Download Negative regulation of defense responses in Arabidopsis by two

The Plant Journal (2006) 48, 647–656
doi: 10.1111/j.1365-313X.2006.02903.x
Negative regulation of defense responses in Arabidopsis by
two NPR1 paralogs
Yuelin Zhang1,2,*, Yu Ti Cheng2, Na Qu1, Qingguo Zhao1, Donglin Bi1 and Xin Li2,3
National Institute of Biological Sciences, #7 Science Park Road, Zhongguancun Life Science Park, Beijing, People’s Republic of
China 102206,
2
Michael Smith Laboratories, Room 301, 2185 East Mall, University of British Columbia, Vancouver, BC V6T 1Z4, Canada, and
3
Department of Botany, Room 3529, 6270 Univ. Blvd., University of British Columbia, Vancouver, BC V6T 1Z4, Canada
1
Received 9 May 2006; revised 7 July 2006; accepted 24 July 2006.
*For correspondence (fax þ86 10 807 28646; e-mail [email protected]).
Summary
NPR1 is required for systemic acquired resistance, and there are five NPR1 paralogs in Arabidopsis. Here we
report knockout analysis of two of these, NPR3 and NPR4. npr3 single mutants have elevated basal PR-1
expression and the npr3 npr4 double mutant shows even higher expression. The double mutant plants also
display enhanced resistance against virulent bacterial and oomycete pathogens. This enhanced disease
resistance is partially dependent on NPR1, can be in part complemented by either wild-type NPR3 or NPR4, and
is not associated with an elevated level of salicylic acid. NPR3 and NPR4 interact with TGA2, TGA3, TGA5 and
TGA6 in yeast two-hybrid assays. Using bimolecular fluorescence complementation analysis, we show that
NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts.
Combined with our previous finding that basal PR-1 levels are also elevated in the tga2 tga5 tga6 triple mutant,
we propose that NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance through
their association with TGA2 and its paralogs.
Keywords: plant disease resistance, PR genes, NPR3, NPR4, TGA transcription factors.
Introduction
Plants have evolved sophisticated defense mechanisms to
deal with constant exposure to a wide range of microbial
pathogens. Systemic acquired resistance (SAR) is a secondary resistance response that is effective against a broad
spectrum of pathogens (Durrant and Dong, 2004). One
important signal molecule in SAR is salicylic acid (SA). The
endogenous level of SA increases upon pathogen infection
(Malamy et al., 1990; Metraux et al., 1990; Rasmussen et al.,
1991). Exogenous application of SA or SA analogs induces
Pathogenesis-related (PR) genes and pathogen resistance,
suggesting that SA is sufficient to induce SAR (Gorlach
et al., 1996; Metraux et al., 1991; White, 1979). Salicylic acid
is also essential for SAR since blocking its accumulation by
expressing the bacterial gene NahG, which encodes the SAdegrading enzyme salicylate hydroxylase, leads to reduced
expression of PR genes and enhanced susceptibility to both
compatible and incompatible pathogens (Delaney et al.,
1994; Gaffney et al., 1993; Nawrath and Metraux, 1999). In
Arabidopsis, mutants unable to synthesize SA after infection
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd
are also more susceptible to both virulent and avirulent
pathogens (Nawrath and Metraux, 1999; Wildermuth et al.,
2001).
NPR1 (also known as NIM1 and SAI1) is required for SAinduced PR gene expression and pathogen resistance in
Arabidopsis (Dong, 2004). It encodes a protein with two
protein–protein interaction domains, a BTB/POZ domain at
the N-terminus and an ankyrin-repeat domain in the central
region (Aravind and Koonin, 1999; Cao et al., 1997; Ryals
et al., 1997). NPR1 accumulates in the nucleus after induction by SA treatment or pathogen infection (Kinkema et al.,
2000). Nuclear localization of NPR1 is required for the
activation of PR-1 expression. An increase in the level of
SA induces redox changes, which appear to regulate the
translocation of the NPR1 protein from the cytoplasm to the
nucleus (Mou et al., 2003). Increasing evidence indicates
that NPR1 functions through its association with TGA
transcription factors. NPR1 interacts with various TGA
transcription factors in the yeast two-hybrid assay as well
647
648 Yuelin Zhang et al.
Results
Identification of knockout mutants for NPR3 and NPR4
Real-time RT-PCR analysis of NPR3 and NPR4 expression
showed that both genes were induced after treatment with
the SA analog INA (Figure 1a,b) or pathogen infection
(Figure 1c,d). Both NPR3 and NPR4 have high sequence
similarity to NPR1 throughout the protein (Figure S1). The
most conserved regions are the four ankyrin repeat domains
corresponding to amino acids 265–393 in NPR1. Both the Nterminal BTB/POZ domain and the C-terminal nuclear
localization signatures are also present in NPR3 and NPR4. In
addition, the intron–exon structures are conserved among
NPR1, NPR3 and NPR4 (Figure 2 and Figure S1). Sequence
analysis indicates that there are orthologs of NPR3 and NPR4
in rice (data not shown).
To identify the functions of NPR3 and NPR4, deletion
mutants of NPR3 and NPR4 were obtained by screening a
fast neutron-mutagenized Arabidopsis population (Li et al.,
2001b). These were named npr3-1 and npr4-3, respectively.
The coding sequences of NPR3 and NPR4 were completely
deleted in the mutants (Figure 2a,c), suggesting that they are
null mutations. The double mutant npr3-1 npr4-3 was
(a)
(b)
1.0
Relative NPR4/ACTIN1
20
10
0
0.8
0.6
0.4
0.2
0
–INA
+INA
(c)
–INA
+INA
(d)
15
Relative NPR4/ACTIN1
0.4
10
5
0.3
0.2
0.1
6
32
.E
S4
M
S4
32
6
oc
k
M
P.
s.
m
.E
oc
k
0
0
P.
s.
m
Relative NPR3/ACTIN1
30
Relative NPR3/ACTIN1
as in planta (Despres et al., 2000; Kim and Delaney, 2002;
Subramaniam et al., 2001; Zhang et al., 1999; Zhou et al.,
2000). In addition, the triple knockout mutant of TGA2, TGA5
and TGA6 has phenotypes similar to npr1 (Zhang et al.,
2003b). Induction of PR-1 expression and pathogen resistance by the SA analog 2,6-dichloroisonicotinic acid (INA)
was blocked in tga6-1 tga2-1 tga5-1, but not in tga6-1 or tga21 tga5-1 plants, suggesting that TGA2, TGA5 and TGA6
encode proteins with redundant and essential functions in
SA signaling.
