Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105 ...€¦ · Supplemental Data. Hsu et al....

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 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.


2


3

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.


4

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.


5

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

0

10

20

30

40

0 2 4 6 8 10Time under submergence (h)

Fold

induc

tion

**

****

**

**

***

Columbiawrky22-1wrky22-2


6

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

10

15

20

25

0

5

10

15

20

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%


7

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.


8

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

****

**

**

**

**

**

**

****

**

**

**

******

***

**

*

*

*

*

**

*

*

*

*

*

* *

*

*

* * *

**

**

**** ** **

****

**

******

** ****

****

****

**

****

****

**

**

**

** **

**

**

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.


9

1 3 6 1 3 6 Time undersubmergence (h)

Fold-change

Fold-change

Fold-change

Columbia wrky22-2

LecRK-I.2LecRK-I.7LecRK-I.8LecRK-II.1LecRK-IV.2LecRK-V.5LecRK-V.7LecRK-V.9LecRK-VI.2LecRK-VI.4LecRK-VII.1LecRK-VII.2LecRK-IX.2LecRK-S.1LecRK-S.2LecRK-S.4

At3g45390At5g60270At5g60280At5g59260At3g53810At3g59700At3g59740At4g29050At5g01540At5g01560At4g04960At4g28350At5g65600At1g15530At2g32800At5g06740

1 2 4 8


Columbia wrky22-2


Columbia wrky22-2

LecRK-I.5LecRK-IV.4LecRK-VI.1LecRK-VIII.1LecRK-VIII.2

At3g45430At4g02420At3g08870At3g53380At5g03140

1 0.5 0.25 0.125

AOBAK1

At5g14760At4g33430

1 2 4 6

A

B

C

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.


10

Supplemental Table 1. List of innate immunity and stress-regulated marker genes.

Gene name Marker Locus Other Name Reference

FRK1 Innate immunity At2g19190 FLG22-INDUCED RECEPTOR-LIKE KINASE 1

Asai et al., 2002; Boudsocq et al., 2010

WRKY29 Innate immunity At4g23550 WRKY DNA-binding protein 29 Asai et al., 2002

GST1 Innate immunity At1g02930 GLUTATHIONE S-TRANSFERASE 1 Asai et al., 2002

PHI-1 Innate immunity At1g35140 PHOSPHATE INDUCED 1 Boudsocq et al., 2010 NHL10 Innate immunity At2g35980 NDR1/HIN1-LIKE 10 Boudsocq et al., 2010 PER62 Innate immunity At5g39580 PEROXIDASE62 Boudsocq et al., 2010 PER4 Innate immunity At1g14540 PEROXIDASE4 Boudsocq et al., 2010

CYP82C2 Innate immunity At4g31970 CYTOCHROME P450, FAMILY 82, SUBFAMILY C, POLYPEPTIDE 2

Boudsocq et al., 2010

CYP81F2 Innate immunity At5g57220 CYTOCHROME P450, FAMILY 81, SUBFAMILY F, POLYPEPTIDE 2

Boudsocq et al., 2010

WAK2 Innate immunity At1g21270 WALL-ASSOCIATEDKINASE 2 Boudsocq et al., 2010

FOX Innate immunity At1g26380 FAD-LINKED OXIDOREDUCTASE Boudsocq et al., 2010

CBP60g Innate immunity At5g26920 CAM-BINDING PROTEIN 60-LIKE G Wang et al., 2009

GST6 Oxidative stress At2g47730 GLUTATHIONE S-TRANSFERASE 6

Asai et al., 2002; Kovtun et al., 2000

RD29A Cold, drought, ABA At5g52310 RESPONSIVE TO

DESSICATION 29A Asai et al., 2002; Kovtun et al., 2000

Asai, T. et al. MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977-983 (2002).

Boudsocq, M. et al. Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464, 418-422

(2010).

