doi: 10.

1038/nature08854

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Supplementary Figure 1| Induction of rapid JAZ1 degradation by JA. a, Schematic

overview of the T-DNA constructed by multisite Gateway cloning with the destination vector

pKCTAP. LB and RB, T-DNA borders; B1, B2, B3 and B4, Gateway recombination sites; eGFP,

enhanced Green Fluorescent Protein under control of the rolD promoter; KmR, kanamycin

resistance marker under control of the nos promoter; fLUC, firefly luciferase. b, Cycloheximide

(CHX) chase experiment. Stably transformed cell cultures expressing fLUC or a JAZ1-fLUC

fusion were treated with 200 µg/mL CHX and 100 µM JA or CHX alone and sampled up to 5 min

after treatments. Average normalized fLUC activities ± s.d. are plotted. Full lines are best-fit

lines.

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Supplementary Figure 2| Induction of NINJA expression by MeJA, but not by ABA. a,

Microarray analysis of AFP and NINJA gene expression response to MeJA and ABA in 7-day-

old Arabidopsis seedlings38. Mean expression values relative to the untreated control are

plotted. Asterisks indicate a significantly differential expression (FDR corrected p-value ≤ 0.05).

b, Real-time qPCR analysis of MYC2, JAZ1 and NINJA expression over time in response to 25

µM MeJA (dashed lines) or mock treatment (solid lines). Fold changes relative to the zero time

point are plotted in function of time (hours).

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Supplementary Figure 3| Interaction of NINJA with JAZ proteins. a, Cotransformation of the

AH109 yeast strain with pGADT7-NINJA and pGBKT7-JAZ1 to JAZ12. Transformants were

spotted on SD-Leu-Trp as a control for growth (-2). To investigate interaction of bait and prey,

yeasts were dropped undiluted or in a 50-fold dilution on selective media additionally lacking His

and Ade (-4). b, Immunoblots with anti-GFP antibody of recovered NINJA-GFP after pull-down

reaction from 35S:NINJA-GFP (N) or Col-0 (C) Arabidopsis plant extracts with recombinant

MBP or MBP-fused proteins (top). Coomassie staining shows the input quantity of recombinant

proteins used (bottom). JAZ3ΔN and JAZ3ΔC represent the C- and N-terminal parts of JAZ3

that lack or hold the TIFY motif, respectively.

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Supplementary Figure 4| Yeast two-hybrid assays with JAZ1, NINJA and TPL. a,

Interaction of NINJA with non-JAZ Group-II TIFY proteins. b, Lack of interaction between JAZ1

and AFP2 or AFP3. c, Interaction of NINJA with JAZ1 derivatives that contain the TIFY motif. d,

Interaction of a JAZ1 fragment containing the TIFY motif and the NINJA domain C. e,

Interaction of TPL with AFP proteins. In all cases the PJ69-4A yeast strain was cotransformed

with Gateway compatible pGBT9 (BD) and pGAD424 (AD) vectors. Transformants were spotted

on SD-Leu-Trp as a control for growth (-2). To investigate interaction of bait and prey, yeasts

were dropped in 10- and 100-fold dilutions on selective media additionally lacking His (-3). The

pGBT9 and pGAD424 vectors were cotransformed as controls to assess auto-activation (bottom

row). Schematic drawings represent TPL, JAZ1, NINJA and derivatives. Numbers indicate

ending amino acids.

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Supplementary Figure 5| Stability and subcellular localization of NINJA-GFP. Laser

scanning confocal microscopy images of Arabidopsis seedling roots expressing JAZ1-GFP (a)

or NINJA-GFP (b). Roots were treated with 25 µM MeJA for the time indicated (minutes) and

stained with propidium iodide to visualize cell borders. GFP fluorescence was observed and

imaged with a Zeiss LSM 510 laser scanning confocal microscope. Scale bars, 50 µm.

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Supplementary Figure 6| Expression of NINJA in transgenic gain-of-function or loss-of-

function Arabidopsis plants. a, RNA gel blot analysis of NINJA expression in 3-week-old wild-

type (Col-0) and two NINJA overexpressing (OE) lines. Total RNA (8 µg) was loaded. b, Real-

time qPCR analysis of NINJA expression in 7-day-old wild-type (Col-0) and two NINJA

knockdown (KD) lines. Data are means ± s.e.m.

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Supplementary Figure 7| NINJA overexpression does not affect JAZ3 stability.

35
Quantification of S-labelled JAZ3 residual protein following incubation with cellular extracts

from wild-type (Col-0) or NINJA OE plants after indicated treatments (50 µM JA and/or 200 µM

MG132) and incubation times.

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Supplementary Figure 8| Upregulation of early MeJA responsive genes in elicited NINJA

KD plants. Shown in heat maps is the MeJA modulated expression in wild-type 7-day-old

seedlings (0.5, 1 and 3 h after treatment) of genes significantly induced in NINJA KD plants

(versus wild-type) treated with 0.5 µM coronatine for 6 h. Data were obtained with the

Expression Browser software (http://bar.toronto.ca). The scale bar indicates the log fold change.

Only genes early induced by MeJA (97 out of 169) are included. A complete list of the genes

that are differentially regulated in coronatine-treated NINJA KD plants is given in Supplemental

Table 4. Many of the genes in this list are also regulated by MeJA at later times (e.g. PDF

genes).

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Supplementary Figure 9│ Presence of EAR domains in NINJA, AFP and AUX/IAA

proteins. Protein sequence alignment of the N-termini of the 16 AUX/IAA proteins shown to

interact with TPL8, NINJA and the four AFP proteins. Depicted are amino acids 1-37, except for

IAA9, IAA11, IAA18, IAA26, IAA27 and AFP1 (all 1-70), AFP2 (47-85) and AFP4 (54-90).