In the Arabidopsis genome, there are five paralogs of
NPR1 (At4g26120/NPR2, At5g45110/NPR3, At4g19660/NPR4,
At3g57130/BOP1 and At2g41370/BOP2). Phylogenetic analysis of the NPR1 protein family revealed that NPR2 is closest
to NPR1 and forms a subgroup with NPR1; NPR3 and NPR4
form a distinctive pair; and BOP1 and BOP2 form another
pair farthest from NPR1 (Hepworth et al., 2005; Liu et al.,
2005). It was previously reported that At3g57130 and
At2g41370 encode BOP1 (Blade-on-petiole, 1) and BOP2,
respectively, which function in the control of growth symmetry (Ha et al., 2004; Hepworth et al., 2005; Norberg et al.,
2005). Phenotypes in the bop1 bop2 double mutant include
leafy petioles, loss of abscission of floral organs and
asymmetric flowers subtended by a bract. BOP1 and BOP2
interact with PERIANTHIA (AtbZip46), a bZip transcription
factor in the TGA subfamily (Hepworth et al., 2005). Here we
report the knockout analysis of At5g45110 (NPR3) and
At4g19660 (NPR4) and their overlapping roles in negative
regulation of plant defense responses.
Figure 1. Expression of NPR3 and NPR4.
(a, b) Real-time RT-PCR analysis of the expression of NPR3 and NPR4 in wildtype plants with or without INA treatment.
(c, d) Real-time RT-PCR analysis of NPR3 and NPR4 expression in wild-type
plants inoculated with P.s.m. ES4326.
For RT-PCR analysis, RNA was extracted 2 days after the treatments from
control plants and plants sprayed with 0.33 mM INA or inoculated with P.s.m.
ES4326 (OD600 ¼ 0.001). The cDNA was reverse transcribed and normalized to
the expression of Actin1. Error bars represent standard deviation from three
measurements. Each experiment was repeated once with similar results.
generated by crossing the two single mutants and screening
for lines carrying both homozygous mutations in the F2
population. In the double deletion mutant, no transcripts of
NPR3 and NPR4 were detected by RT-PCR (data not shown).
Neither the single nor the double mutant plants have
obvious developmental phenotypes.
Insertion mutants for NPR3 and NPR4 were also obtained
from the Arabidopsis Stock Center (Alonso et al., 2003).
SALK_043055 was named npr3-2 and SALK_098460 was
previously named npr4-2 (Liu et al., 2005). The locations of
the T-DNA insertions in npr3-2 and npr4-2 are shown in
Figure 2(b) and (d). Both T-DNA insertions are in exons.
npr3 and npr4 mutants have elevated basal PR gene
expression
To determine whether basal PR-1 expression was altered in
the npr3 and npr4 mutants, seedlings of wild-type and
mutant plants were grown on MS medium and their PR-1
expression was determined with real-time PCR. As shown in
Figure 3(a), single mutants for NPR3 have slightly increased
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
Negative regulation of defense responses in Arabidopsis 649
(a)
NPR3
At5g45110
At5g45120
TAC clone
K17O22
Deletion
49832–56001
(b)
ATG
NPR3
At5g45110
SALK_043055
Insertion
(c)
NPR4
At4g19650 At4g1966
0
BAC clone
T16H5
Deletion
311–9188
(d)
ATG
NPR4
At4g19660
SALK_098460
Insertion
Figure 2. Molecular lesions in the NPR3 and NPR4 mutants. The white boxes
represent exons, and the gray boxes represent genes.
(a) Deletion in npr3-1.
(b) Location of T-DNA insertion in npr3-2.
(c) Deletion in npr4-3.
(d) Location of T-DNA insertion in npr4-2.
basal PR-1 expression. PR-1 expression in the npr3-1 npr4-3
double mutant is dramatically increased (Figure 3a). Previously the tga2-1 tga5-1 tga6-1 triple mutant was also reported to have elevated PR-1 expression. Because background
PR-1 expression is often very low for plate-grown seedlings,
and varies depending on the growth conditions, the npr3-1
npr4-3 double mutant and the tga2-1 tga5-1 tga6-1 triple
mutant were grown on the same MS plate to directly compare the PR-1 level in these plants. As shown in Figure 3(b),
PR-1 expression in the npr3-1 npr4-3 double mutant was
comparable with that in the tga2-1 tga5-1 tga6-1 triple mutant. We also found that PR-2 and PR-5 expression levels in
the npr3-1 npr4-3 double mutant plants were significantly
higher than those in the wild-type plants (Figure 3c,d).
To test whether PR-1 expression in the npr3-1 npr4-3
double mutant can be induced by SA, we grew the mutant
and wild-type plants on MS plates with or without the SA
analog INA. As shown in Figure 3(b), treatment with INA
further induced the expression of PR-1 in the double
mutant. The expression level of PR-1 in the INA-treated
double mutant is very similar to that in the wild type. We
also tested the soil-grown plants for the induction of PR-1
by INA. One day after induction, PR-1 expression increased
about sevenfold in the npr3-1 npr4-3 double mutant and
less than twofold in the wild-type plants (Figure 3e,f),
suggesting that induction of PR-1 in the double mutant is
quicker than that in the wild-type plants. Two days after
INA induction, the PR-1 level is similar in the npr3-1 npr4-3
and wild-type plants.
The npr3-1 npr4-3 double mutant shows enhanced resistance against virulent pathogens
To test whether NPR3 and NPR4 affect the bacterial disease
resistance response, single and double mutants were challenged with Pseudomonas syringae pv. maculicola (P.s.m.)
ES4326, which is virulent on the Columbia ecotype. Figure 4
shows that while there is no significant difference in bacterial growth between wild type and npr3-1 or npr4-3, about
tenfold less bacterial growth was observed in the npr3-1
npr4-3 double mutant. Similar results were obtained when
the plants were challenged with Pseudomonas syringae pv.
tomato (P.s.t.) DC3000 (Figure S2).
