1 Supporting Information Supplemental Table S1. Flowering time in plants grown in long-day condition (16hr- light/8hr-dark). For each genotype (n>10) grown in long-day condition, the number of rosette leaves and the days post planting required for the appearance of the first flower bud were recorded. Data from two independent experiments were shown. Statistical analysis was performed with Student’s t-test (StatView 5.0.1). Letters indicate significant difference among the samples (P<0.05). Experiment 1 Experiment 2 Leaf number at bolting Flowering time (days post planting) Leaf number at bolting Flowering time (days post planting) Col 11.0±0.2 d 22.0±0.4 g 11.0±0.2 c 21.5±0.4 de win3-1 8.5±0.3 c 17.6±0.2 d 7.9±0.3 b 17.3±0.3 c acd6-1 10.4±0.2 d 19.9±0.2 e 10.8±0.2 c 20.8±0.4 d npr1-1 6.8±0.2 b 15.6±0.2 b 6.3±0.2 a 15.7±0.2 b sid2-1 10.2±0.3 d 22.4±0.3 g 10.7±0.3 c 22.1±0.3 e pad4-1 10.5±0.3 d 22.2±0.2 g 11.1±0.3 c 22.3±0.3 e acd6-1win3-1 8.7±0.2 c 16.7±0.2 c 8.2±0.3 b 17.2±0.3 c acd6-1npr1-1 10.5±0.4 d 22.9±0.2 g 11.1±0.3 c 21.8±0.2 de acd6-1sid2-1 10.1±0.3 d 22.8±0.1 g 10.8±0.3 c 21.6±0.3 de acd6-1pad4-1 10.2±0.2 d 21.3±0.2 f 10.7±0.4 c 20.7±0.3 d acd6-1win3-1npr1-1 6.1±0.2 a 13.4±0.2 a 5.9±0.2 a 13.6±0.2 a acd6-1win3-1sid2-1 8.2±0.2 c 16.4±0.2 c 7.8±0.4 b 16.9±0.2 c acd6-1win3-1pad4-1 8.6±0.3 c 16.5±0.2 c 7.9±0.4 b 16.9±0.2 c
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1
Supporting Information
Supplemental Table S1. Flowering time in plants grown in long-day condition (16hr-
light/8hr-dark). For each genotype (n>10) grown in long-day condition, the
number of rosette leaves and the days post planting required for the appearance of
the first flower bud were recorded. Data from two independent experiments were
shown. Statistical analysis was performed with Student’s t-test (StatView 5.0.1).
Letters indicate significant difference among the samples (P<0.05).
Experiment 1 Experiment 2
Leaf number
at bolting
Flowering time
(days post planting)
Leaf number
at bolting
Flowering time
(days post planting)
Col 11.0±0.2 d 22.0±0.4 g 11.0±0.2 c 21.5±0.4 de
win3-1 8.5±0.3 c 17.6±0.2 d 7.9±0.3 b 17.3±0.3 c
acd6-1 10.4±0.2 d 19.9±0.2 e 10.8±0.2 c 20.8±0.4 d
npr1-1 6.8±0.2 b 15.6±0.2 b 6.3±0.2 a 15.7±0.2 b
sid2-1 10.2±0.3 d 22.4±0.3 g 10.7±0.3 c 22.1±0.3 e
pad4-1 10.5±0.3 d 22.2±0.2 g 11.1±0.3 c 22.3±0.3 e
acd6-1win3-1 8.7±0.2 c 16.7±0.2 c 8.2±0.3 b 17.2±0.3 c
acd6-1npr1-1 10.5±0.4 d 22.9±0.2 g 11.1±0.3 c 21.8±0.2 de
acd6-1sid2-1 10.1±0.3 d 22.8±0.1 g 10.8±0.3 c 21.6±0.3 de
acd6-1pad4-1 10.2±0.2 d 21.3±0.2 f 10.7±0.4 c 20.7±0.3 d
acd6-1win3-1npr1-1 6.1±0.2 a 13.4±0.2 a 5.9±0.2 a 13.6±0.2 a
acd6-1win3-1sid2-1 8.2±0.2 c 16.4±0.2 c 7.8±0.4 b 16.9±0.2 c
acd6-1win3-1pad4-1 8.6±0.3 c 16.5±0.2 c 7.9±0.4 b 16.9±0.2 c
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Supplemental Table S2. Primer sets used in northern blotting and RT-PCR.