Wang, L. et al. Arabidopsis CaM binding protein CBP60g contributes to MAMP-induced SA accumulation and is

involved in disease resistance against Pseudomonas syringae. PLoS Pathog 5, e1000301 (2009).

Kovtun, Y., Chiu, W.-L., Tena, G. & Sheen, J. Functional analysis of oxidative stress-activated mitogen-activated

protein kinase cascade in plants. Proc Natl Acad Sci USA 97, 2940-2945 (2000).


11

Supplemental Table 2. Differential expression of selected submergence-responsive genes in

WRKY22 knock-out plants

Log2 ratio (submergence/control) Columbia wrky22-2 Gene name Probe Time(h) 1 3 6 1 3 6 Description Immunity related FRK1 A_84_P14446* 0.3 4.6 4.8 -0.5 1.8 2.1 FLG22-induced receptor-like kinase 1 MLO12 A_84_P20166* 2.1 3.4 3.6 1.8 1.2 2.7 MLO-like protein 12 MLO12 A_84_P836887* 2.3 3.7 3.7 2.0 2.1 2.9 MLO-like protein 12 RLP33 A_84_P852482 0.7 4.1 3.9 0.1 2.5 2.1 receptor like protein 33 RLP46 A_84_P837183 0.1 1.9 2.6 0.0 0.9 0.7 receptor like protein 46 AT5G44910 A_84_P13086* 2.0 4.1 4.5 1.4 2.8 3.8 Toll-Interleukin-Resistance domain-containing protein AT5G41740 A_84_P22532 3.7 2.2 2.3 3.4 1.7 0.3 TIR-NBS-LRR class disease resistance protein AT1G51820 A_84_P13200* 3.1 4.4 3.7 2.6 3.5 2.3 putative LRR receptor-like protein kinase HLECRK A_84_P10912 1.2 2.4 3.2 1.1 1.6 1.7 lectin-receptor kinase EP3 A_84_P13726 0.5 1.9 2.4 0.5 1.2 2.1 chitinase PP2-A7 A_84_P305590* 1.1 2.8 3.4 0.5 2.0 1.3 phloem protein 2-A7 AT3G29970 A_84_P131666 2.0 6.3 7.9 1.9 5.4 5.7 B12D protein AT2G17740 A_84_P220868 1.9 5.4 6.7 1.4 3.1 4.4 cysteine/histidine-rich C1 domain-containing protein Transcription factors WRKY22 A_84_P16606* 4.9 4.4 3.9 0.8 0.7 0.8 WRKY transcription factor 22 WRKY49 A_84_P192244* 0.4 2.3 2.7 -0.1 0.0 1.3 WRKY transcription factor 49 WRKY53 A_84_P763290 3.4 3.3 3.2 2.8 2.4 3.3 WRKY transcription factor 53 MYB15 A_84_P870349 2.2 3.3 5.1 1.2 1.5 3.5 myb domain protein 15 AT1G64380 A_84_P18782 1.4 2.3 2.0 0.9 0.9 1.2 ethylene-responsive transcription factor ERF061 AT5G61890 A_84_P837351 0.2 1.8 3.0 0.3 0.7 1.5 ethylene-responsive transcription factor ERF114 AT5G61890 A_84_P15041* 0.5 5.6 6.4 0.0 2.4 4.2 ethylene-responsive transcription factor ERF114 Carbohydrate & amino acid metabolism related SUS2 A_84_P830821* 0.8 4.6 4.0 1.4 4.8 2.3 sucrose synthase 2 AT1G22650 A_84_P13295* 0.7 1.1 1.7 0.7 -0.9 0.4 putative neutral invertase PDC3 A_84_P13921 0.6 1.5 2.3 0.9 2.6 0.8 pyruvate decarboxylase 3 AT5G01320 A_84_P310283* 4.3 5.7 6.7 3.9 5.6 4.7 pyruvate decarboxylase GAD4 A_84_P305850 2.1 2.5 2.4 1.8 1.4 1.1 glutamate decarboxylase 4 GLT1 A_84_P838187* -0.1 -0.8 1.2 -0.6 -0.1 0.1 glutamate synthase 1 FAMT A_84_P813757* 2.3 0.8 -1.1 0.8 -1.2 -1.4 farnesoic acid carboxyl-O-methyltransferase Redox related NUDX25 A_84_P22822* 0.6 2.1 2.8 0.4 1.0 1.6 nudix hydrolase 25 CYP76C2 A_84_P11607* -0.2 2.5 2.9 1.0 0.5 1.7 cytochrome P450, family 76, subfamily C2 CYP710A3 A_84_P22962* 0.2 3.8 4.7 0.5 3.5 2.6 cytochrome P450, family 710, subfamily A GSTU11 A_84_P18100 4.0 4.8 5.8 3.1 3.3 3.8 glutathione S-transferase TAU 11 AT5G58660 A_84_P834496* 0.8 2.2 2.4 0.9 1.4 1.6 oxidoreductase, 2OG-Fe(II) oxygenase family protein AT4G31760 A_84_P17653* 0.6 3.0 3.1 0.8 2.0 1.0 peroxidase 46 (PER46) AT5G39580 A_84_P16848 4.2 4.5 4.6 4.0 3.1 4.3 peroxidase 62 (PER62) AT2G38240 A_84_P13535* 3.0 2.8 2.9 1.7 2.6 1.1 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase-like protein AT2G36690 A_84_P13494* 2.5 3.9 3.9 2.3 2.0 2.6 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase-like protein Transporters WR3/NRT3.1 A_84_P241903 0.2 2.0 2.4 0.2 1.5 1.6 nitrate transmembrane transporter OPT1 A_84_P10290* -0.2 2.2 3.0 0.2 0.7 1.6 oligopeptide transporter 1 OPT6 A_84_P18581 0.1 2.6 2.7 0.3 1.9 2.1 oligopeptide transporter 6 OPT5 A_84_P15738 0.0 1.1 1.8 0.3 0.6 0.1 oligopeptide transporter 5 OPT8 A_84_P17845 0.3 1.9 1.7 0.3 0.9 1.0 oligopeptide transporter 8 AT2G29870 A_84_P68924* 4.8 7.6 8.3 4.4 6.9 6.0 aquaporin NIP