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Supplementary Figure 10│ In planta NINJA-TPL interaction with Bimolecular

Fluorescence Complementation in a tobacco transient expression system. a, TPL-SPYCE

and NINJA-SPYNE b, NINJA-SPYCE and TPL-SPYNE c, TPL-SPYCE and TPL-SPYNE d, The

two halves of the BiFC complex co-transformed showing no YFP fluorescence. Scale bars, 10

µm.

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Supplementary Table 1| MALDI-TOF/TOF-MS identification of JAZ1 interactors.

Bait Prey PMF data MSMS data
Found/ Protein RMS error Sequence Unique Total Peptide Variable
1 2 3 4
Bait Specifications Prey Locus Prey Name 3 exp Score E-value (ppm) coverage % Peptides Ion Score Number Start End Observed Mr(Exp) Mr(Calc) Delta Miss Ions Score Expect Peptide Modification
JAZ1 Mock AT4G28910 NINJA 3 158 5,20E-12 4 37 11 75 1 276 292 1618,82 1617,81 1617,81 0,00 0 51 1,83E-04 K.DGSGGIVALSQSPFAGR.V
2 73 89 1908,86 1907,86 1907,86 -0,01 0 9 1,42E+00 R.SDSGQQPPQNFFNDLSK.A
3 387 403 2031,89 2030,88 2030,88 0,00 0 3 2,82E+00 K.IVCACHGSHMSPEEFVR.H Oxidation (M)
4 179 197 2124,10 2123,09 2123,10 -0,01 0 13 9,31E-01 K.EVVRPPTDTNIVDNLTGQR.R
JAZ1 Mock AT5G46760 MYC3 1 216 8,20E-18 9 21 9 165 1 192 201 1034,56 1033,55 1033,56 0,00 0 43 1,33E-03 R.AGQGQIYGLK.T
2 324 333 1091,59 1090,58 1090,60 -0,02 0 31 2,43E-02 K.DLTFQGGLLK.S
3 518 526 1102,60 1101,60 1101,60 0,00 0 14 1,23E+00 K.IIGWDVMIR.V
4 518 526 1118,60 1117,59 1117,60 0,00 0 27 5,40E-02 K.IIGWDVMIR.V Oxidation (M)
5 570 581 1467,66 1466,66 1466,66 0,00 0 18 2,34E-01 K.MGSQFFNHDQLK.V Oxidation (M)
6 447 461 1606,85 1605,84 1605,86 -0,02 0 40 1,98E-03 K.ASLLGDAISYINELK.S
7 355 370 1712,79 1711,79 1711,78 0,00 0 7 3,23E+00 K.GSNNDEGMLSFSTVVR.S
JAZ1 Mock AT5G20900 JAZ12 1 173 1,60E-13 6 39 7 114 1 161 170 1196,59 1195,58 1195,59 0,00 1 45 8,22E-04 K.NPYPTSDFKK.T
2 92 106 1529,79 1528,79 1528,78 0,00 0 51 1,67E-04 K.NSTSISPVSSPALNR.A
3 171 187 1804,88 1803,87 1803,89 -0,02 1 15 5,34E-01 K.TDVPTGNVSIKEEFPTA.
4 107 133 2864,41 2863,40 2863,40 0,00 0 2 7,46E+00 R.APSFSSTSNVASPAAQPFPIQPISFCR.S
JAZ1 +JA (1min) AT5G46760 MYC3 2 133 1,60E-09 6 16 8 97 1 192 201 1034,56 1033,55 1033,56 0,00 0 44 1,15E-03 R.AGQGQIYGLK.T
2 324 333 1091,60 1090,59 1090,60 -0,01 0 40 2,62E-03 K.DLTFQGGLLK.S
3 447 461 1606,86 1605,85 1605,86 -0,01 0 13 8,68E-01 K.ASLLGDAISYINELK.S
JAZ1 +JA (1min) AT4G28910 NINJA 2 108 5,20E-07 4 40 13
JAZ1 +JA (1min) AT5G20900 JAZ12 2 97 7,30E-06 4 36 5 43 1 92 106 1529,79 1528,78 1528,78 0,00 0 42 1,62E-03 K.NSTSISPVSSPALNR.A
2 107 133 2864,40 2863,39 2863,40 -0,01 0 2 7,66E+00 R.APSFSSTSNVASPAAQPFPIQPISFCR.S
JAZ1 +JA (1min) AT2G39940 COI1 2 65 1,10E-02 6 17 8 33 1 263 270 1055,53 1054,52 1054,53 0,00 0 0 1,72E+01 K.YMNLVFPR.K Oxidation (M)
2 517 524 1084,56 1083,55 1083,55 0,00 0 28 2,70E-02 R.YLWVQGYR.A
3 447 466 2138,08 2137,07 2137,08 -0,01 0 5 6,44E+00 R.QGGLTDLGLSYIGQYSPNVR.W
1
Number of times that a prey was found in 3 independent TAP experiments with the respective bait.
2
The E-value in Mascot corresponds to the number of times one would expect this score by chance alone.
3
Unique peptides found per identification. Peptides not present in control TAP experiments.
4
Number of peptides from which a sequence was obtained.
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Supplementary Table 2| Primary root length in 9-day-old Arabidopsis seedlings

Treatment 1 Genotype n 2 Average %3 SD SE LSmean 4 SE LSmean Interaction 5 SE Interaction unadjusted p FDR 6
(mm) p-value

