material, samples collected from the distant zone of
the transformants consistently included necrotic tissue
14 days after virus infection due to lesion expansion. At
14 days post-infection, lesions on nahG expressing plants
from line 201–10 had reached an average diameter of
7.3 ± 0.6 mm and were still growing (Fig. 5B,C), whereas
lesions on wild-type plants remained restricted to
4.2 ± 0.5 mm. Proteins were extracted from the samples
and analyzed for the expression of PR-1 proteins.
Significant amounts of PR-1 proteins were detected in
the samples from the narrow zone around lesions
collected from tissue of nontransformed plants as well
as from nahG transformants (Fig. 6B, lanes 1,3,5,7).
However, PR-1 proteins were never detected in tissue
further away from the center of the necrotic lesions in
control plants (Fig. 6B, lanes 6 and 8), whereas high levels
of PR-1 proteins had accumulated at 14 days post-infec-
tion in the samples from the distant zone around lesions
in nahG expressing plants (Fig. 6B, lane 4). Therefore,
although depleted from endogenous SA and thus inca-
pable to restrict lesion growth, tobacco leaf tissue
undergoing necrosis is able to support efficient induction
of PR-1 proteins.
Fig. 5. Phenotype of local lesions in transgenic tobacco plants containing

a 35S[nahG] chimeric gene. The phenotype of lesions induced by TMV
infection in plant line 201–10 is shown. The bars correspond to 10 mm.
(A) The photograph was taken at 7 days post-infection. At this time
point, lesions on nahG expressing plants are not different in size or
phenotype from lesions on control plants. (B) The same leaf as
depicted in A is shown. The photograph was taken at 14 days post-
infection. (C) Close-up demonstrating discontinuous enlargement of
lesions in nahG transformants. The photograph was taken at
14 days post-infection.
Fig. 6. Expression of PR-1 proteins in transgenic tobacco plants con-
taining a 35S[nahG] chimeric gene. (A) Expression of PR-1 proteins in
SA treated transformants. Leaf disks were cut from wild-type plants
(SNN) or from progeny plants of lines 201–10 or 201–4. Equal
amounts of protein from disks incubated for 3 days on water (0), on
1m
M
(1), or on 5 m
M
SA (5) were analyzed for the accumulation of
PR-1 proteins by immunodetection. (B) Expression of PR-1 proteins in
transformants displaying the HR. A wild-type plant (SNN) and a
progeny plant from line 201–10, which exhibited lesion expansion as
depicted in Fig. 5 and impaired accumulation of PR-1 proteins in
response to SA as depicted in A, were infected with TMV. Leaf sam-
ples were collected 7 and 14 days post-infection from tissue around
single lesions. Leaf material of zone 1 (lanes 1,3,5,7) included necrotic
tissue from the lesions, whereas leaf material of zone 2 was collected in
a region 7–14 mm away from the center of the lesions. Due to the
expansion of local lesions in nahG expressing plants, sample 2 from line
201–10 contained necrotic tissue 14 days post-infection (lane 4), but

not sample 2 from the wild-type plant (lane 8). Equal amounts of
protein were analyzed for the accumulation of PR-1 proteins by
immunodetection.
4882 R. Gru
¨
ner et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Discussion
Reporter gene expression from the tobacco 0.9 kb PR-1a
upstream region has been studied extensively by several
groups [7,39,40]. Although clearly inducible in plants treated
with SA, during the HR, and during adult stages of
development, the 0.9 kb PR-1a upstream region constitutes
a rather variable promoter of only intermediate strength. By
using a PR-1 reporter protein, we here demonstrate that a
longer upstream region, the )1533 bp PR-1a promoter,
yields regulated gene expression on the same order of
magnitude as observed with the endogenous PR-1a gene
(Fig. 1). Likewise, GUS reporter gene expression from the
)1533 bp PR-1a promoter proved to be stronger and more
reliable on average than expression from the 0.9 kb
promoter (Fig. 3) [7]. On the other hand, gene expression
from the )1533 bp PR-1a promoter, as expression from the
short )906 bp PR-1a promoter, is controlled in a similar
waybythesamecis-acting sequence, the as-1-like element
(Fig. 2) [22]. Therefore, the tobacco )1533 bp PR-1a
upstream sequence represents a strong and highly inducible
promoter region whose transcriptional activity largely relies
on the as-1-like element.
To further characterize the significance of the as-1-like
element for gene expression, we have inserted an authentic