12

AT4G15236 A_84_P94319 2.9 2.9 3.1 2.3 1.3 0.7 ABC transporter G family member 43 Heat Shock proteins HSP23.6-MITO A_84_P15731* 2.2 5.4 5.8 1.7 3.3 5.1 small heat shock protein 23.6 HSP21 A_84_P13852* 2.0 5.8 5.6 1.4 2.8 4.9 heat shock protein 21 AT1G53540 A_84_P10555* 2.2 4.0 3.5 1.6 1.8 2.7 HSP20-like chaperone Protein kinases MAPKKK15 A_84_P23526* 2.5 4.2 4.0 2.3 2.2 2.4 mitogen-activated protein kinase kinase kinase 15 MPK11 A_84_P10559 5.4 4.0 4.8 4.7 2.5 3.6 mitogen-activated protein kinase 11 WAKL2 A_84_P15224 4.1 4.3 3.2 3.9 3.3 1.5 wall-associated receptor kinase-like 2 WAKL6 A_84_P17200 3.3 4.2 3.1 2.6 3.4 1.4 wall associated kinase-like 6 WAKL10 A_84_P12477 4.6 3.2 3.3 3.9 1.5 2.5 wall-associated receptor kinase-like 10 AT4G11890 A_84_P10967* 3.4 6.1 6.4 2.7 4.0 4.4 protein kinase family protein U-box proteins PUB22 A_84_P826034 2.4 2.9 3.2 2.3 2.5 2.4 E3 ubiquitin-protein ligase PUB22 AT1G53490 A_84_P725341* -0.2 1.2 1.2 0.0 0.9 0.2 RING/U-box domain-containing protein AT2G42360 A_84_P829746 1.6 3.0 3.9 1.1 2.1 3.3 E3 ubiquitin-protein ligase ATL41 Hormone biosynthesis related PSK1 A_84_P22860 0.6 1.7 2.4 0.6 1.0 1.5 phytosulfokine-beta ACS7 A_84_P17628* 3.3 4.1 4.5 2.8 2.5 3.9 1-aminocyclopropane-1-carboxylate synthase 7 YUC5 A_84_P23485 2.1 2.4 4.0 1.9 2.4 2.5 YUCCA family monooxygenase Others JAZ8 A_84_P595141* 2.4 1.9 2.1 3.4 1.0 1.3 protein TIFY 5A (JAZ8) SMU1 A_84_P14297* -0.1 0.8 1.5 0.0 0.7 0.7 WD40 repeat-containing protein SMU1 AT3G03270 A_84_P598098* 3.1 5.7 6.3 2.7 3.7 5.4 adenine nucleotide alpha hydrolases-like protein AT1G10340 A_84_P233559* 2.6 3.2 2.7 2.4 2.1 2.0 ankyrin repeat-containing protein