Ler 20 27.95 2.8 0.6 3.3 0.03

tpl-1 21 27.3 4.7 1.0 3.3 0.03
Col-0 21 30.7 4.3 0.9 3.4 0.03
Control NINJAOE1 22 28.0 2.9 0.6 3.3 0.03
NINJAOE2 18 30.0 4.4 1.0 3.4 0.03
NINJAKD1 21 27.6 4.3 0.9 3.3 0.03
NINJAKD2 23 25.3 5.9 1.2 3.2 0.03
Ler 18 8.8 69 0.9 0.2 2.2 0.03
tpl-1 18 7.1 74 1.1 0.3 1.9 0.03 -0.2 0.1 0.004 0.004
Col-0 35 9.0 71 1.5 0.2 2.2 0.03
10 µM JA NINJAOE1 20 11.3 60 1.5 0.3 2.4 0.03 0.3 0.1 <0.0001 <0.0001
NINJAOE2 21 10.9 64 1.2 0.3 2.4 0.03 0.2 0.1 0.001 0.002
NINJAKD1 21 7.5 73 0.9 0.2 2.0 0.03 -0.1 0.1 0.322 0.403
NINJAKD2 23 7.34 71 0.9 0.2 2.0 0.03 0.0 0.1 0.814 0.904
Ler 23 7.0 75 0.6 0.1 1.9 0.03
tpl-1 18 4.7 83 0.6 0.2 1.6 0.03 -0.3 0.1 <0.0001 <0.0001
Col-0 27 80
8.0 74 10
1.0 02
0.2 21
2.1 0 03
0.03
50 µM JA NINJAOE1 34 9.4 66 0.9 0.2 2.2 0.03 0.3 0.1 <0.0001 <0.0001
NINJAOE2 30 8.9 70 1.0 0.2 2.2 0.03 0.1 0.1 0.036 0.051
NINJAKD1 25 5.8 79 1.1 0.2 1.7 0.03 -0.2 0.1 0.001 0.002
NINJAKD2 26 6.4 75 0.6 0.1 1.9 0.03 0.0 0.1 0.979 0.979
1
Seeds were germinated on Johnson's medium supplemented with jasmonic acid (JA) at the indicated concentration.
2
Number of individual seedlings measured
3
Percentage of growth reduction as compared to control treatment
4
Least square mean (ln transformed)
5
Calculated based on ln transformed data
6
False Discovery Rate corrected p-value, significant values are indicated in bold

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Supplementary Table 3| Differentially expressed genes in NINJAKD vs wild-type plants1

AGI2 logFC3 p-value4 Annotation

AT1G06620 1,18 0,003 2-oxoglutarate-dependent dioxygenase
AT1G14250 1,59 0,001 nucleoside phosphatase family protein
AT1G18710 1,44 0,001 AtMYB47
AT1G19670 1,14 0,005 ATCLH1 (CORONATINE-INDUCED PROTEIN 1)
AT1G24070 1,09 0,014 ATCSLA10 (Cellulose synthase-like A10)
AT1G52000 2,1 0,000 jacalin lectin family protein [AT1G52000.1]
AT1G52030 1,79 0,000 MBP2 (MYROSINASE-BINDING PROTEIN 2)
AT1G52040 2,12 0,000 MBP1 (MYROSINASE-BINDING PROTEIN 1)
AT1G52400 2,66 0,000 BGL1 (BETA-GLUCOSIDASE HOMOLOG 1)
AT1G52410 1,88 0,000 TSA1 (TSK-ASSOCIATING PROTEIN 1)
AT1G52890 1,14 0,006 ANAC019 (Arabidopsis NAC domain containing protein 19)
AT1G53480 3,72 0,000 unknown protein
AT1G54010 1,64 0,000 myrosinase-associated protein
AT1G54020 2,3 0,000 myrosinase-associated protein
AT1G61120 1,33 0,001 terpene synthase/cyclase family protein
AT1G62660 1,06 0,024 beta-fructosidase (BFRUCT3)
AT1G72120 1,09 0,007 transporter
AT1G73325 1,19 0,002 trypsin and protease inhibitor family protein
AT1G80120 1,12 0,005 unknown protein
AT2G02990 1,06 0,025 RNS1 (RIBONUCLEASE 1)
AT2G16367 1,98 0,000 Defensin-like (DEFL) family protein
AT2G24850 1,02 0,004 TAT3 (TYROSINE AMINOTRANSFERASE 3)
AT2G26010 2,36 0,000 PDF1.3 (plant defensin 1.3)
AT2G26020 2,17 0,000 PDF1.2b (plant defensin 1.2b)
AT2G34600 2,24 0,000 JAZ7/TIFY5B
AT2G36080 1,06 0,013 DNA-binding protein
AT2G37620 1,15 0,004 ACT1 (ACTIN 1)
AT2G38240 1,33 0,001 oxidoreductase
AT2G39030 2,51 0,000 GCN5-related N-acetyltransferase (GNAT)
AT2G43510 1,31 0,003 ATTI1 (ARABIDOPSIS THALIANA TRYPSIN INHIBITOR PROTEIN 1)
AT2G44578 1,5 0,000 protein binding / zinc ion binding
AT3G01345 4,18 0,000 Expressed protein [AT3G01345.1]
AT3G04720 1,02 0,041 PR4 (PATHOGENESIS-RELATED 4)
AT3G11480 1,09 0,006 BSMT1; S-adenosylmethionine-dependent methyltransferase
AT3G12145 1,19 0,003 FLR1 (FLOR1)
AT3G16470 1,03 0,019 JASMONATE RESPONSIVE 1
AT3G25760 1,46 0,001 AOC1 (ALLENE OXIDE CYCLASE 1)
AT3G25770 1,02 0,017 AOC2 (ALLENE OXIDE CYCLASE 2)
AT3G28220 2,36 0,000 meprin and TRAF homology domain-containing protein
AT3G28270 1,38 0,001 UPF0496 protein
AT3G42658 1,51 0,000 transposable element gene
AT3G51450 1,07 0,009 strictosidine synthase family protein
AT3G54990 1,18 0,002 SMZ (SCHLAFMUTZE)
AT3G55970 1,55 0,001 oxidoreductase
AT4G02360 1,01 0,017 unknown protein
AT4G04223 1,08 0,027 unknown
AT4G07812 1,83 0,000 unknown pseudogene
AT4G11290 1,02 0,009 peroxidase
AT4G11320 1,81 0,000 cysteine proteinase
AT4G12480 1,41 0,001 pEARLI 1
AT4G12490 1,64 0,001 lipid transfer protein (LTP) family protein
AT4G12500 1,53 0,001 lipid transfer protein (LTP) family protein
AT4G15610 1,01 0,041 Integral membrane family protein
AT4G16590 1,26 0,005 ATCSLA01 (Cellulose synthase-like A1)
AT4G27860 1,24 0,004 integral membrane family protein [AT4G27860.1]
AT5G05600 2,13 0,000 oxidoreductase