To confirm whether the enhanced resistance phenotype
observed in the double mutant is caused by mutations in
NPR3 and NPR4, a cDNA clone of NPR3 under the control of
the CaMV 35S promoter and a genomic clone of NPR4 were
constructed and transformed into the npr3-1 npr4-3 double
mutant. As shown in Figure 4, both the NPR3 cDNA clone
and the NPR4 genomic clone can suppress the enhanced
resistance phenotype in the npr3-1 npr4-3 double mutant,
indicating that the enhanced resistance phenotype observed
in the double mutant is caused by the mutations in both
NPR3 and NPR4. In addition, we constructed another double
mutant, npr3-2 npr4-2, using the T-DNA insertion mutant
alleles. Similar to npr3-1 npr4-3, the npr3-2 npr4-2 double
mutant also displayed enhanced resistance to P.s.m. ES4326
(Figure S3), further confirming that NPR3 and NPR4 perform
overlapping functions and that loss of the function in both
genes leads to enhanced resistance to P.s.m. ES4326.
To determine whether mutations in NPR3 and NPR4
confer enhanced resistance against the virulent oomycete
pathogen Hyaloperonospora parasitica Noco2, the single
and double mutants were infected with H. parasitica Noco2
conidiospores at a concentration of 5000 spores ml)1. To
assess disease severity, conidiospores were collected from
the plants 6 days post-inoculation, and quantified with a
hemocytometer. As shown in Figure 5, npr3-1 and npr3-2
single mutants are significantly more resistant to H. parasitica Noco2. Resistance in the npr3-1 npr4-3 double mutant is
further enhanced compared to the npr3 single mutants,
indicating that both the npr3-1 and the npr4-3 mutation
contribute to the enhanced resistance. In addition, the
enhanced disease resistance phenotype in the npr3-1 npr43 double mutant can be partially rescued by the wild-type
NPR3 or NPR4 genes (Figure 5).
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
650 Yuelin Zhang et al.
Relative PR-2/ACTIN1
MS+INA
160
10000
120
0
(e)
Relative PR-1/ACTIN1
400
300
200
100
np
r4
-3
200
100
0
Col
800
600
400
200
0
Day 0 Day 1 Day 2
C
600
npr3-1 npr4-3
400
200
0
Day 0 Day 1 Day 2
np
r3
-1
C
ol
0
300
(f)
Relative PR-1/ACTIN1
(d)
400
np
r3
-1
tg
a2
np
r3
-1
tg
a5
C
np ol
r4
np 3
r4
np 2
r4
np -3
r3
-2
np
r3
-1
1
40
Co
l
np
r4
-3
10
80
np
r3
-1
100
tg
np
a6
r3
-1
np
r4
-3
1000
Relative PR-5/ACTIN1
(c)
MS
ol
np
r4
-3
(b)
Relative PR-1/ACTIN1
Relative PR-1/ACTIN1
(a)
Figure 3. Analysis of PR gene expression in npr3 and npr4 mutants.
(a) PR-1 expression in npr3 single, npr4 single and npr3-1 npr4-3 double mutants. The relative expression levels of PR-1 were normalized to the expression of Actin1
(multiplied by 1000 for clarity).
(b) Induction of PR-1 expression in wild-type and npr3-1 npr4-3 plants grown on MS plates with or without INA. The relative expression levels of PR-1 were
normalized to the expression of Actin1.
(c) PR-2 expression in the npr3-1 npr4-3 double mutant. The relative expression levels of PR-2 were normalized to the expression of Actin1 (multiplied by 1000 for
clarity).
(d) PR-5 expression in the npr3-1 npr4-3 double mutant. The relative expression levels of PR-5 were normalized to the expression of Actin1 (multiplied by 1000 for
clarity).
(e, f) Induction of PR-1 expression in wild-type and npr3-1 npr4-3 plants grown on soil. The relative expression levels of PR-1 were normalized to the expression of
Actin1.
Plants were grown on soil and 20-day-old seedlings were treated with 0.33 mM INA. Samples were taken 0 h (day 0), 24 h (day 1) and 48 h (day 2) after the application
of INA.
The RNAs were reverse transcribed to obtain total cDNA and the cDNAs were used to determine the relative expression levels of PR-1, PR-2 and PR-5 expression in
wild-type and mutant plants by real-time PCR using SYBR Green I chemistry. Error bars represent the standard deviation from three measurements. Each
experiment was repeated once with similar results. For (a–d), total RNA was extracted from 20-day-old seedlings grown on MS plates with or without 50 lM INA
under a 16-h day/8-h night cycle.
The level of SA is not elevated in the npr3-1 npr4-3 double
mutant
Most mutants with constitutive PR gene expression have
increased SA levels (Bowling et al., 1994, 1997; Clarke
et al., 1998; Li et al., 2001a; Maleck et al., 2002; Rate et al.,
1999; Shirano et al., 2002). To test whether the constitutive
PR-1 expression and pathogen resistance in the npr3-1
npr4-3 double mutant is due to an elevated level of SA, the
level of SA was determined in the double mutant. As
shown in Figure 6(a), the total SA level of npr3-1 npr4-3
plants is not significantly higher than that of wild-type
plants. To determine whether accumulation of SA in the
npr3-1 npr4-3 double mutant is affected after avirulent
pathogen induction, leaves of wild-type and mutant plants
were challenged with P.s.t. DC3000 carrying AvrRpt2 and
collected for SA measurement. As shown in Figure 6(b),
the double mutant accumulated a similar amount of total
SA as the wild-type plants. The levels of free SA in the
npr3-1 npr4-3 plants were also not increased compared
with those in the wild-type plants under induced or uninduced conditions (data not shown). Thus the enhanced
resistance in npr3-1 npr4-3 is not caused by increased
endogenous SA.
