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Supporting Information Legends
Figure S1. Map-Based Cloning of isn2
(A) A schematic diagram of the map-based cloning process. Homozygous isn2 npr1, which is in the
Col-0 genetic background, was crossed to the polymorphic ecotype Ler. A total of 48 F2 progeny
homozygous for isn2 were used to determine the approximate position of the isn2 mutation using bulked
segregant analysis. The isn2 mutation was linked to the markers F21M12 and CIW12 on the north arm
of chromosome 1. Out of a total mapping population of 1586 plants homozygous for isn2, two were
heterozygous at M1, and four were heterozygous at M5. The heterozygotes found by these two markers
were mutually exclusive. No heterozygotes were found at M2, M3, and M4 (Table S1). cM,
centimorgan; Rec., number of recombinants.
(B) A single base substitution of A for G was found at the end of the third intron and can be detected in
isn2 by a cleaved amplified polymorphic sequences (CAPS) marker. The PCR products amplified from
isn2 and Col-0 genomic DNA were digested with Mse I and separated in an agarose gel. The primers
are listed in Table S1.
(C) Luminescence from Psm ES4326-infected Col-0, npr1, isn2 npr1, F1 (of a cross between isn2 npr1
and aba3-1 npr1), aba3-1 npr1 measured with an SA biosensor-based method (Marek et al., 2010).
Values are the mean of eight independent samples with standard deviation (SD).
(D) Morphology of three-week-old npr1, isn2 npr1, F1 from a cross of isn2 npr1 and aba3-1 npr1, and
aba3-1 npr1 plants.
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(E) ABA was extracted from four-week-old Col-0, aba3-1, and isn2 plants and measured with ELISA.
Data represent the mean of three independent samples with SD. Asterisks indicate significant
differences between aba3-1 and Col-0, and isn2 and Col-0 (p < 0.05, Student’s t test). FW, fresh
weight.
(F) Morphology of npr1, isn2 npr1, and isn2 npr1 plants treated with 10 M ABA. The isn2 npr1
plants were treated with water (Mock) or ABA for six days.
Figure S2. Dependence of aba3-Mediated Resistance on SA
Plants were inoculated with a suspension of Psm ES4326 (OD600 = 0.0001). The in planta bacterial
titers were determined three days postinoculation. Data represent the mean of eight independent
samples with SD. Different letters above the bars indicate significant differences (p < 0.05, one-way
ANOVA).
Figure S3. Effects of ABA on NPR1 Gene Expression
Leaves of four-week-old soil-grown Col-0, ProNPR1:Myc-NPR1, and Pro35S:NPR1-GFP plants were
treated with 80 M ABA for 24 hours. Total RNA was extracted from the treated leaf tissues and
analyzed for gene expression by qPCR. Expression of the tested gene was normalized against
constitutively expressed UBQ5. Data represent the mean of three independent biological replicates with
SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t test).
(A) Expression levels of the ABA-responsive gene RAB18 in Col-0 leaves treated with (+) or without (-)
ABA.
(B) Expression levels of NPR1 in Col-0 leaves treated with or without ABA.
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(C) Expression levels of Myc-NPR1 in ProNPR1:Myc-NPR1 leaves treated with or without ABA.
(D) Expression levels of NPR1-GFP in Pro35S:NPR1-GFP leaves treated with or without ABA.
Figure S4. Effects of ABA on Pathogen Growth in Pro35S:NPR1-GFP and Pro35S:npr1C156A-GFP
Plants
Four-week-old soil-grown Pro35S:NPR1-GFP and Pro35S:npr1C156A-GFP plants were inoculated
with Psm ES4326 (OD600 = 0.0001) with (+) or without (-) 80 M ABA. The in planta bacterial titers
were determined 3 days post-inoculation. Data represent the mean of eight independent samples with
SD. The asterisk shows significant interaction between genotype and treatment (Two-way ANOVA, p =
0.0104), indicating that ABA treatment enhanced susceptibility to Psm ES4326 to a greater extent in the
Pro35S:npr1C156A-GFP plants than in the Pro35S:NPR1-GFP plants. cfu: colony-forming units.
Figure S5. Antagonism between SA and ABA on NPR1-GFP Protein Stability
For (A) to (C), three to four-week-old soil-grown Pro35S:NPR1-GFP plants were treated with SA and
ABA in different combinations. Total protein was extracted from leaf tissues collected at the indicated
time below and analyzed by reducing SDS-PAGE and immunoblotting with anti-GFP antibody.
Ponceau S staining of Rubisco confirmed equal loading.
(A) NPR1-GFP protein levels in plants treated with (+) or without (-) 80 M ABA for 24 hours prior to
treatment with or without 1 mM SA for another 24 hours. Please note that the NPR1-GFP protein levels
in Pro35S:NPR1-GFP plants can be strongly induced by 1 mM SA (Tada et al., 2008), but only slightly
by 0.5 mM of the SA analogs 2,6-dichloroisonicotinic acid (INA) (Mou et al., 2003).
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(B) NPR1-GFP protein levels in plants treated with or without 80 M ABA and/or 1 mM SA for 24
hours.
(C) NPR1-GFP protein levels in plants treated with or without 1 mM SA for 24 hours prior to treatment
with or without 80 M ABA for another 24 hours.
(D) Pro35S:NPR1-GFP plants were treated as in (A) to (C) and leaf tissues were viewed with a
fluorescence microscope.