LITERATURE CITED
Ishikawa M, Kiba T, Chua NH (2006) The Arabidopsis SPA1 gene is required for circadian clock function and photoperiodic flowering. Plant J 46: 736-746
Mockler TC, Yu X, Shalitin D, Parikh D, Michael TP, Liou J, Huang J, Smith Z, Alonso JM, Ecker JR, Chory J, Lin C (2004) Regulation of flowering time in Arabidopsis by K homology domain proteins. Proc Natl Acad Sci U S A 101: 12759-12764
Gene Forward primer (5' to 3') Reverse primer (5' to 3') Reference
Primer sets used to make probes for northern blotting EF1a GCTGTCCTTATCATTGACTCCACC TCATACCAGTCTCAACACGTCC Lu et al., 2009
PDF1.2 AATACACACGATTTAGCACCA TCATGGCTAAGTTTGCTTCCA Lu et al., 2009
PR1 CACATAATTCCCACGAGGATC GTAGGTGCTCTTGTTCTTCCC Lu et al., 2009
WIN3 CCGTTGTCACTGGTTCAATGGGACAGT ACTGAGGCGCGTTGTTGTAGAAACCAG Lee et al., 2007
Primer sets used in RT-PCR
CO CATTAACCATAACGCATACATTTC CTCCTCGGCTTCGATTTCTC Ishikawa et al., 2006
FLC ATGGGAAGAAAAAAACTAGAAATCAA CTAATTAAGTAGTGGGAGAGTCAC Mockler et al., 2004
FT TAAGCAGAGTTGTTGGAGACG TCTAAAGTCTTCTTCCTCCGCAG Ishikawa et al., 2006
SOC1 ATGGTGAGGGGCAAAACTCAGATGAA TTCATGAGATCCCCACTTTTCAGAGA Mockler et al., 2004
ACTIN GATGAACCAGAAGGATGCATATG GAGCTTCTCCTTGATGTCTCTTAC This paper
Wang et al. Fig. S1
B A Col-0 win3-1 fls2
Figure S1. The win3-1 mutant is less responsive to flg22 treatment in root growth assay. Surface sterilized Col-0, win3-1, and fls2 (SALK_062054) seeds were grown on MS media supplemented with 1% sucrose (pH 5.7) in a tissue culture chamber with a 12hr-light and 12hr-dark cycle and 22oC. Five day-old Seedlings were transferred to 24-well tissue culture plates with sterile water plus or minus 2µM flg22. (A) Picture of the seedlings. The seedlings were photographed five days post treatment. (B) The folding difference of root length. The seedlings (n=4) were measured for the root length five days post treatment. The folding difference was calculated by water-treated root length/flg22-treated root length in each genotype. The data shown in panel (B) is the average of two independent experiments.
0
1
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3 - + - + - +
Fold
ing
diffe
renc
e of
root
leng
th
Wang et al. Fig. S2
B
A Col-0 pbs3-1 pbs3-2
0
Dis
ease
ratin
g
1
2
3
4
5
a
b b
Figure S2. The pbs3 mutants are more susceptible to Botrytis infection. 25-day-old Col-0, pbs3-1 (a second allele of WIN3), and pbs3-2 (the same mutant as win3-1; the pbs3-2 seed was provided by Mary Wildermuth’s laboratory at UC Berkley) grown in 12hr-light and 12hr-dark condition were sprayed with Botrytis spore suspension (2 ×105 spores/ml) and covered to maintain high humidity. (A) Disease symptoms. Photos were taken two days after infection with Botrytis. (B) Disease ratings. A previously described scale (0= no disease to 6 = extensive disease) was used to rate disease symptoms of the infected plants (Genger et al., 2008). Statistical analysis was performed with Student's t-test (StatView 5.0.1). Letters indicate significant difference among the samples (P<0.05).
PR1
PDF1.2
rRNA
WIN3
Figure S3. WIN3 expression in SA mutants . Total RNA was extracted from 25-day old plants for northern blot analysis. Probes specific to PR1, PDF1;2, and WIN3 were used to study expression of these genes. EF1α was used as a loading control. These experiments were repeated twice with similar results.
Wang et al. Fig. S3
Wang et al. Fig. S4
A
Figure S4. Expression of CO and SOC1 is constant in win3-1 and npr1-1 mutants in long-day condition (16hr-light and 8hr-dark). (A) Gene expression in 16-day old plants. (B) Gene expression in 25-day old plants. Plant tissue was harvested at 4 pm of a day (10 hrs after light was on in the chamber). Total RNA was extracted from the indicated genotypes and reverse-transcribed with the First Strand cDNA Synthesis kit (Fermentas) according to the manufacturer’s instructions. RT-PCR of 25 and 30 cycles showed similar results of gene expression and only the result from 30-cycle PCR was shown. ACTIN was used as a loading control. The RT-PCR product sizes are: 300 bp for CO; 536bp for SOC1, and 513bp for ACTIN. The size of the corresponding genomic fragment for each gene that can also be amplified with the RT-PCR primers is: 2275bp for SOC1 and 588bp for ACTIN. Note the absence of SOC1 genomic fragment due to its large size that was not amplified from the genomic DNA template under the RT-PCR condition. For CO, the primers anchor on the junctions of two exons, therefore, no PCR product should be amplified with the genomic DNA template. These experiments were repeated two times with similar results.