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AT5G10300 1,01 0,024 hydrolase

AT5G13220 1,5 0,000 JAS1/JAZ10/TIFY9
AT5G24420 1,55 0,000 glucosamine/galactosamine-6-phosphate isomerase-related
AT5G24770 1,3 0,001 VSP2 (VEGETATIVE STORAGE PROTEIN 2)
AT5G24780 1,75 0,001 VSP1 (VEGETATIVE STORAGE PROTEIN 1)
AT5G26270 1,05 0,019 unknown protein
AT5G44420 1,95 0,000 PDF1.2
AT5G44430 2,48 0,000 PDF1.2c (
AT5G47220 1,05 0,025 ATERF-2/ATERF2/ERF2 (ETHYLENE RESPONSE FACTOR 2)
AT5G63970 1,03 0,008 copine-related
AT5G67080 1,01 0,039 MAPKKK19
CO048493 1,6 0,000 cDNA clone tk49b11

AT1G35230 -1,03 0,028 AGP5 (ARABINOGALACTAN-PROTEIN 5)

AT4G15110 -1,07 0,084 CYP97B3
AT5G35935 -1,15 0,024 transposable element gene
ATCG00840 -1,2 0,129 chloroplast ribosomal protein L23
AT5G35935 -1,29 0,017 transposable element gene
ATCG00070 -1,7 0,013 PSII K protein
AT4G28910 -1,76 0,000 NINJA (RNAi target)
1
False discovery rate (FDR) corrected RankProduct p-value < 0.05 and logFC >1 or <-1
2
Arabidopsis Genome Initiative identifier, loci in bold are used in this order for figure 1e
3
log Fold Change of the means of four biological replicated experiments
4
False discovery rate (FDR) corrected RankProduct p-value

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Supplementary Table 4| Differentially expressed genes in NINJAKD vs wild-type plants treated with
0.5 µM coronatine1
AGI2 logFC3 p-value4 Annotation
AT1G02920 1,12 0,042 ATGSTF7 (GLUTATHIONE S-TRANSFERASE 11)
AT1G06620 13 0,009 2-oxoglutarate-dependent dioxygenase
AT1G07930 1,59 0,015 elongation factor 1-alpha
AT1G14120 1,74 0,001 2-oxoglutarate-dependent dioxygenase
AT1G14240 1,71 0,001 nucleoside phosphatase family protein
AT1G14250 1,19 0,042 nucleoside phosphatase family protein
AT1G15520 1,56 0,009 ATPDR12 (PLEIOTROPIC DRUG RESISTANCE 12)
AT1G15670 1,59 0,001 kelch repeat-containing F-box family protein
AT1G17380 18 0,008 JAZ5/TIFY11A
AT1G17420 2,11 0,000 LOX3 (Lipoxygenase 3)
AT1G18710 2,16 0,000 AtMYB47
AT1G19680 1 0,050 protein binding / zinc ion binding
AT1G24070 1,31 0,008 ATCSLA10 (Cellulose synthase-like A10)
AT1G30135 1,31 0,011 JAZ8/TIFY5A
AT1G33960 1,58 0,015 AIG1 (AVRRPT2-INDUCED GENE 1)
AT1G35230 11 0,017 AGP5 (ARABINOGALACTAN-PROTEIN 5)
AT1G35580 19 0,018 CINV1 (CYTOSOLIC INVERTASE 1)
AT1G52000 2,57 0,000 jacalin lectin family protein [AT1G52000.1]
AT1G52030 1,89 0,000 MBP2 (MYROSINASE-BINDING PROTEIN 2)
AT1G52040 2,47 0,000 MBP1 (MYROSINASE-BINDING PROTEIN 1)
AT1G52380 1,1 0,015 Ran-binding protein 1 domain-containing protein
AT1G52400 1,81 0,001 BGL1 (BETA-GLUCOSIDASE HOMOLOG 1)
AT1G52410 1,46 0,004 TSA1 (TSK-ASSOCIATING PROTEIN 1)
AT1G53480 3,08 0,000 unknown protein
AT1G54000 1,59 0,004 myrosinase-associated protein
AT1G54010 1,33 0,006 myrosinase-associated protein
AT1G54020 29 0,000 myrosinase-associated protein
AT1G54030 1,41 0,004 GDSL-motif lipase
AT1G55490 1,02 0,032 CPN60B (CHAPERONIN 60 BETA)
AT1G58270 1,36 0,008 ZW9
AT1G61120 21 0,000 terpene synthase/cyclase family protein
AT1G61800 1,56 0,006 GPT2 (glucose-6-phosphate/phosphate translocator 2)
AT1G64160 1,3 0,006 disease resistance-responsive family protein
AT1G68620 11 0,049 hydrolase
AT1G72260 1,7 0,001 THI2.1 (THIONIN 2.1)
AT1G72450 1,16 0,021 JAZ6/TIFY11B (JASMONATE-ZIM-DOMAIN PROTEIN 6)
AT1G79690 1,04 0,023 ATNUDT3 (Arabidopsis thaliana Nudix hydrolase homolog 3)
AT1G80110 1,02 0,050 ARABIDOPSIS THALIANA PHLOEM PROTEIN 2-B11 (ATPP2-B11)
AT2G01450 1,3 0,049 ATMPK17 (Arabidopsis thaliana MAP kinase 17)
AT2G03980 1,41 0,004 GDSL-motif lipase/hydrolase family protein
AT2G13810 1,44 0,031 ALD1 (AGD2-LIKE DEFENSE RESPONSE PROTEIN1)
AT2G15760 1,63 0,004 calmodulin-binding protein
AT2G16367 1,86 0,001 Defensin-like (DEFL) family protein
AT2G16720 1,44 0,004 MYB7 (myb domain protein 7)