Footnote: The numbers indicate submergence treatment-to-control ratios of expression within each strain. Asterisk

indicates the top 20 genes according to the raw intensity ratios or normalized ratios from each time point.


13

Supplemental Table 3. Potential direct targets of WRKY22 by ChIP-chip and ChIP-Q-PCR

experiments

Log2 ratio

(submergence/control)

Gene name Percent positive

experimentsa

W-box numberb

Fold enrichment

in ChIP-Q-PCRc 1 h 3 h 6 h Description

Up-regulated genes AT4G24310 67% 4 2.3±0.9; 2.4±1.0 1.0 1.4 1.6 Unknown protein AT5G01720 67% 0 0.8±0.5; 1.1±1.0 0.2 1.5 1.8 F-box family protein (FBL3) TRE1 67% 2 3.3±1.5 0.8 2.4 2.0 Trehalase 1 VPS60.1 67% 3 1.1±1.0; 0.7±0.6 1.5 0.4 0.7 VPS60.1 AT1G02470 50% 0 -0.4 -0.4 1.3 Unknown protein AT1G03590 50% 3 0.8 1.6 1.5 Catalytic/ protein serine/threonine phosphatase AT1G07728 50% 1 0.6 1.3 1.3 OtherRNA AT1G10340 50% 4 2.6±0.7; 3.8±1.1 2.1 2.8 2.4 Ankyrin repeat family protein AT1G19540 50% 0 6.6±2.8 0.9 1.4 1.2 NmrA-like negative transcriptional regulator family protein AT2G18690 50% 2 2.3±1.0 3.7 3.7 3.6 Unknown protein AT2G39100 50% 2 0.4 1.4 1.5 C3HC4-type RING finger AT3G02070 50% 3 6.1±3.2; 6.7±4.6 1.4 1.9 2.1 OTU-like cysteine protease family protein AT3G02340 50% 0 0.8 1.5 0.9 C3HC4-type RING finger AT3G19660 50% 2 3.6±0.8; 2.4±1.5 0.7 1.3 1.6 Unknown protein AT5G19230 50% 3 2.2 2.1 1.1 Unknown protein AT5G39050 50% 2 0.3 1.3 1.4 Transferase, transferring acyl groups AT5G44910 50% 6 7.3±4.1 1.8 4.0 4.3 Toll-Interleukin-Resistance domain-containing protein AT5G59550 50% 5 2.7 0.9 1.0 C3HC4-type RING finger AT5G66675 50% 1 1.8 0.8 0.7 Unknown protein ACS7 50% 1 4.2±2.7 3.3 4.1 4.5 1-Amino-cyclopropane-1-carboxylate synthase 7 CENP-C 50% 0 0.0 0.7 1.6 Centromere protein C ERF1 50% 1 2.2±1.1; 1.3±0.6 2.3 1.6 1.5 Ethylene responsive element binding factor 1 ERO2 50% 2 74.8±39.7 0.4 1.7 2.0 Endoplasmic reticulum oxidoreductins 2 EXO70B1 50% 3 1.9 1.3 1.0 Exocyst subunit EXO70 family protein B1 GCP2 50% 0 0.1 0.7 1.7 Gamma-tubulin complex protein 2 