as-1 element from the CaMV 35S RNA promoter in place
of the as-1-like sequence in the PR-1a promoter. GUS
reporter gene expression from the as-1 carrying long and
shorter PR-1a upstream sequences was monitored in
transgenic tobacco plants in comparison to the wild-type
promoter regions in response to SA, the HR and develop-
mental cues. Clear induction of gene expression was
retained with the mutant promoter regions towards the
diverse stimuli (Figs 3 and 4 and data not shown). Thus, as-1
from a viral promoter is able to fully replace the as-1-like
element in the tobacco PR-1a promoter. These findings
would imply that the cauliflower mosaic virus, in order to
enable transcription of its genes in planta, has adopted a
cis-acting element from a plant gene active during the
defense response. Furthermore, our results demonstrate
that different as-1 related elements can be functional in the
context of the PR-1a promoter, suggesting that the same or
similar factors are involved in transcription via as-1 and the
as-1-like element in the complex PR-1a upstream region
in planta. Our conclusion is consistent with previous reports
showing that TGA transcription factors, which were
originally isolated via physical interaction with as-1 [25],
bind in vitro to as-1-like elements present in the tobacco PR-
1a and the Arabidopsis PR-1 upstream regions [22,27–29].
Most importantly, however, our data show that reporter
gene induction from an as-1 containing PR-1a promoter is
even stronger on average than reporter gene induction from
the wild-type promoter in SA treated and in mature
untreated plants (Fig. 3 and data not shown; threefold
increase of SA responsive reporter gene expression with the

as-1 upstream region). On the contrary, reporter gene
expression was not markedly affected in plants with the
)1533as-1[GUS] chimeric gene during the HR (Fig. 4).
Therefore, the as-1 containing mutant promoter appears to
be regulated differently from the wild-type promoter in
response to different stimuli. To account for a differential
inducibility of the PR-1a wild-type and the as-1 carrying
mutant promoter regions by diverse stimuli, it seems
plausible to speculate that gene expression from the PR-1a
promoter occurs through different pathways activated by
exogenous application of SA and by signals elicited during
the HR, respectively. Given this case, different, albeit
related, transcription complexes could form on the PR-1a
promoter in response to varying stimuli. These complexes
may include TGA factors to mediate physical association of
alternating transcription complexes with the PR-1a
upstream region via the as-1-like element. In this scenario,
variation of the as-1-like element within the PR-1a promoter
could directly affect gene expression by differential inter-
action of the mutated promoter region with different
transcription complexes.
To seek for support for our model of distinct signal
transduction pathways leading to the independent activa-
tion of the tobacco PR-1a gene by SA and by an unknown
HR released signal, we have made use of transgenic tobacco
plants expressing the nahG gene from P. putida. The nahG
gene codes for salicylate hydroxylase (SAH), which converts
SA to the inactive compound catechol. It has been shown
previously that tobacco plants expressing the nahG gene are
barely able to induce SA levels above the background in