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AT2G17500 2,08 0,000 auxin efflux carrier family protein

AT2G18690 15 0,030 unknown protein
AT2G20340 1,45 0,004 tyrosine decarboxylase
AT2G22200 1,12 0,024 AP2 domain-containing transcription factor
AT2G22330 1,1 0,030 CYP79B3 (cytochrome P450, family 79, subfamily B, polypeptide 3)
AT2G22770 1,32 0,015 NAI1; bHLH transcription factor
AT2G24850 1,58 0,008 TAT3 (TYROSINE AMINOTRANSFERASE 3)
AT2G25450 1,34 0,010 2-oxoglutarate-dependent dioxygenase
AT2G26560 1,01 0,025 PLP2 (PHOSPHOLIPASE A 2A)
AT2G27690 1,09 0,039 CYP94C1 (cytochrome P450, family 94, subfamily C, polypeptide 1)
AT2G29350 1,54 0,008 SAG13 (senescence-associated gene 13)
AT2G29440 1,49 0,004 ATGSTU6 (GLUTATHIONE S-TRANSFERASE 24)
AT2G29460 1,67 0,001 ATGSTU4 (GLUTATHIONE S-TRANSFERASE 22)
AT2G30720 1,13 0,012 thioesterase family protein
AT2G30770 1,48 0,008 CYP71A13
AT2G30830 1,14 0,027 2-oxoglutarate-dependent dioxygenase
AT2G30950 1,32 0,017 VAR2 (VARIEGATED 2)
AT2G33070 1,42 0,006 jacalin lectin family protein
AT2G34600 12 0,019 JAZ7/TIFY5B
AT2G37025 1,02 0,027 TRFL8 (TRF-LIKE 8)
AT2G37620 1,38 0,009 ACT1 (ACTIN 1)
AT2G38240 1,03 0,048 oxidoreductase
AT2G39330 12 0,031 jacalin lectin family protein
AT2G39770 1,36 0,017 CYT1 (CYTOKINESIS DEFECTIVE 1)
AT2G46680 1,06 0,017 ATHB-7 (ARABIDOPSIS THALIANA HOMEOBOX 7)
AT3G01345 3,84 0,000 Expressed protein [AT3G01345.1]
AT3G09940 1,99 0,000 ATMDAR3/MDHAR (MONODEHYDROASCORBATE REDUCTASE)
AT3G09950 1,39 0,007 unknown protein
AT3G11340 2,06 0,000 UDP-glucoronosyl/UDP-glucosyl transferase family protein
AT3G11480 2,57 0,000 BSMT1; S-adenosylmethionine-dependent methyltransferase
AT3G12145 1,3 0,009 FLR1 (FLOR1)
AT3G14310 1,4 0,027 ATPME3 (Arabidopsis thaliana pectin methylesterase 3)
AT3G15500 11 0,017 ATNAC3 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 55)
AT3G15730 1,31 0,024 PLDALPHA1 (PHOSPHOLIPASE D ALPHA 1)
AT3G15950 16 0,006 TSA1-LIKE
AT3G16400 1,96 0,000 ATMLP-470 (MYROSINASE-BINDING PROTEIN-LIKE PROTEIN-470)
AT3G16420 1,11 0,022 PBP1 (PYK10-BINDING PROTEIN 1)
AT3G16460 1,08 0,022 jacalin lectin family protein
AT3G16470 2,08 0,000 JASMONATE RESPONSIVE 1
AT3G20370 1,07 0,024 meprin and TRAF homology domain-containing protein
AT3G22160 1,17 0,021 VQ motif-containing protein
AT3G22275 2,33 0,000 unknown protein
AT3G22740 1,58 0,004 HMT3 (Homocysteine S-methyltransferase 3)
AT3G25180 1,59 0,005 CYP82G1
AT3G26330 13 0,017 CYP71B37
AT3G26830 1,64 0,004 PAD3 (PHYTOALEXIN DEFICIENT 3)
AT3G26840 12 0,008 esterase/lipase/thioesterase family protein

