Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447 1 Supplemental Figure 1. Induction of DAMP markers, PROPEP2 and PROPEP3, in response to submergence. (A) Expression profiling of DAMP marker genes upon submergence. Gene expression was determined by microarray analysis in nine-day-old wild-type Arabidopsis (Columbia) seedlings with four independent biological replicates. The color scale indicates treatment-to-control ratio of expression in log 2 or in fold-induction. (B) Induction of PROPEP2 and PROPEP3 upon submergence. Gene expression was validated by Q-RT-PCR in nine-day-old wild-type Arabidopsis (Columbia) seedlings from at least four independent biological replicates. The data represent means ± SD from five to six independent repeats.
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Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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Supplemental Figure 1. Induction of DAMP markers, PROPEP2 and PROPEP3, in response to submergence. (A) Expression profiling of DAMP marker genes upon submergence. Gene expression was determined by microarray analysis in nine-day-old wild-type Arabidopsis (Columbia) seedlings with four independent biological replicates. The color scale indicates treatment-to-control ratio of expression in log2 or in fold-induction. (B) Induction of PROPEP2 and PROPEP3 upon submergence. Gene expression was validated by Q-RT-PCR in nine-day-old wild-type Arabidopsis (Columbia) seedlings from at least four independent biological replicates. The data represent means ± SD from five to six independent repeats.
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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Supplemental Figure 2. Expression of innate immunity marker genes, PR genes, submergence-early-induced WRKYs and submergence-late-induced WRKYs upon abiotic and biotic stresses. Gene lists from Fig. 1. were submitted to Genevestigator (Hruz et al., 2008) to request all available expression data on studies related to abiotic (A) and biotic (B) stresses. The color scale shown indicates log2 expression ratio (treatment/control). A. brassicicola, Alternaria brassicicola; B. cinerea, Botrytis cinerea; B. garminis, Blumeria graminis; B. tabaci, Bemisia tabaci, the silverleaf whitefly; CaLCuV, Cabbage leaf curl virus; E. cichoracearum, Erysiphe cichoracearum; E. coli, Escherichia coli; E. orontii, Erysiphe orontii; G. cichoracearum, Golovinomyces cichoracearum; G. rosea, Gigaspora rosea; H. schachtii, Heterodera schachtii, a plant pathogenic nematode; M. incognita, Meloidogyne incognita, a plant pathogenic nematode; M. persicae, Myzus persicae, the green peach aphid; P. infestans, Phytophthora infestans; P. parasitica, Phytophthora parasitica; P. syringae, Pseudomonas syringae; TuMV, Turnip mosaic virus; EF-Tu, elongation factor thermo unstable; FLG22, a flagellin fragment; NPP1, Phytophthora protein 1; HrpZ, a harpin protein; LPS, Lipopolysaccharides; OGs, oligogalacturonides.
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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1 cm
2 cm
A
B
Control
2hsubmergence
Control
2hsubmergence
Supplemental Figure 3. Plant immunity is triggered by short submergence. Disease symptoms assessed 5 days post-inoculation (dpi) with Pseudomonas syringae in five-week-old (A) and three-week-old (B) submerged and control Columbia plants. Short submergence treatment is submerged for 2 hours followed by 1 hour of recovery under light.
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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Supplemental Figure 4. Isolation of WRKY22 T-DNA insertion mutants.
(A) Genomic structure of WRKY22. Orange arrows, blue lines and green lines indicate exons, introns and UTRs, respectively. Thick black arrows indicate T-DNA insertions. Small black arrows indicate primers. Small red arrows indicate primers for Q-RT-PCR. (B) Homozygosity tests for SALK_047120 and SALK_098205. Genomic DNA of the wild-type Arabidopsis (Columbia), SALK_047120 and SALK_098205 were amplified by PCR using LBb1 and RP primers for T-DNA inserted alleles and RP and LP primers for no T-DNA inserted alleles. (C) WRKY22 transcript levels in Columbia and WRKY22 T-DNA insertion mutants. Nine-day-old wild-type seedlings were submerged for up to 9 hours and RNA was isolated at specific time points (0, 1, 3, 6 and 9 h). Transcript levels were detected by Q-RT-PCR using specific primers (AtWRKY22-1 and AtWRKY22-4r in (A)). TUBULIN mRNA was used as an internal control. The data represent means ± S.D. from six independent biological replicates. Statistical differences between Columbia and WRKY22 mutants are detected with Student’s t-tests. *, P < 0.05; **, P < 0.01.