response to TMV infection [16,19]. Curiously, plants
incapable to accumulate SA to normal levels due to nahG
expression exhibit a phenotype of enlarged local lesions
upon infection with avirulent pathogens, whereas wild-type
plants are able to restrict lesion size more rigorously. This
phenotype has been reported for nahG expressing tobacco
and tomato plants after infection with TMV, and for
Arabidopsis plants after infection with avirulent bacterial
and fungal pathogens [16,17,19,42,43]. Our tobacco plants
transformed with a 35S[nahG] chimeric gene displayed the
expected phenotype of enlarged local lesions after TMV
infection. Once formed, lesions kept on expanding for
several weeks and typically reached diameters of up to
22 mm 8 weeks after virus inoculation on progeny plants of
line 201–10 (Fig. 5 and data not shown). These results
clearly indicate that the transformants, unlike wild-type
plants, did not contain sufficient SA to restrict lesion
growth. Further proof that the nahG transformants used in
this study were severely impaired in the transmission of the
SA signal came from their lack to induce PR-1 proteins to
normal levels after exposure of leaf disks to high concen-
trations of SA (Fig. 6A). Consistently, total SA levels in
TMV infected leaves from nahG transformants were shown
to be even lower than the levels detected in noninfected
tissue from control plants. Plants handicapped in the
accumulation of SA were infected with TMV, and PR-1
proteins were monitored in the necrotic tissue induced by
the spreading lesions. Whereas lesions and the accumulation
of PR-1 proteins remained strictly localized in infected wild-
type plants over a period of 14 days, the extension and the

level of PR-1 protein synthesis increased significantly in the
nahG transgenic plants in parallel with the induction of
necrosis by the expanding lesions (Fig. 6B). Our finding of
substantial amounts of PR-1 proteins in nahG plants is
consistent with other observations. Friedrich et al. [19]
report that SAR associated genes like PR-1 are clearly
expressed in the inoculated leaves of TMV infected nahG
transgenic plants exhibiting drastically reduced levels of SA.
Taken together, tobacco plants, although expressing active
Ó FEBS 2003 Analysis of PR-1a gene regulation (Eur. J. Biochem. 270) 4883
SAH, are able to induce PR-1 proteins in tissue undergoing
necrosis, demonstrating that the expression of PR-1 proteins
to high levels during the HR does not depend solely on the
accumulation or transmission of the signal molecule SA.
Based on our findings we propose a new model for the
activation of the tobacco PR-1a gene by SA and during the
HR. Foremost, our model implies at least two distinct signal
transduction pathways leading independently from each
other to PR-1a gene induction, an SA dependent pathway
and a pathway relying on unknown signals produced during
the HR (Fig. 7). As activation of the PR-1a gene strictly
depends on the as-1-like element [22], we further suggest
that the two independent signaling cascades lead to the
formation of related, albeit different, transcription com-
plexes, which are likely to include TGA factors. Thus, gene
activation via distinct signal transduction events would
converge on the as-1-like element in the tobacco PR-1a
promoter (Fig. 7). Our model is consistent with the
existence of small protein families of plant TGA factors.
TGA factors from Arabidopsis and tobacco share an

extremely conserved basic domain by which they are able
to interact with different as-1 related target sequences
in vitro [22,32,44,45]. Furthermore, using chromatin immu-
noprecipitation assays, it has been shown recently that
TGA2 and TGA3 are recruited in vivo to the Arabidopsis
PR-1 promoter in response to a stimulus induction pathway
involving SA and NPR1/NIM1 [46]. TGA factors differ,
however, clearly in other biochemical properties. Some
TGA factors activate transcription in yeast via their
N-terminal domains, whereas others do not seem to have
an intrinsic transactivation potential [32]. Also, some TGA
factors interact in the yeast two-hybrid system with NPR1/
NIM1, while others fail to do so [27–29,32]. Therefore, it
seems plausible that different TGA factors, although
binding to the same cis-acting element, are engaged in
variant transcription complexes that are induced in plants in
response to different stimuli, as proposed in our model
(Fig. 7).
Our model is in conflict with previous conceptions in at
least one important aspect. Generally, SA accumulation in
local and systemic tissues of infected plants is considered to
be a product of processes induced by the HR and is thought
to be responsible for PR-1 gene induction during both the
HR and SAR [20,21,41]. Yet, plants transformed with the
nahG gene clearly demonstrate that PR-1 protein accumu-
lation can occur to high levels in necrotic tissue depleted
from SA (Fig. 6) [19]. However, in nahG transgenic plants,
PR-1 gene expression is abolished in the uninoculated leaves
of tobacco plants displaying the HR after TMV infection
[19]. Therefore, active SAH is able to fully suppress the SA