PLL5 50% 3 0.6 1.6 2.1 Catalytic/ protein serine/threonine phosphatase PUB24 50% 1 6.8±3.0; 3.2±0.3 2.2 1.5 1.5 PLANT U-BOX 24; ubiquitin-protein ligase UGT71B6 50% 1 1.9 2.5 1.7 UDP-glucosyl transferase 71B6 UPM1 50% 0 0.2 1.6 2.9 Urophorphyrin methylase 1 Down-regulated genes AT1G27480 67% 1 -0.96 -1.68 -2.17 Lecithin:cholesterol acyltransferase family protein FAH1 67% 1 -1.18 -1.44 -1.82 Ferulate 5-hydroxylase PDF1B 67% 0 -0.19 -0.53 -1.25 Peptide deformylase 1B PSBTN 67% 0 -0.31 -0.63 -1.61 photosystem II subunit T RLK 67% 1 -1.75 -2.15 -2.18 Receptor-like kinase AT1G06000 50% 0 -1.08 -2.08 -2.01 UDP-glucosyl transferase family protein AT1G15260 50% 1 -1.96 -1.71 -2.02 Unknown protein AT1G52220 50% 0 -0.04 -0.77 -1.93 Unknown protein AT2G32480 50% 1 -0.93 -1.49 -1.99 Serin protease AT2G32560 50% 1 -0.50 -1.42 -1.90 F-box family protein AT3G12320 50% 1 -1.23 -2.01 -1.80 Unknown protein AT3G14067 50% 0 -0.40 -1.59 -2.45 Subtilase family protein AT4G24090 50% 4 -0.31 -0.79 -1.50 Unknown protein AT4G38690 50% 0 -0.45 -1.25 -1.90 1-phosphatidylinositol phosphodiesterase-related AT5G57785 50% 2 -0.64 -2.08 -2.66 Unknown protein


14

ATEXLA2 50% 1 0.59 -1.15 -1.91 Expansin-like A2 CYP706A6 50% 1 -2.38 -3.19 -3.46 CYP706A6 DRB3 50% 2 -0.95 -1.35 -1.60 DsRNA-binding protein 3 FD1 50% 1 -0.58 -1.41 -1.99 Ferredoxin 1 KT2 50% 1 -0.75 -1.28 -1.68 Potassium transporter 2 PGLP1 50% 2 -0.36 -0.73 -1.28 2-phosphoglycolate phosphatase 1 PSAH2 50% 0 -0.36 -1.45 -2.60 Photosystem I subunit H2 Selected un-regulated gene ATL5 100% 1 0.11 -0.22 -0.09 A. thaliana ribosomal protein L5 GER1 100% 2 -0.50 -0.81 -1.42 Germin-like protein 1 GRP23 100% 1 -0.24 -0.34 -0.35 Glycine-rich protein 23 AT2G31610 83% 2 -0.06 -0.13 -0.16 40S ribosomal protein S3 (RPS3A) ATRPAC42 83% 1 -0.39 -0.31 0.16 DNA-directed RNA polymerase/ protein dimerization EIF3E 83% 0 -0.27 -0.15 0.06 Eukaryotic translation initiation factor 3E INO80 83% 0 0.00 0.10 0.00 INO80 ORTHOLOG; helicase/ nucleic acid binding UGT71C2 83% 1 0.68 0.91 0.71 UDP-glucosyl transferase 71C2