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 doi: 10.1038/nature08854 SUPPLEMENTARY INFORMATION

AT3G27415 2,03 0,000 unknown protein

AT3G27809 1 0,011 unknown protein
AT3G28220 1,32 0,009 meprin and TRAF homology domain-containing protein
AT3G28270 22 0,000 UPF0496 protein
AT3G28290 1,73 0,001 AT14A
AT3G42658 1,5 0,008 transposable element gene
AT3G45130 1,46 0,004 LAS1 (Lanosterol synthase 1)
AT3G45140 1,87 0,006 LOX2 (LIPOXYGENASE 2)
AT3G47340 1,51 0,005 ASN1 (DARK INDUCIBLE 6)
AT3G50480 12 0,046 HR4 (HOMOLOG OF RPW8 4)
AT3G50930 1,17 0,019 AAA-type ATPase family protein
AT3G54990 1,46 0,005 SMZ (SCHLAFMUTZE)
AT3G56200 16 0,031 short-chain dehydrogenase/reductase
AT3G57260 1,16 0,049 BGL2 (PATHOGENESIS-RELATED PROTEIN 2)
AT3G59970 1,48 0,017 MTHFR1 (METHYLENETETRAHYDROFOLATE REDUCTASE 1)
AT4G03400 1 0,009 DFL2 (DWARF IN LIGHT 2)
AT4G03420 18 0,009 unknown protein
AT4G04840 1,39 0,008 methionine sulfoxide reductase domain-containing protein
AT4G07812 1,79 0,001 unknown pseudogene
AT4G10500 1,39 0,047 oxidoreductase
AT4G11310 1,18 0,028 cysteine proteinase
AT4G11320 1,61 0,003 cysteine proteinase
AT4G14365 1,16 0,025 zinc finger (C3HC4-type RING finger) family protein
AT4G16730 1,4 0,005 lyase/ magnesium ion binding
AT4G17500 1,51 0,004 ATERF-1 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 1)
AT4G18950 1,3 0,019 ankyrin protein kinase
AT4G21830 1,93 0,000 METHIONINE SULFOXIDE REDUCTASE B7 (ATMSRB7)
AT4G21840 1,7 0,004 methionine sulfoxide reductase domain-containing protein
AT4G21910 1,19 0,013 MATE efflux family protein
AT4G23420 1,1 0,017 short-chain dehydrogenase/reductase
AT4G23600 1,06 0,049 CORI3 (CORONATINE INDUCED 1, JASMONIC ACID RESPONSIVE 2)
AT4G24190 1,58 0,006 SHD (SHEPHERD)
AT4G25780 1,06 0,032 pathogenesis-related protein
AT4G27860 1,42 0,006 integral membrane family protein [AT4G27860.1]
AT4G30270 1,04 0,031 MERI5B (MERISTEM-5)
AT4G31500 18 0,009 CYP83B1
AT4G33050 1,18 0,009 EDA39
AT4G36950 15 0,017 O-methyltransferase family 2 protein
AT4G37990 1,56 0,003 MAPKKK21
AT4G39030 1,14 0,039 EDS5 (ENHANCED DISEASE SUSCEPTIBILITY 5)
AT5G01900 1,41 0,008 WRKY62 (WRKY DNA-binding protein 62)
AT5G02500 1,64 0,025 heat shock cognate 70 kDa protein 1
AT5G02940 16 0,010 phosphotransferase-related
AT5G04590 1,06 0,047 SIR (sulfite reductase)
AT5G07350 1,03 0,017 tudor domain-containing protein
AT5G09440 1,1 0,024 phosphate-responsive protein
AT5G12340 1,32 0,010 unknown protein

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 doi: 10.1038/nature08854 SUPPLEMENTARY INFORMATION

AT5G13220 1,8 0,000 JAS1/JAZ10/TIFY9

AT5G13320 1,55 0,019 PBS3 (AVRPPHB SUSCEPTIBLE 3)
AT5G17990 1,45 0,005 TRP1 (TRYPTOPHAN BIOSYNTHESIS 1)
AT5G22630 1,52 0,006 ADT5 (AROGENATE DEHYDRATASE 5)
AT5G24420 1,64 0,001 glucosamine/galactosamine-6-phosphate isomerase-related
AT5G25250 1,11 0,033 VSP2 (VEGETATIVE STORAGE PROTEIN 2)
AT5G26270 1,03 0,047 unknown protein
AT5G27380 1,4 0,031 GSH2/GSHB (GLUTATHIONE SYNTHETASE 2)
AT5G28237 1,06 0,041 tryptophan synthase
AT5G28540 1,04 0,040 BIP1 (LUMINAL BINDING PROTEIN)
AT5G41120 1,03 0,025 esterase/lipase/thioesterase family protein
AT5G42020 1,08 0,031 BIP (LUMINAL BINDING PROTEIN)
AT5G43745 1,63 0,003 phosphotransferase-related
AT5G47220 1,11 0,019 ATERF-2/ATERF2/ERF2 (ETHYLENE RESPONSE FACTOR 2)
AT5G51070 15 0,030 ERD1 (EARLY RESPONSIVE TO DEHYDRATION 1)
AT5G60890 1 0,018 ATMYB34/ATR1/MYB34
AT5G61790 1,08 0,032 calnexin 1 (CNX1)
AT5G61890 2,38 0,000 AP2 domain-containing transcription factor
AT5G63450 17 0,009 CYP94B1
AT5G63970 1,71 0,001 copine-related
AT5G65380 1,53 0,005 ripening-responsive protein
AT5G67080 28 0,000 MAPKKK19
ATCG00070 1,15 0,013 PSII K protein
ATCG00640 1,04 0,041 chloroplast ribosomal protein L33
ATCG00650 1,01 0,010 chloroplast ribosomal protein S18
ATCG00770 1,33 0,012 chloroplast ribosomal protein S8
ATCG00810 1,18 0,030 chloroplast ribosomal protein L22
BP839700 1,44 0,030 cDNA clone RAFL22-27-O05
CO048493 1,51 0,004 cDNA clone tk49b11
H36258 1,49 0,006 Cysteine proteinase At4g11320 precursor
N37480 1,02 0,016 ADP glucose pyrophosphorylase small subunit