dependent pathway of systemic PR-1 gene induction in
tobacco, but fails to fully suppress HR dependent PR-1 gene
activation (Fig. 7), although SA levels are diminished to
those in noninfected wild-type plants. Apart from the results
with nahG transgenic plants, there are other indications in
favor of our model. In Arabidopsis plants insensitive to SA
due to a mutation in the NPR1/NIM1 gene, PR gene
expression is blocked in systemic tissues after induction by a
pathogen. However, in local tissues, PR gene expression,
including PR-1, is not affected substantially by npr1 [21].
Thus, as PR-1 gene induction is abolished nearly completely
in the npr1 mutant in response to SA [10], local induction of
PR-1 in infected tissue must be independent of SA in
Arabidopsis as well. A recent observation made by Fan and
Dong [31] is intriguingly relevant to our model. The authors
found that a chimeric transcription factor consisting of a
fusion between Arabidopsis TGA2 and the GAL4 DNA
binding domain is able to activate a GAL4 responsive
reporter gene in Arabidopsis plants after exposure to SA.
Yet, when a bacterial pathogen was used to infect transgenic
plants with the TGA2::GAL4 effector/reporter gene system,
only little reporter gene induction was observed, although
infection can cause a significant induction of the endo-
genous PR genes and also of a PR reporter gene.
In conclusion, based on our findings that an as-1 carrying
PR-1a promoter is differentially regulated from the wild-
type promoter in response to different stimuli and that PR-1
proteins are induced to high levels in necrotic tissue depleted
from endogenous SA, we suggest that the tobacco PR-1a
gene can be activated by at least two distinct signal

transduction pathways which both rely on the as-1-like
element in a similar way. Whereas one pathway is triggered
Fig. 7. Proposed model for the induction of the tobacco PR-1a gene via
two distinct signal transduction pathways elicited during the HR and by
SA, respectively, that converge on the as-1-like element. TMV infection
of tobacco (cv. Samsun NN) triggers the activation of a signal cascade
leading to necrosis. This pathway still operates in transgenic plants
expressing a 35S[nahG] chimeric gene and leads to high level induction
of PR-1 proteins. Application of exogenous SA triggers the activation
of a signal cascade leading to TMV resistance and moderate
induction of PR-1 proteins independent of necrosis. This pathway
mimics SAR (SAR-like response). SA dependent PR-1 gene activation
is (nearly) abolished in transgenic plants expressing a 35S[nahG] chi-
meric gene. As mutation of the as-1-like element markedly reduces
reporter gene expression in SA treated as well as in TMV infected
plants exhibiting the HR (Fig. 2) [22], both pathways mediate indu-
cible gene expression through the same cis-acting element in the PR-1a
promoter. On the other hand, an as-1 containing PR-1a promoter
responds differently from the wild-type promoter towards SA and the
HR. Therefore, different, albeit related, transcription factor complexes
interacting with the as-1-like element seem to be involved in the HR
and the SA dependent signal transduction cascades leading to PR-1a
gene activation in tobacco.
4884 R. Gru
¨
ner et al.(Eur. J. Biochem. 270) Ó FEBS 2003
by SA accumulating in noninfected tissues during the SAR,
the other pathway is induced by a yet unknown signal
molecule formed in tissues undergoing necrosis during the
HR. The identification of factors whose expression is

regulated solely by SA or by HR released signals will
support our hypothesis.
Acknowledgements
We would like to thank Bernhard Roth for communicating SA levels in
tobacco tissue; Ivana Glocova for help with the analysis of transgenic
plants; Maren Babbick for providing photographs of nahG plants; Ingrid
Priessnitz-Hohos for transformation of tobacco plants; and Jochen
Grob for technical assistance. This work was supported by grants from
Genzentrum Mu
¨
nchen and from Fonds der Chemischen Industrie.
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