Figure S6. SA Induces NPR1 Protein Accumulation in a Concentration-Dependent Manner
Myc-NPR1 protein levels in four-week-old soil-grown ProNPR1:Myc-NPR1 plants treated with the
indicated concentrations of SA for 24 hours. Total protein was extracted and analyzed by reducing
SDS-PAGE and immunoblotting with anti-Myc antibody. The Ponceau S-stained Rubisco large subunit
served as a loading control.
Figure S7. Effects of Mock Treatment on NPR1 Protein Levels
Leaves of four-week-old soil-grown ProNPR1:Myc-NPR1 plants were infiltrated with 10 mM MgCl2.
Total protein was extracted from leaf tissues collected at the indicated time points and analyzed by
reducing SDS-PAGE and immunoblotting with anti-Myc antibody. The Ponceau S-stained Rubisco
large subunit served as a loading control.
Figure S8. Accumulation of Total SA in Local and Adjacent Tissues
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(A) Dynamic changes in total SA levels in the local tissues. Four-week-old soil-grown wild-type Col-0
and aba3 plants were inoculated with Psm ES4326 (OD600 = 0.001) and the inoculated leaves (local
tissues) were collected at the indicated time points for total SA measurement (SAG, 2-O-b-Dglucosylsalicylic acid).
(B) Dynamic changes in total SA levels in the adjacent tissues. Half leaves of four-week-old soil-grown
Col-0 and aba3 plants were inoculated with Psm ES4326 (OD600 = 0.002) and the uninoculated halves
(adjacent tissues) were collected at the indicated time points for total SA measurement.
Data represent the mean of three independent biological replicates with SD, and an asterisk indicates
significant difference between Col-0 and aba3 (p < 0.05, Student’s t test).
Figure S9. Effects of a Low Concentration of ABA on Psm ES4326 Growth
(A) Five-week-old soil-grown ProNPR1:Myc-NPR1 aba3 plants were inoculated with Psm ES4326
(OD600 = 0.0001) with or without the indicated concentration of ABA. The in planta bacterial titers
were determined three days postinoculation. Data represent the mean of eight independent samples with
SD. Different letters above the bars indicate significant differences (p < 0.05, one-way ANOVA).
(B) Five-week-old soil-grown ProNPR1:Myc-NPR1 aba3 plants were inoculated with Psm ES4326
(OD600 = 0.0001) with or without 10 M ABA. The in planta bacterial titers were determined
immediately (0 hr) and 24 hours later. Data represent the mean of eight independent samples with SD.
(C) Expression levels of PR1, WRKY18, WRKY38, and WRKY62 in Figure 9B were normalized against
the bacterial numbers at 24 hr in (B). An asterisk indicates significant difference between the samples
with and without ABA addition (p < 0.05, Student’s t test).
Figure S10. Effects of Basal SA and ABA on NPR1 Protein Homeostasis
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Total protein was extracted from four to five-week-old soil-grown Col-0, sid2, aba3, and aba3 sid2
plants and analyzed by reducing SDS-PAGE and immunoblotting with anti-NPR1 antibody. The
asterisk indicates a nonspecific band used as the loading control, which was detected by a polyclonal
anti-GFP antibody.
Figure S11. Working Model for Dynamic Changes in Cellular Levels of SA, ABA, NPR1 protein, and
PR Gene Transcription During Plant Immune Responses
Under non-inducing conditions, the balance between basal SA and ABA maintains basal levels of NPR1
protein and PR gene transcription. This is important because both constitutive defense responses
(caused by increased SA or decreased ABA) and compromised immunity (caused by decreased SA or
increased ABA) are detrimental to plant growth and development (Heidel et al., 2004; Jones and Dangl,
2006). Upon pathogen infection, plant cells enter three sequential phases: activation phase, inactivation
phase, and restoration phase. In the activation phase, cellular SA levels increase, which induces NPR1
gene transcription, stabilizes NPR1 protein against ABA-promoted degradation probably by
phosphorylation, and activates NPR1 for transcription initiation through an unknown mechanism. After
initiating transcription, NPR1 is likely labeled as “exhausted” and then removed by transcriptioncoupled degradation, which allows for a new round of transcription initiation. In the inactivation phase,
cellular SA levels decrease sharply and ABA levels increase. Increased levels of ABA reduce cellular
NPR1 abundance, which decreases PR gene transcription. Finally, in the restoration phase, SA
concentrations continue to decrease and high levels of ABA drive NPR1 clearance, which inactivates PR
gene transcription. During this phase, ABA levels also decrease, resulting in a new balance between SA
and ABA at the end of the restoration phase.
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Figure S12. Immunoblot Detection of NPR1, NPR1-GFP, Myc-NPR1, and the Unspecific Band Used as
Loading Controls
(A) Total protein was extracted from four-week-old soil-grown wild-type Col-0 and npr1-3 plants and
analyzed by reducing SDS-PAGE and immunoblotting with the newly developed anti-NPR1 antibody.
(B) Four-week-old soil-grown ProNPR1:Myc-NPR1 plants were treated with defined concentrations of
SA and ABA for 24 hours. Total protein was extracted and analyzed by reducing SDS-PAGE and
immunoblotting with an anti-Myc antibody plus a polyclonal anti-GFP antibody.
(C) Four-week-old soil-grown 35S:NPR1-GFP plants were treated with or without 1 mM SA or 80 M
ABA for 24 hours. Total protein was extracted and analyzed by reducing SDS-PAGE and
immunoblotting with the polyclonal anti-GFP antibody. The intensity of the nonspecific band detected
by the polyclonal anti-GFP antibody was not altered by SA or ABA treatment.
Table S1. Primers used in this study.
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