Disease resistance in npr3-1 npr4-3 is partially dependent on
NPR1
To test the relationship between NPR3, NPR4 and NPR1, a
triple mutant of npr3-1 npr4-3 npr1-1 was constructed. As
shown in Figure 7(a), constitutive PR-1 expression in the
npr3-1 npr4-3 double mutant was only partially affected by
the npr1-1 mutation. When triple mutant plants were infected with P.s.m. ES4326, the resistance in npr3-1 npr4-3 was
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
Negative regulation of defense responses in Arabidopsis 651
P.s.m. ES4326
Day 0
P.p. Noco2
8
Day 3
5
7
4.5
6
Spores per plant (x104)
Log (cfu mg FW–1)
4
3.5
3
2.5
2
1.5
1
5
4
3
2
0.5
1
Figure 4. Growth of P.s.m. ES4326 in wild type, npr3-1, npr4-3, npr3-1 npr4-3
double mutant and npr3-1 npr4-3 transformed with either NPR4 genomic
clone (NPR4g#15) or 35S-NPR3 cDNA clone (NPR3c#6).
The complementing lines are BASTA-resistant T2 plants from the designated
lines. The plants were infiltrated with a bacterial suspension of
OD600 ¼ 0.0001. The error bars represent the standard deviation of four
replicates. The experiment was repeated three times with similar results. Cfu,
colony-forming units.
also partly compromised (Figure 7b). These data suggest
that the enhanced resistance mechanisms being activated in
npr3-1 npr4-3 are partially dependent on NPR1, thus both
NPR1-dependent and NPR1-independent pathways are
activated in the npr3-1 npr4-3 double mutant.
NPR3 and NPR4 interact with TGA transcription factors in the
yeast two-hybrid assay
To identify proteins that interact with NPR4, a yeast
two-hybrid screen of an Arabidopsis cDNA library was
performed using NPR4 as bait. Among the positive clones
identified and sequenced, three contained truncated cDNAs
of TGA2, TGA3 or TGA6. A comparison of the sequences of
the recovered cDNA clones with the full-length cDNAs
showed that the TGA2 clone starts from nucleotide 265 of
At5g06950 (accession number BT006134), the TGA3 clone
starts from nucleotide 588 of At1g22070 (accession number
NM_102057) and the TGA6 clone starts from nucleotide
466 of At3g12250 (accession number NM_112061). Yeast
two-hybrid assays were subsequently performed using
NPR3 or NPR4 as bait and TGA1 to TGA6 as prey. As shown
in Figure 8, NPR3 interacts with TGA2, TGA3, TGA5 and
TGA6, but not TGA1 or TGA4 in the yeast two-hybrid assay.
Similar results were observed when NPR4 were used as
bait except that weak interaction was observed between
NPR4 and TGA1.
C
ol
r3
-1
np
r3
-2
np
r4
-3
n
np
pr
4r3
2
-1
np
r4
N
PR 3
3c
#6
N
PR
4g
#1
3
0
np
Col
npr
3-1
npr
npr
43-1
npr 3
4- 3
NP
R4g
#15
NP
R3c
#6
Col
npr
3-1
npr
npr
4
-3
3-1
npr
4-3
NP
R4g
#15
NP
R3c
#6
0
Figure 5. Growth of H. parasitica Noco2 on npr3 single, npr4 single, npr3-1
npr4-3, and npr3-1 npr4-3 double mutant plants transformed with the 35SNPR3 cDNA clone (NPR3c#6) or npr3-1 npr4-3 plants transformed with the
genomic clone of NPR4 (NPR4g#13).
The complementing lines are BASTA-resistant T2 plants from the designated
lines. Two-week-old soil-grown seedlings were sprayed with H. parasitica
Noco2 spores at a concentration of 5000 spores ml)1 of water. After 6 days of
incubation under 90% humidity and 16-h day/8-h night cycles, five plants were
randomly harvested into 1 ml of water and conidiospores on the plants were
vortexed off and counted using a hemocytometer. Each treatment contains
four replicates, and the numbers represent the average spore counts on one
plant. The error bars represent the standard deviation of four replicates. The
experiment was repeated twice with similar results.
Bimolecular fluorescence complementation analysis of
NPR3 and TGA2 interaction
To test whether NPR3 and TGA2 associate with each other
in vivo, we used a bimolecular fluorescence complementation (BiFC) approach for visualization of protein–protein
interactions in plant cells. In this method, two non-fluorescent fragments (YFPN and YFPC) of yellow fluorescent protein (YFP) are fused to two different proteins separately
(Walter et al., 2004). When these two proteins associate with
each other, a fluorescent YFP complex is formed. We first
made constructs to express NPR3–YFPN and TGA2–YFPC
fusion proteins. In both cases, the YFP fragments are located
at the C-terminus of the fusion proteins.
When the NPR3–YFPN and TGA2–YFPC constructs were
co-bombarded to onion epidermal cells, YFP fluorescence
was observed, predominantly in the nucleus (Figure 9a).
When Arabidopsis mesophyll protoplasts were transiently
transfected with the NPR3–YFPN and TGA2–YFPC constructs,
YFP fluorescence was also observed in the nucleus of the
transfected protoplasts (Figure 9b), suggesting that NPR3
and TGA2 associate with each other in vivo in the nucleus.
No yellow fluorescence was observed when NPR3–YFPN or
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
652 Yuelin Zhang et al.
(a)
(a)
Uninduced total SA
0.8
100000
Relative PR-1/ACTIN1
0.7
0.5
0.4
0.3
0.2
10000
1000
100
10
0.1
Local total SA
(b)
6
npr
1-1
npr
4-3
Day 0
6
Day 3
5
4
TGA2–YFPC was co-transfected with the empty YFPC or YFPN
vectors (data not shown).
Discussion
Systemic acquired resistance is an important pathway in
plant innate immunity, and NPR1 is a key positive regulator
of SAR (Cao et al., 1997). Since there are five paralogs of
NPR1 in the Arabidopsis genome, their functions were
intriguing. Here we present the knockout analysis of NPR3
and NPR4. Our data suggest that these genes function as
negative regulators of plant defense. The npr3-1 single
mutant displayed elevated PR-1 level and enhanced resistance to H. parasitica Noco2. In npr4-3 plants, no obvious
enhanced resistance phenotype was observed. The combination of npr3-1 and npr4-3 leads to much higher PR-1
expression and a dramatic increase in resistance to both the
virulent bacterial pathogen P.s.m. ES4326 and the oomycete
pathogen H. parasitica Noco2.