RP & LP
RP & LBb1
Col Col047120 098205SALK SALK
(wrky22-1)
SALK_047120-LP
SALK_047120-RP
SALK_098201-LP SALK_098201-RP
3' UTR5' UTR
SALK_047120 T-DNASALK_047120 T-DNALBb1
SALK_098205 T-DNASALK_098205 T-DNA
LBb1
AtWRKY22-1 AtWRKY22-4r
(wrky22-1)
(wrky22-2)
(wrky22-2)
A
B C
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0 2 4 6 8 10Time under submergence (h)
Fold
induc
tion
**
****
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***
Columbiawrky22-1wrky22-2
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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Supplemental Figure 5. WRKY22 mediates basal resistance and submergence-triggered resistance to Pseudomonas syringae. Five-week-old wild-type (Columbia) plants and WRKY22 T-DNA insertion mutants (wrky22-1 and wrky22-2) that grew in pots were submerged in the dark for 12 h (and control plants were left in the dark only for 12h) followed by 1 h recovery under light before dip-inoculation with 1×107 cfu/ml Pst DC3000. (A) Disease symptoms in control and submerged Columbia, wrky22-1, and wrky22-2 plants at 4 dpi. Scale bar: 2 cm. (B) Levels of resistance in control and submerged Columbia, wrky22-1, and wrky22-2 plants. The levels of resistance were defined using a damage index based on the necrotic and chlorotic area of leaves ( black, 100% leaf area; dark gray, equal to or more than 50% leaf area; light gray, less than 50% leaf area; white, no damage observed). The data represent means ± SD from eight independent repeats. Pair wise statistical differences between Columbia and WRKY22 mutants are detected with Student’s t-tests for the sum of 100% and ≥50% indexes. *, P < 0.05; **, P < 0.01.
2 cm
Control
wrky22-1 wrky22-2Columbia
Submerged
A
B Damaging index
SubmergedControl
0
5
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25
0
5
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15
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25
Columbia
wrky22-
1
wrky22-
2Colu
mbia
wrky22-
1
wrky22-
2
0<50%≧50%100%
** **
Leaf
num
ber
Leaf
num
ber
**
0<50%≧50%100%
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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A
B
Supplemental Figure 6. Transgenic c-myc tagged WRKY22 are induced by submergence at transcript and protein levels. (A)Transcript levels were detected by RT-PCR using specific primers in nine-day-old wild-type Arabidopsis (Columbia) and WRKY22-18×c-myc in wrky22-2 seedlings under submergence. ACTIN mRNA was used as an internal control. (B) Protein levels of c-myc tagged WRKY22 and Tubulin were detected by Western blotting using specific antibodies against c-myc and Tubulin, respectively.
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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NUDX25 At2g42360
WR3/NRT3.1 MYB15Transporters Transcrption factors
U-box proteinRedox related
Columbia
wrky22-1wrky22-2
024681012
0 2 4 6 8 10
01234567
020406080
100120140160180
02468
101214
0 2 4 6 8 10
Fold
indu
ctio
nFo
ld in
duct
ion
Time under submergence (h)
Time under submergence (h)
*
*
*
***
****
****
****
****
**
** ****
******
****
******
**** **** **
******
**
0123456
Columbiawrky22-ko1wrky22-ko2
ERO2 TRE1
0 02468
1012
PUB24
12345678
02468
1012141618
At1g10340
At3g19660
At3g02070 At1g19540
0123456
0123456
012345678
0 2 4 6 8 1001234567
0 2 4 6 8 10
At4g24310 At2g18690
05
101520253035
0 2 4 6 8 10
****
**
**
**
**
**
**
****
**
**
**
******
***
**
*
*
*
*
**
*
*
*
*
*
* *
*
*
* * *
**
**
**** ** **
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** **
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**
A WRKY22 targets from expression array
B WRKY22 targets from ChI P
Supplemental Figure 7. Effects of WRKY22 knock-out on transcript levels of WRKY22 target genes. WRKY22 target genes were selected from expression array data (A) and ChIP experiments (B). Nine-day-old Columbia, wrky22-1, and wrky22-2 seedlings were submerged up to 9 hours and collected at specific time points (0, 1, 3, 6 and 9 h). Transcript levels were detected by Q-RT-PCR using specific primers. TUBULIN mRNA was used as an internal control. The data represent means ± SD from four to eight independent biological replicates. *, P < 0.05 and **, P < 0.01 in Student’s t-test.
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
Supplemental Figure 8. Regulation of LecRKs and stomatal-immunity-associated genes under submergence. Expression of LecRKs is upregulated (A) or downregulated (B) by submergence. (C) Expression of L-ASPARTATE OXIDASE (AO) and BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) under submergence. Gene expression was determined by microarray analysis in nine-day-old wild-type (Columbia) plants and a WRKY22 T-DNA insertion mutant (wrky22-2) from four independent biological replicates. Listed LecRKs were screened with > 2-fold induction or repression in expression at any one time point from 1 to 6 hours under submergence in the wild-type Columbia strain. Arrows indicate that genes are known to mediate stomatal innate immunity.
Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105/tpc.113.114447
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Supplemental Table 1. List of innate immunity and stress-regulated marker genes.