Footnotes: Fourteen-day-old Columbia plants and wrky22-2 plants expressing WRKY22-18xc-myc were submerged

for 3 hours. The WRKY22 interacting DNA of these plants were enriched with anti-c-myc antibody by ChIP

protocols as described in materials and methods, followed by detection with microarrays. Peaks of enriched DNA

fragments were identified by default settings in NimbleScan software. These peaks were further filtered by

presenting between +300 to -1200 of a promoter with FDR < 0.1. The promoters that harbored a filtered peak over

three times in six independent experiments were selected. The selected promoter list was classified with its gene

expression by showing > 2 or < 0.5 fold induction (Log2 > 1 or < -1) in any time point under submergence

treatments in Columbia expression array data. For selecting un-regulated genes, the promoters that harbored a

filtered peak over five times in six independent experiments were showed in this table.

aPercent positive experiment indicates percentage of presence of the filtered peaks in six independent experiments.

bW-box number indicates that the number of defined canonical W-box (TTGACY) present in upstream -1000 of

these genes.

cFold enrichment in ChIP-Q-PCR indicates the mean ± S.D. of fold-enrichment (c-myc tagged WRKY22 in wrky22-

2 IP/ Columbia IP) from three independent biological replicates. Two numbers indicates data from two sets of

primers for different location of a promoter.


15

Supplemental Table 4. Primer lists.

Gene name AGI number Forward primer (5' to 3') Forward primer (3' to 5')

Primers for genomic DNA PCR SALK_047120-RP CAAACCAAAATCAGACAGAATCAC SALK_047120-LP GACTGTACTGCTGACGGATTATTC SALK_098205-RP GAAACAATAGGAAGAAAACCCG SALK_098205-LP TCTCTGTTTTTATTTGGTTGCTTG LBb1.3 ATTTTGCCGATTTCGGAAC Primers for Q-RT-PCR TUB3 At5g62700 GGTTGGTTTTGCTCCTCTCACC TAGCGTCCGTGCCTTGGGTC