1
1-1
npr
1-1
3-1
npr
4-3
npr
4-3
npr
Col
0
3-1
Figure 6. Total SA in uninfected and infected local leaves of Col, npr3-1,
npr4-3 and the npr3-1 npr4-3 double mutant plants.
(a) Total SA from uninfected leaf tissue of 4-week-old soil-grown plants.
(b) Total SA from the local leaves of 4-week-old soil-grown plants infected
with P.s.t. DC3000 AvrRpt2 at a dose of OD600 ¼ 0.2. Half of a leaf was
inoculated and the whole leaf was collected 3 days after infection. The error
bars represent the standard deviation of four replicates. The experiment was
repeated once with similar results.
2
npr
npr3-1 npr4-3
npr
Col
3
Col
3-1
npr
n
p
r
3-1
4-3
npr
4-3
npr
1-1
npr
1-1
0
4
npr
2
Log (cfu mg FW–1)
µg SA / g tissue
(b)
npr
3-1
np
r3
npr
3-1
-1
np
r4
Col
-3
np
r4
-3
1
np
r3
-1
Co
l
0
npr
4-3
µg SA / g tissue
0.6
Figure 7. Analysis of the npr3-1 npr4-3 npr1-1 triple mutant.
(a) PR-1 expression in npr3-1 npr4-3 npr1-1 mutant plants determined by realtime PCR. The relative expression levels of PR-1 were normalized to the
expression of Actin1; error bars represent standard deviation from three
measurements. The experiment was repeated once with similar results.
(b) Growth of P.s.m. ES4326 in the npr3-1 npr4-3 npr1-1 triple mutant. The
plants were infiltrated with a bacterial suspension of OD600 ¼ 0.0001. The
error bars represent the standard deviation of four replicates. The experiment
was repeated three times with similar results. Cfu, colony-forming units.
Previously Liu et al. (2005) analyzed the npr4 single
mutants for altered resistance to pathogens. Similar to our
results, they did not observe any obvious difference in
resistance or susceptibility to H. parasitica Noco2 in the npr4
single mutants compared to the wild-type plants. Interestingly, they found that the npr4 single mutants are more
susceptible to P.s.t. DC3000. However, under our growth
conditions, no enhanced susceptibility to P.s.m. ES4326 or
P.s.t. DC3000 was observed in npr4-3 or npr4-2 in multiple
repeated experiments, although npr4-2 is the same mutant
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
Negative regulation of defense responses in Arabidopsis 653
(a)
(b)
Figure 8. Yeast two-hybrid interactions between TGA transcription factors
and NPR3 or NPR4.
Yeast cells carrying the TGA cDNA clones and either pBI880 vector control or
indicated bait were grown for 2 days at 30C on medium lacking tryptophan
and leucine, and the b-galactosidase reporter expression was subsequently
assayed using the X-gal filter assay.
allele as used in the study by Liu et al. (2005). The effect of
the npr4-2 mutation on disease resistance can only be
observed when combined with the npr3-2 mutation. Since
npr4 single mutants have an elevated PR-1 expression level
and enhanced resistance to both H. parasitica and P.s.m.
ES4326 when combined with npr3 knockout mutants, it is
very unlikely that NPR4 is a positive regulator of disease
resistance and required for disease resistance as suggested
by Liu et al. (2005).
In Arabidopsis, a large number of mutants that display
constitutive PR gene expression and pathogen resistance
have been identified (Bowling et al., 1994, 1997; Clarke et al.,
1998; Li et al., 2001a; Maleck et al., 2002; Rate et al., 1999;
Shirano et al., 2002). Most of these mutants constitutively
accumulate high levels of SA in the absence of pathogen
infection and this is often required for enhanced pathogen
resistance. In contrast, the npr3-1 npr4-3 double mutant
does not accumulate higher levels of SA than wild-type
plants with or without pathogen induction, but still expresses elevated levels of PR genes and enhanced resistance to
both P.s.m. ES4326 and H. parasitica Noco2. Since induction
of PR-1 is quicker in the double mutant, the enhanced
disease resistance phenotype is probably caused by an
enhanced capability of the plants to activate infectioninduced defense responses, a process called priming
(Conrath et al., 2002). As PR-1 is a hallmark of the SA
signaling pathway (Uknes et al., 1992) and SA synthesis is
not affected in the npr3-1 npr4-3 double mutant, our data
suggest that NPR3 and NPR4 may regulate defense
responses downstream of SA. Previous studies have suggested that both NPR1-dependent and NPR1-independent
Figure 9. Bimolecular fluorescence complementation visualization of NPR3
and TGA2 interaction.
(a) Epifluorescence (I) and bright field (II) images of onion epidermal cells cobombarded with constructs expressing the NPR3–YFPN and TGA2–YFPC
fusion proteins.
(b) Epifluorescence (I), bright field (II), chloroplast autofluorescence (III) and
merged (IV) images of Arabidopsis mesophyll protoplasts co-transfected with
constructs expressing the NPR3–YFPN and TGA2–YFPC fusion proteins.
resistance pathways exist downstream of SA synthesis (Li
et al., 2001a; Zhang et al., 2003a). Our analysis of the npr3-1
npr4-3 npr1-1 triple mutant indicates that the enhanced
resistance in the npr3-1 npr4-3 plants is a combination of
both NPR1-dependent and NPR1-independent resistance.
Like NPR1, NPR3 and NPR4 have no obvious biochemical
functions except for the presence of two protein–protein
interaction domains, suggesting that their functions may be
to interact with other proteins as adaptors. NPR1 is located
in the cytoplasm as an oligomer under non-inducing conditions and Cys216 is one of the cysteines that is required for
keeping NPR1 in the cytoplasm (Mou et al., 2003). Sequence
comparison between NPR1 and its paralogs indicate that
this cysteine residue is not conserved in the NPR3 and NPR4
proteins, suggesting that NPR3 and NPR4 may be regulated
differently. In the roots, the NPR4–GFP fusion protein was
found to localize in the nucleus (Figure S4). It remains to be
determined whether SA induction affects the localization of
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
654 Yuelin Zhang et al.
NPR3 and NPR4. We found that TGA2, TGA3, TGA5 and
TGA6 interact with NPR3 and NPR4 in yeast two-hybrid
assays (Figure 8). It was further demonstrated that NPR3
interacts with TGA2 in the nucleus of onion epidermal cells
and Arabidopsis mesophyll protoplasts using BiFC analysis.