AK119050 -1,03 0,019 Arabidopsis thaliana mRNA for unknown protein

AT1G07490 -1,03 0,027 DVL9/RTFL3 (ROTUNDIFOLIA LIKE 3)
AT1G15405 -1,02 0,018 other RNA [AT1G15405.1]
AT1G17090 -1,05 0,021 unknown protein
AT1G22160 -1,02 0,019 senescence-associated protein-related
AT1G54580 -1,05 0,019 ACP2 (ACYL CARRIER PROTEIN 2)
AT1G56650 -1,02 0,019 PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1)
AT2G04110 -1,16 0,004 pseudogene of unknown protein
AT2G37950 -1,08 0,010 zinc finger (C3HC4-type RING finger)
AT3G25597 -1,08 0,003 unknown protein
AT3G55290 -1,05 0,015 short-chain dehydrogenase/reductase
AT4G10290 -1,85 0,000 unknown protein
AT4G11911 -1,14 0,005 similar to ATNYE1/NYE1 (NON-YELLOWING 1)
AT4G15110 -18 0,002 CYP97B3
AT4G15210 -17 0,002 ATBETA-AMY (BETA-AMYLASE)

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 doi: 10.1038/nature08854 SUPPLEMENTARY INFORMATION

AT4G21870 -1,01 0,040 heat shock protein (HSP26.5-P)

AT4G28910 -2,65 0,000 NINJA (RNAi target)
AT4G29610 -1,47 0,000 cytidine deaminase
AT4G35160 -12 0,004 O-methyltransferase family 2 protein
AT5G02380 -1,12 0,002 MT2B (METALLOTHIONEIN 2B)
AT5G17220 -1,65 0,000 ATGSTF12 (GLUTATHIONE S-TRANSFERASE 26)
AT5G35935 -1,4 0,002 transposable element gene
AT5G41761 -1,1 0,013 unknown protein
AT5G49525 -1,17 0,003 unknown protein
AT5G58310 -1,01 0,022 hydrolase
AT5G63500 -1,15 0,002 unknown protein
AY090965 -1,46 0,000 unknown
1
False discovery rate (FDR) corrected RankProduct p-value < 0.05 and logFC >1 or <-1
2
Arabidopsis Genome Initiative identifier, loci in bold are used in this order for figure S7
3
log Fold Change of the means of four biological replicated experiments
4
False discovery rate (FDR) corrected RankProduct p-value

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 Supplementary Table 5| MALDI-TOF/TOF-MS identification of NINJA interactors.

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Bait Prey PMF data MSMS data
Found/ Protein RMS error Sequence Unique Total Peptide Variable
3 4
Bait Specifications Prey Locus Prey Name 3 exp1 Score E-value2 (ppm) coverage % Peptides Ion Score Number Start End Observed Mr(Exp) Mr(Calc) Delta Miss Ions Score Expect Peptide Modification
NINJA Mock AT5G27030 TPR3 3 429 4,10E-39 8 19 13 375 1 429 443 1743,91 1742,91 1742,91 0,00 0 88 2,60E-08 K.HLIQLYAFSGPNDLR.Q
doi: 10.1038/nature08854