WRKY6 At1g62300 GGTGGCTGATACAATAACGGCA GCCGTCGTGGTGGTTCGT

WRKY8 At5g46350 CCGATCTCATCATCAATACTCCAAG GCGTATGGCACACGGAAGAGAT

WRKY11 At4g31550 CTCAAATCGTTCCTCCACCGTCT GCCGACGACATGAATGAGGAGTT

WRKY15 At2g23320 CTGATGATTCGTCCATGTTGATTG AGATCAGCGGCGGAGAGAGA

WRKY17 At2g24570 CTTCTTTCATGTCTTCTGCGATCAC GTCACTGGCACCGCTGGAG

WRKY25 At2g30250 CCTAAGCCTGAGTTCACCAAGAGAC CAACAGAAGCAGGATTAAACAAGCG

WRKY26 At5g07100 CCAAATCCCGACCCCAAGAA TTTGCTCTTTATGTCTCTGTTTTTCCA

WRKY27 At5g52830 CACACTCATCCACGTCCTACTCACC AACGGGCTGAGATTTGTTACGAGT

WRKY29 At4g23550 CGGAGATGGAGACAAGTGGCTT TGTGAGGATCGTTTGTGTGGAGAA

WRKY45 At3g01970 TGCCGTTGATAAACCCTCTGATAA CAAAAGGGAGGGAAGATGTGC

WRKY48 At5g49520 CGTGGACACATCGGAATGCT AGGGATGGAGAATGATGATGATGAC

WRKY49 At5g43290 TGCCAACAAGTTCGTCACATCA GGGTGGGTCAGGGGTTCG

WRKY63 At1g66600 CCTTTGGGGTGCATGATAATACG CCTCCATGAGAACAGCTTGTTCAC

WRKY18 At4g31800 ACAAGCCAGGGTGGGTCAAG AACGCTAGTCTATGACAGCCATTAACC

WRKY22 At4g01250 CGTCCTCTTTCTCTCTCTGCTTCTTC CCATGCCCAGACATCGGAGTTTA

WRKY28 At4g18170 CATGATATGTTTAGGACGGCAGC CACACTACCATAACCACTTTGTCCATATC

WRKY30 At5g24110 CCCTTTGGAATCCCCGTTTG GAATAGAACCCACCAAATCCTCCA

WRKY33 At2g38470 AAACTTTGTTGGTGGTGGATTCTCTA ACGAATCGAAAAATGAGGTCTCC

WRKY40 At1g80840 TCTCCTTCACGACAGTCTCTTCTCTCT TTGGCGGAGTGTCGGAGAGTAG

WRKY46 At2g46400 GAAACCGACCAAGTCCGAAGA CCACACACCGTTCTCAATCTCAT

WRKY53 At4g23810 GGAGAAGCGACAAGACACCAGA TATCCTTTGGCTTTTGGGTAATGG

PROPEP2 At5g64890 ATGATGTCGTTGTGTTGCTTAGGG GCGTTGGGTACACTATTAGTTTGGC

PROPEP3 At5g64905 TCAACGATGGAGAATCTCAGAAATGG CAAAGAACGTAAATGGGATCAAAGAACC

ACS7 At4g26200 GATACCATTGTGGAAGGGCTTAA CCCTGCGTTCCCTTTCAAA

FRK1 At2g19190 GCTTGTACCGAGCACACTTCTG CAGTCACTATGCCATAAACAATCTGTT

MYB15 At3g23250 AGTGAGATGGACTTCTGGTTCGA ACCATTTCACTTTACTCCTCAAACATG

At2g42360 GCCTCCCCGTCATTTTCCG GCAGCCACGCCATCAGTG

At5g43580 GCAGTTATCATTTTGGATGGAACA GTTGACCCAAACGTAAACACGAG

MLO12 At2g39200 TCAGCAATAGTCATCATCCCCAAG TGCTCAACAATCTCCTTCTCTTCTCTAAC

NRT3.1 At5g50200 CTTGGACTCTTGAGCGTGACATC TGCTCTGTCCATAGGCAACTTCA


16

PSK1 At1g13590 CCTCCTAAACCATCTCCCACCA GCACTCTACTCCATCTCTGTCACCTT

NUDX25 At1g30110 GATTCAGAGTTTGCGGAGTGGA ATCCACTGCTTGCTCTACCACTTCT

EP3 At3g54420 TCCAGTCATATCCTTCAAAACCG GCACCAAAACCTTGAGAGATAACAG

OPT1 At5g55930 GGTTGTCCCGTGTTTTAAGAATTG GCCATCAACAGTTACCCCTTTC

At5g44910 AGTGGGAGGATTCATTGAGTTCAA CGTGGTCTTCGGAGGTTAAGC

ERO2 At2g38960 ATGAATCTTCTCCACCGATTATCTGA GCCGCAGGACTCATGTCAT

TRE1 At4g24040 CTCATTGCTCCCGCTGGAATC TCGGAGAATCCCATTGTTGTCCT

PUB24 At3g11840 CTTGCTCATAGGGGCGGAATC TCGTCTGCCGCAGGTGAAA

At1g10340 GTTTAAGAAGATGAATCGTGTAGGGGATA GGAGACGGCTAAATCTGAACTGGTT

At3g02070 TCAGTTTGTGATAAGTCTACCGTCTCATAC GTTACAGCTCAAGCGAGTTCGTAAG

At1g19540 TCTCTACATTCACCCGCCCAA TCCTCCCACAAACCAACCATATC

At3g19660 TCGCCATCCGTCGTAAGAAGA CAGGACGATCAGCGACCTTTAAG

At4g24310 TGCTCCTCACGCTTCTCTCCAT AGCCTTGACGCTGTCAGTGAAG

At2g18690 TGAGTCGCTCAGGGATGTGGAA AAGGCAATCATTCCGTGAGAGTAGT

Primers for RT-PCR transgenic c-myc tagged WRKY22 CGTCCTCTTTCTCTCTCTGCTTCTTC CACCACTAGCAGCAGAACCG