Thus it is likely NPR3 regulates PR gene expression through
interaction with TGA factors in the nucleus.
We previously reported that the tga6-1 tga2-1 tga5-1 triple
mutant displayed multiple phenotypes. The loss of SAR
responses in the tga triple mutant resembles that in the npr1
mutant plants, but the elevated basal PR-1 expression in the
tga triple mutant was not observed in the npr1 mutants,
suggesting that suppression of basal PR-1 expression by
these TGA transcription factors is facilitated by other factors
(Zhang et al., 2003b). Since the npr3-1 npr4-3 double mutant
also accumulates high levels of basal PR-1, and NPR3
interacts with TGA2, TGA5 and TGA6 in the yeast two-hybrid
assays and associates with TGA2 in the nucleus of Arabidopsis mesophyll protoplasts, it is plausible to hypothesize that
NPR3 and NPR4 negatively regulate PR-1 expression and
pathogen resistance through association with TGA2 and its
paralogs. Similar to the npr3-1 npr4-3 npr1-1 triple mutant,
the tga triple mutant did not display enhanced disease
susceptibility to P.s.m. ES4326. It is most likely that the
enhanced disease susceptibility associated with the loss of
response to SA was overcome by enhanced resistance
associated with the elevated basal PR gene expression.
Our study on the npr3 and npr4 single and double mutants
provides strong evidence that NPR3 and NPR4 are negative
regulators of plant defense responses. This is a major step
toward understanding their functions in regulating plant
defense responses downstream of SA synthesis. It remains
to be determined how NPR3 and NPR4 negatively regulate
PR gene expression and pathogen resistance. It is possible
that NPR3 and NPR4 bind to TGA2, TGA5 and TGA6 and
compete with NPR1 for interactions with the TGA factors
under non-inducing conditions. When complexed with
NPR3 or NPR4, these TGA factors cannot induce PR gene
expression. Inactivating both NPR3 and NPR4 leads to
activation of TGA2, TGA5 and TGA6 and expression of PR
genes. Alternatively, NPR3 and NPR4 may function as cofactors of TGA2, TGA5 and TGA6, and are required for the
function of these TGA factors. In this scenario, TGA2, TGA5
and TGA6 might be the transcription factors required for the
expression of a negative regulator of PR gene expression
and pathogen resistance.
Experimental procedures
Mutant isolation
The npr3-1 and npr4-3 mutants were identified by screening a fast
neutron-mutagenized population by PCR (Li et al., 2001b). The npr32 (SALK_043055) and npr4-2 (SALK_098460) mutants were obtained
from the Arabidopsis Stock Center (ABRC). Homozygous plants
were identified by PCR using primers flanking the insertion.
The double mutants npr3-1 npr4-3 and npr3-2 npr4-4 were
obtained by crossing the two single mutants, selfing F1, and
screening in the F2 population for plants with an absence of both
genes. The triple mutant npr3-1 npr4-3 npr1-1 was obtained by
crossing npr1-1 with the double mutant npr3-1 npr4-3, selfing F1 and
screening for the triple mutant in the F2 population with primers
specific for the three mutations. Two independent triple mutant
lines were obtained from 72 F2 plants. Both lines have similar
phenotypes and one line was used for detailed analysis.
Expression analysis and pathogen infections
All plants were grown at 22C under 16-h light/8-h dark cycles.
Infection of plants with P.s.m. ES4326 and H. parasitica Noco2 was
carried out as previously described (Li et al., 2001a). Ribonucleic
acid expression was carried out as described earlier (Zhang et al.,
2003b). Levels of SA were determined using a 1:5 scaled-down
protocol as previously described (Li et al., 1999).
Complementation of the npr3-1 npr4-3 double mutant
A 3.8-kb fragment (T16H5, nucleotides 5136–8952, accession number
AL024486) containing NPR4 was amplified by PCR from wild-type
genomic DNA and cloned into the binary vector pGreen229 (Hellens
et al., 2000) to create pG229NPR4g. The full-length cDNA of NPR3
was amplified by PCR and cloned into a modified pGreen229 vector
under the control of the CaMV 35S promoter. To confirm that the
phenotypes observed in the npr3-1 npr4-3 double mutant or the
npr3-1 npr4-3 npr1-1 triple mutant were caused by mutations in
NPR3 and NPR4, the cDNA clone of NPR3 and genomic clone of NPR4
were transformed into Agrobacterium and subsequently into the
double or triple mutant by floral dipping (Clough and Bent, 1998).
Yeast two-hybrid screen
The plasmid vectors pBI880 and pBI881 and the prey Arabidopsis
library were obtained from Dr William Crosby (University of Windsor, Ontario, Canada). The full-length NPR3 and NPR4 cDNAs were
amplified by PCR and cloned into pBI880. The yeast two-hybrid
screen was performed as previously described (Kohalmi et al.,
1997) except that we used the yeast strain PJ694a. Transformants
containing both the pBI880NPR4 bait plasmid and the library plasmids were plated on medium lacking tryptophan, leucine and histidine and supplemented with 5 mM 3-amino-1¢,2¢,4¢-triazole to
select colonies that express the HIS3 reporter gene. The X-gal filter
assay was subsequently performed to monitor the expression of the
lacZ reporter gene. For the two-hybrid assays, cells were co-transformed with the bait and the prey plasmids, and the transformants
were analyzed by X-gal filter assay.
Bimolecular fluorescence complementation analysis of
NPR3 and TGA2
The TGA2 cDNA was amplified by PCR and cloned into pUCSPYCE that contained the 35S promoter and C-terminal region of
YFP to obtain pTGA2•YCE. Meanwhile, the NPR3 cDNA was
amplified by PCR and cloned into pUC-SPYNE that contained the
35S promoter and N-terminal region of YFP to obtain pNPR3•YNE.