2 507 520 1582,81 1581,81 1581,80 0,00 0 93 1,10E-08 K.ENIQFIFSTAIDGK.I

3 756 778 2344,29 2343,28 2343,29 -0,01 0 139 7,70E-14 K.VVQLLYTNSGAGILALGSNGIQR.L
4 967 982 1769,95 1768,94 1768,97 -0,03 0 56 4,00E-05 R.ILVVHETQLAVFDASK.M
NINJA Mock AT1G15750 TOPLESS 2 362 2,10E-32 7 15 12 321 1 114 125 1476,81 1475,8 1475,8 0,01 0 48 2,60E-04 K.EITQLLTLENFR.E
2 368 388 2227,21 2226,2 2226,2 0,00 0 134 3,70E-13 K.QTLLLVGTNVGDIGLWEVGSR.E
3 518 531 1582,81 1581,81 1581,8 0,00 0 93 1,10E-08 K.ENIQFIFSTALDGK.I
4 858 867 1231,63 1230,62 1230,61 0,00 0 47 5,30E-04 K.ISLFNMMTFK.T
NINJA Mock AT4G14720 PPD2 2 99 4,60E-06 6 30 6 67 1 18 29 1417,75 1416,74 1416,75 0,00 0 41 2,40E-03 K.LLTEEDISQLTR.E
2 76 86 1217,69 1216,68 1216,69 -0,01 0 26 3,90E-02 K.ILVSQPPNPPR.V
NINJA Mock AT3G16830 TPR2 1 298 5,20E-26 4 9 9 275 1 114 125 1476,81 1475,8 1475,80 0,01 0 48 2,60E-04 K.EITQLLTLENFR.E
2 431 444 1671,86 1670,85 1670,85 0,00 0 47 4,10E-04 K.HLIHVYAYQGSDLR.Q
3 508 521 1582,81 1581,81 1581,80 0,00 0 93 1,10E-08 K.ENIQFIFSTALDGK.I
4 768 790 2344,29 2343,28 2343,30 -0,02 1 26 1,30E-02 K.VARLLYTNSGVGVLALGSNGVQR.L
5 771 790 2018,1 2017,09 2017,10 0,00 0 61 7,40E-06 R.LLYTNSGVGVLALGSNGVQR.L
NINJA Mock AT5G46760 MYC3 1 91 2,70E-05 2 6 3 83 1 447 461 1606,87 1605,86 1605,86 0,00 0 73 8,90E-07 K.ASLLGDAISYINELK.S
2 518 526 1102,61 1101,6 1101,60 0,00 0 11 2,50E+00 K.IIGWDVMIR.V
3 518 526 1118,61 1117,6 1117,60 0,00 0 9 3,50E+00 K.IIGWDVMIR.V Oxidation (M)
NINJA Mock AT5G20900 JAZ12 1 65 9,70E-03 1 14 1 58 1 107 133 2864,4 2863,39 2863,40 -0,01 0 58 2,00E-05 R.APSFSSTSNVASPAAQPFPIQPISFCR.S
NINJA +JA (1min) AT5G27030 TPR3 3 371 2,60E-33 6 23 17 290 1 150 158 1072,61 1071,61 1071,61 0,00 0 38 4,20E-03 K.LIEANPLFR.D
2 309 324 1781,95 1780,94 1780,95 0,00 0 53 8,40E-05 R.LRPAPSVEEVTYPAPR.Q
3 429 443 1743,91 1742,91 1742,91 0,00 0 82 1,00E-07 K.HLIQLYAFSGPNDLR.Q
4 756 778 2344,28 2343,28 2343,29 -0,01 0 117 1,10E-11 K.VVQLLYTNSGAGILALGSNGIQR.L
NINJA +JA (1min) AT1G15750 TOPLESS 2 214 1,30E-17 8 20 19 131 1 150 158 1072,62 1071,61 1071,61 0,00 0 24 9,51E-02 K.LIEANPLFR.D
2 159 167 1117,62 1116,62 1116,63 -0,01 1 14 1,03E+00 R.DKLQFPTLR.N
3 534 543 1212,54 1211,54 1211,54 0,00 0 51 1,07E-04 K.AWLYDNMGSR.V
4 114 125 1476,80 1475,79 1475,80 -0,01 0 42 1,31E-03 K.EITQLLTLENFR.E
NINJA +JA (1min) AT3G16830 TPR2 1 124 1,30E-08 5 7 6 118 1 114 125 1476,81 1475,8 1475,80 0,00 0 62 1,20E-05 K.EITQLLTLENFR.E
2 150 158 1072,61 1071,61 1071,61 0,00 0 38 4,20E-03 K.LIEANPLFR.E
3 768 790 2344,28 2343,28 2343,30 -0,03 1 18 9,90E-02 K.VARLLYTNSGVGVLALGSNGVQR.L
NINJA +JA (1h) AT5G27030 TPR3 3 382 2,10E-34 5 20 17 302 1 45 62 2029,98 2028,97 2028,97 0,01 0 77 3,50E-07 K.VLAGEWDDVETYLSGFTK.V
2 150 158 1072,61 1071,61 1071,61 0,00 0 24 9,50E-02 K.LIEANPLFR.D
3 413 428 1708,87 1707,87 1707,86 0,00 0 55 5,60E-05 R.VAWSPDGNFIGVAFTK.H
4 429 443 1743,92 1742,91 1742,91 0,00 0 94 6,30E-09 K.HLIQLYAFSGPNDLR.Q
5 756 778 2344,29 2343,28 2343,29 -0,01 0 51 5,10E-05 K.VVQLLYTNSGAGILALGSNGIQR.L
NINJA +JA (1h) AT1G15750 TOPLESS 3 263 1,60E-22 7 14 12 224 1 114 125 1476,81 1475,81 1475,80 0,01 0 36 4,10E-03 K.EITQLLTLENFR.E
2 368 388 2227,21 2226,2 2226,20 0,00 0 70 2,00E-06 K.QTLLLVGTNVGDIGLWEVGSR.E
3 518 531 1582,81 1581,8 1581,80 0,00 0 118 1,30E-11 K.ENIQFIFSTALDGK.I
NINJA +JA (1h) AT3G16830 TPR2 1 223 1,60E-18 3 12 11 188 1 114 125 1476,81 1475,81 1475,80 0,01 0 36 4,10E-03 K.EITQLLTLENFR.E
2 508 521 1582,81 1581,8 1581,80 0,00 0 70 2,00E-06 K.ENIQFIFSTALDGK.I
3 768 790 2344,3 2343,29 2343,30 -0,01 1 16 1,20E-01 K.VARLLYTNSGVGVLALGSNGVQR.L
4 771 790 2018,1 2017,09 2017,10 -0,01 0 65 2,70E-06 R.LLYTNSGVGVLALGSNGVQR.L
1
Number of times that a prey was found in 3 independent TAP experiments with the respective bait.
2
The E-value in Mascot corresponds to the number of times one would expect this score by chance alone.
3
Unique peptides found per identification. Peptides not present in control TAP experiments.
4
Number of peptides from which a sequence was obtained.
SUPPLEMENTARY INFORMATION

20