WRKY22 At4g01250 CGTCCTCTTTCTCTCTCTGCTTCTTC TATGCTCCGCCGTGTAAGTGAC

ACTIN 8 At1g49240 CGACTACGAGCAAGAGATGGAGACCT GGCTGGATTCGCTGGAGATGA

Primers for ChIP-Q-PCR At1g10340-p region1 TGGATATGCGGGATCTAATAATGG GCCAAGTTCTACCTCAAAACGATATG

At1g10340-p region2 AGAGGAACCTGCGTTTTAGTTTGT ATATTCGTTGACTTCGTAAGCAGAAG

At1g19540-p TTGGTTGTGTGAGGGTGTAATTTACTT AGCACACGCTATTTCATCAAGGA

At2g18690-p CGGTCAGGAAGTCATCATTTTTAGACT GAAACCAAGACTTAGACAAATCCAAGA

At3g02070-p region1 CGTTTGTGCTGGATGAAGTTGAC CATCACCTGATCTCTGAAGCCTCTCT

At3g02070-p region2 TGGAATGTGCCAATAAAAAAGAAGG AGACAATGGCAGTTGGAACCG

At3g19660-p region1 CCAAGTGTTGTAAGTGATGCAAAAT ATAATGGAGAAGCCGGAGATGTT

At3g19660-p region2 ACTGATGACATCTACAGCAGTTGACTACT CGATATTGCCACTAGTTGTCCTTCTT

At4g24310-p region1 GCATCCAAATACCTAGAAAAAAAAGAAG AACCCTGGCTAAGGTAAATAAAACCG

At4g24310-p region2 AAGAAGGAATCAAAAGAGACCCATTG GTGTAGAAGATGGTTGTTGACGTGAT

At5g01720-p region1 TGGAAGAGCAAGGGCAATTAAGTAA ACAGTTTGTTTTCTGTGGGAGAGAGA

At5g01720-p region2 TGGAGAAACGGCCAGAAATATT ACGCATACATGTGTCTTATAGCGTTA

At5g44910-p ATGAAGTTGTTGAGTGTCCCTAAAAACA GACCACGACTACCGAGCTGCT

ACS7-p At4g26200 CCTAAAGATGAACCACCGAGAAGT GTGTCATTGACCTGCAAATGGA

ERF1-p region1 At3g23240 GAGTCCACTTAGAAATTATAAGATGGGAA TGAATCTAGAAGAAAATAAGATGGCGAT

ERF1-p region2 At3g23240 TTTTGTTGAGTTGATCCAGCAGTT GAACAGAAATAACAAGATCGATGTGAA

ERO2-p At2g38950 GAATCGCAAAGGATGGACAAGAGT TCCCCAAGACATACACAGCATTCC

PUB24-p region1 At3g11840 CTTTATCTGCCCAATCTCACTAGAGATC GGTCGTAAGTGATGCCAGAAACG

PUB24-p region2 At3g11840 TGTTTGCTGACCATGAGTTGAATAGAG ACTCGGCTTTAGAACATGGTCTGAA

TRE1-p At4g24040 TCCAACTGCTGAAGAAGCAAGTATG GCCAAGAATTATCAAAGAGACAGAGA

VPS60.1-p region1 At3g10640 CAAGGAGGTGGGAGCTTTTCAC GATTCTGCGGCCTGACTTATCG


17

VPS60.1-p region2 At3g10640 AGCTTCTTTTAGTTGACCAATTCCAT AGAAGGCTTCTCTAATAAAAGGATTGAGA

Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105 ...€¦ · Supplemental Data. Hsu et al....

Documents

Transcript of Supplemental Data. Hsu et al. Plant Cell. (2013). 10.1105 ...€¦ · Supplemental Data. Hsu et al....