Both plasmids were confirmed by sequencing and purified by
CsCl gradient.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
Negative regulation of defense responses in Arabidopsis 655
For transient expression of the NPR3-YFPN and TGA2-YFPC
fusion protein in onion epidermal cells, pNPR3•YNE and
pTGA2•YCE were bombarded into onion epidermal cells according
to a previously described protocol (von Arnim and Deng, 1994). For
transient expression of the NPR3–YFPN and TGA2–YFPC fusion
proteins, pNPR3•YNE and pTGA2•YCE were co-transfected into
Arabidopsis mesophyll protoplasts according to a previously described protocol (Sheen, 2001). Yellow fluorescent protein fluorescence was observed by confocal microscopy.
Acknowledgements
We thank Gary Wong, Arthur Lau and Lu Qun for their excellent
technical assistance; Zhang Jie, Quan Ruidang and Dr Yan Guo for
help on BiFC experiments; Maxygen Inc. (especially Dr Mike Lassner)
for providing us with the npr3-1 and npr4-3 mutants; ABRC for npr3-2
and npr4-2 mutant seeds; Dr William Crosby for providing the yeast
two-hybrid library, Dr Phil Hieter for yeast strain PJ694a and Dr Jim
Kronstad, Sandra Goritschnig and Kristoffer Palma for critical reading of the manuscript. We are grateful for financial support to Y.Z.
from the Natural Sciences and Engineering Research Council of
Canada (NSERC) and the Chinese Ministry of Science and Technology; and funds to X.L. from UBC Michael Smith Laboratories,
NSERC, Canadian Foundation for Innovation (CFI), BCKDF and the
UBC Blussum Fund.
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Alignment of NPR1, NPR3 and NPR4.
Figure S2. Growth of P.s.t DC 3000 in wild type, npr3 single, npr4
single and npr3 npr4 double mutants.
Figure S3. Growth of P.s.m. ES4326 in wild type, npr3 single, npr4
single, and npr3 npr4 double mutant plants.
Figure S4. Analysis of tansgenic plants expressing the NPR4-GFP
fusion proteins.
This material is available as part of the online article from http://
www.blackwell-synergy.com.
References
Alonso, J.M., Stepanova, A.N., Leisse, T.J. et al. (2003) Genomewide insertional mutagenesis of Arabidopsis thaliana. Science,
301, 653–657.
Aravind, L. and Koonin, E.V. (1999) Fold prediction and evolutionary
analysis of the POZ domain: structural and evolutionary relationship with the potassium channel tetramerization domain. J.
Mol. Biol. 285, 1353–1361.
von Arnim, A.G. and Deng, X.W. (1994) Light inactivation of Arabidopsis photomorphogenic repressor COP1 involves a cellspecific regulation of its nucleocytoplasmic partitioning. Cell, 79,
1035–1045.
Bowling, S.A., Guo, A., Cao, H., Gordon, A.S., Klessig, D.F. and
Dong, X. (1994) A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. Plant Cell, 6,
1845–1857.
Bowling, S.A., Clarke, J.D., Liu, Y., Klessig, D.F. and Dong, X. (1997)
The cpr5 mutant of Arabidopsis expresses both NPR1-dependent
and NPR1-independent resistance. Plant Cell, 9, 1573–1584.
Cao, H., Glazebrook, J., Clarke, J.D., Volko, S. and Dong, X. (1997)
The Arabidopsis NPR1 gene that controls systemic acquired
resistance encodes a novel protein containing ankyrin repeats.
Cell, 88, 57–63.
Clarke, J.D., Liu, Y., Klessig, D.F. and Dong, X. (1998) Uncoupling PR
gene expression from NPR1 and bacterial resistance: characterization of the dominant Arabidopsis cpr6-1 mutant. Plant Cell, 10,
557–569.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16, 735–743.
Conrath, U., Pieterse, C.M. and Mauch-Mani, B. (2002) Priming in
plant–pathogen interactions. Trends Plant Sci. 7, 210–216.
Delaney, T.P., Uknes, S., Vernooij, B. et al. (1994) A central role
of salicylic acid in plant disease resistance. Science, 266, 1247–
1250.
Despres, C., DeLong, C., Glaze, S., Liu, E. and Fobert, P.R. (2000) The
Arabidopsis NPR1/NIM1 protein enhances the DNA binding
activity of a subgroup of the TGA family of bZIP transcription
factors. Plant Cell, 12, 279–290.
Dong, X. (2004) NPR1, all things considered. Curr. Opin. Plant Biol.
7, 547–552.
Durrant, W.E. and Dong, X. (2004) Systemic acquired resistance.
Annu. Rev. Phytopathol. 42, 185–209.
Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes,
S., Ward, E., Kessmann, H. and Ryals, J. (1993) Requirement of
salicylic acid for the induction of systemic acquired resistance.
Science, 261, 754–756.
Gorlach, J., Volrath, S., Knauf-Beiter, G. et al. (1996) Benzothiadiazole, a novel class of inducers of systemic acquired resistance,
activates gene expression and disease resistance in wheat. Plant
Cell, 8, 629–643.
Ha, C.M., Jun, J.H., Nam, H.G. and Fletcher, J.C. (2004) BLADE-ONPETIOLE1 encodes a BTB/POZ domain protein required for leaf
morphogenesis in Arabidopsis thaliana. Plant Cell Physiol. 45,
1361–1370.
Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S. and Mullineaux,
P.M. (2000) pGreen: a versatile and flexible binary Ti vector for
Agrobacterium-mediated plant transformation. Plant Mol. Biol.
42, 819–832.
Hepworth, S.R., Zhang, Y., McKim, S., Li, X. and Haughn, G.W.
(2005) BLADE-ON-PETIOLE-dependent signaling controls leaf and
floral patterning in Arabidopsis. Plant Cell, 17, 1434–1448.
Kim, H.S. and Delaney, T.P. (2002) Over-expression of TGA5, which
encodes a bZIP transcription factor that interacts with NIM1/
NPR1, confers SAR-independent resistance in Arabidopsis thaliana to Peronospora parasitica. Plant J. 32, 151–163.
Kinkema, M., Fan, W. and Dong, X. (2000) Nuclear localization of
NPR1 is required for activation of PR gene expression. Plant Cell,
12, 2339–2350.
Kohalmi, S.E., Nowak, J. and Crosby, W.L. (1997) The yeast twohybrid system. In Differentially Expressed Genes in Plants: A
Bench Manual (Hansen, E. and Harper, G., eds) London: Taylor
and Francis, pp. 63–82.
Li, X., Zhang, Y., Clarke, J.D., Li, Y. and Dong, X. (1999) Identification
and cloning of a negative regulator of systemic acquired resistance, SNI1, through a screen for suppressors of npr1-1. Cell, 98,
329–339.
Li, X., Clarke, J.D., Zhang, Y. and Dong, X. (2001a) Activation of an
EDS1-mediated R-gene pathway in the snc1 mutant leads to
constitutive, NPR1-independent pathogen resistance. Mol. Plant
Microbe Interact. 14, 1131–1139.
Li, X., Song, Y., Century, K., Straight, S., Ronald, P., Dong, X.,
Lassner, M. and Zhang, Y. (2001b) A fast neutron deletion mutagenesis-based reverse genetics system for plants. Plant J. 27,
235–242.
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 647–656
656 Yuelin Zhang et al.
Liu, G., Holub, E.B., Alonso, J.M., Ecker, J.R. and Fobert, P.R. (2005)
An Arabidopsis NPR1-like gene, NPR4, is required for disease
resistance. Plant J. 41, 304–318.
Malamy, J., Carr, J.P., Klessig, D.F. and Raskin, I. (1990) Salicylic
acid: a likely endogenous signal in the resistance response of
tobacco to viral infection. Science, 250, 1002–1004.
Maleck, K., Neuenschwander, U., Cade, R.M., Dietrich, R.A., Dangl,
J.L. and Ryals, J.A. (2002) Isolation and characterization of broadspectrum disease-resistant Arabidopsis mutants. Genetics, 160,
1661–1671.
Metraux, J.P., Signer, H., Ryals, J., Ward, E., Wyss-Benz, M.,
Gaudin, J., Raschdorf, K., Schmid, E., Blum, W. and Inverardi, B.
(1990) Increase in salicylic acid at the onset of systemic acquired
resistance in cucumber. Science, 250, 1004–1006.
Metraux, J.P., Ahl-Goy, P., Staub, T., Speich, J., Steinemann, A.,
Ryals, J. and Ward, E. (1991) Induced resistance in cucumber in
response to 2,6-dichloroisonicotinic acid and pathogens. In
Advances in Molecular Genetics of Plant–Microbe Interactions
(Hennecke, H. and Verma, D.P.S., eds) Dordrecht, The
Netherlands: Kluwer Academic Publishers, pp. 432–439.
Mou, Z., Fan, W. and Dong, X. (2003) Inducers of plant systemic
acquired resistance regulate NPR1 function through redox changes. Cell, 113, 935–944.
Nawrath, C. and Metraux, J.P. (1999) Salicylic acid inductiondeficient mutants of Arabidopsis express PR-2 and PR-5 and
accumulate high levels of camalexin after pathogen inoculation.
Plant Cell, 11, 1393–1404.
Norberg, M., Holmlund, M. and Nilsson, O. (2005) The BLADE ON
PETIOLE genes act redundantly to control the growth and
development of lateral organs. Development, 132, 2203–2213.
Rasmussen, J.B., Hammerschmidt, R. and Zook, M.N. (1991) Systemic induction of salicylic acid accumulation in cucumber after
inoculation with Pseudomonas syringae pv. syringae. Plant
Physiol. 97, 1342–1347.
Rate, D.N., Cuenca, J.V., Bowman, G.R., Guttman, D.S. and
Greenberg, J.T. (1999) The gain-of-function Arabidopsis acd6
mutant reveals novel regulation and function of the salicylic acid
signaling pathway in controlling cell death, defenses, and cell
growth. Plant Cell, 11, 1695–1708.
Ryals, J., Weymann, K., Lawton, K. et al. (1997) The Arabidopsis
NIM1 protein shows homology to the mammalian transcription
factor inhibitor I kappa B. Plant Cell, 9, 425–439.
Sheen, J. (2001) Signal transduction in maize and Arabidopsis
mesophyll protoplasts. Plant Physiol. 127, 1466–1475.
Shirano, Y., Kachroo, P., Shah, J. and Klessig, D.F. (2002) A gain-offunction mutation in an Arabidopsis Toll Interleukin1 receptornucleotide binding site-leucine-rich repeat type R gene triggers
defense responses and results in enhanced disease resistance.
Plant Cell, 14, 3149–3162.
Subramaniam, R., Desveaux, D., Spickler, C., Michnick, S.W. and
Brisson, N. (2001) Direct visualization of protein interactions in
plant cells. Nat. Biotechnol. 19, 769–772.
Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S.,
Dincher, S., Chandler, D., Slusarenko, A., Ward, E. and Ryals, J.
(1992) Acquired resistance in Arabidopsis. Plant Cell, 4, 645–
656.
Walter, M., Chaban, C., Schutze, K. et al. (2004) Visualization of
protein interactions in living plant cells using bimolecular
fluorescence complementation. Plant J. 40, 428–438.
White, R.F. (1979) Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco. Virology, 99, 410–
412.
Wildermuth, M.C., Dewdney, J., Wu, G. and Ausubel, F.M. (2001)
Isochorismate synthase is required to synthesize salicylic acid for
plant defence. Nature, 414, 562–565.
Zhang, Y., Fan, W., Kinkema, M., Li, X. and Dong, X. (1999)
Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid
induction of the PR-1 gene. Proc. Natl Acad. Sci. USA, 96, 6523–
6528.
Zhang, Y., Goritschnig, S., Dong, X. and Li, X. (2003a) A gainof-function mutation in a plant disease resistance gene leads
to constitutive activation of downstream signal transduction
pathways in suppressor of npr1-1, constitutive 1. Plant Cell, 15,
2636–2646.
Zhang, Y., Tessaro, M.J., Lassner, M. and Li, X. (2003b) Knockout
analysis of Arabidopsis transcription factors TGA2, TGA5, and
TGA6 reveals their redundant and essential roles in systemic
acquired resistance. Plant Cell, 15, 2647–2653.
Zhou, J.M., Trifa, Y., Silva, H., Pontier, D., Lam, E., Shah, J. and
Klessig, D.F. (2000) NPR1 differentially interacts with members of
the TGA/OBF family of transcription factors that bind an element
of the PR-1 gene required for induction by salicylic acid. Mol.
Plant Microbe Interact. 13, 191–202.
ª 2006 The Authors
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