Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

TTCCAAGAACACAAGCGACTT

ELENA1

A

B

Supplemental Figure 1. Expression level of selected ELENA1 knock down lines by artificial miRNA.

(Supports Figure 2)

(A) Schematic diagram and amiRNA sequences used to knock down ELENA1 expression. Three tandem

copies of the indicated sequence were individually overexpressed under a 35S promoter for the generation

of knock down lines. (B) Time course expression of ELENA1 in two independent knock-down mutants upon

elf18 treatment (#10 and #20). Transcript levels were normalized to ACT2 expression levels. Bars represent

average ±SD (n = 3 independent seedling pools).

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

A B

**

**

****

** ** ** **

Supplemental Figure 2. ELENA1 and PR1 expression level in selected ELENA1 overexpressing lines.

(Supports Figure 2)

(A) R l ti i f ELENA1 i 4 diff t 35S ELENA1 t i li d l diti (B)(A) Relative expression of ELENA1 in 4 different 35S:ELENA1 transgenic lines under normal condition. (B)

Relative expression of PR1 in the four 35S:ELENA1 transgenic lines after 5 µM elf18 treatment. Transcript

levels were normalized to ACT2 expression levels. Bars represent average ±SD (n = 3 independent seedling

pools). Asterisks indicate statistically significant difference compared with WT (Col-0). **P < 0.01; two-tailed t

test.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

A B

ORF No. Size (bp) aa No.

1 18 5

2 132 43

3 27 8

A B

3 27 8

4 90 29

5 39 12

6 111 36

7 102 33

8 9 2

Supplemental Figure 3. Predicted ORFs in ELENA1 transcript. (Supports Figure 3)

(A) ELENA1 nucleotide sequence and predicted ORFs (under lines) (B) Table showing predicted peptide

length in each ORF. Five start codons were mutated in ELENA1_5M (gray background) and 8 start codons

were mutated in ELENA1_8M mutants.

A BWT_1hr WT_6hr

1 253 9520

(n=1,632)

WT_6hr(n=1,450)

1 048 39668

Up-regulated genes in WT (4,030) Down-regulated genes in WT (3,756)WT_0hr_rep1WT_0hr_rep2WT_0hr_rep3OX_0hr_rep1OX_0hr_rep2

(n=2,133) (n=1,142)

WT_1hr

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

5351,253 95

(n=2,662)WT_12h

325 492

1,310

2091,048 396

(n=2,244)WT_12h

307 777

951

WT_1hr(n=2,274)

WT_6hr(n=1,284)

WT_1hr(n=1,491)

WT_6hr(n=1,254)

Up-regulated genes in OX (3,850) Down-regulated genes in OX (3,286)

OX_0hr_rep3WT_1hr_rep1WT_1hr_rep2WT_1hr_rep3OX_1hr_rep1OX_1hr_rep2OX_1hr_rep3WT_6hr_rep1WT_6hr_rep2WT_6hr_rep3OX_6hr_rep1OX 6h 2

C defense responseinnate immune response

6031,271 112

(n=2,427)WT_12h

40

360 529

935

212968 210

(n=2,083)WT_12h

25

286 807

778

OX_6hr_rep2OX_6hr_rep3WT_12hr_rep1WT_12hr_rep2WT_12hr_rep3OX_12hr_rep1OX_12hr_rep2OX_12hr_rep3

pimmune system process

immune responseresponse to chitin

amino acid transportamine transport

response to other organismcarboxylic acid transport

organic acid transportresponse to stress

response to biotic stimulusresponse to stimulus

multi-organism process

OXWT

defense response, incompatible interaction

0 10 20 30 40 50 60 70P value (-log10)

D

5

10

15

20

5

10

15

20

5

10

15

20

ls (O

X_12

hr)

ls (O

X_6h

r)

s (O

X_1h

r)

ELENA1 ELENA1 ELENA1

-20

-15

-10

-5

0

-20 -10 0 10 20-20

-15

-10

-5

0

-20 -10 0 10 20-20

-15

-10

-5

0

-20 -10 0 10 20Expression levels (WT_1hr) Expression levels (WT_6hr) Expression levels (WT_12hr)

Expr

essi

on le

ve

Expr

essi

on le

ve

Expr

essi

on le

vels

Supplemental Figure 4. Global view of ssRNA-seq results. (Supports Figure 4)

(A) Correlation analysis of all ssRNA-seq samples from normal condition (0h) and elf18-treated conditions (1 h, 6 h

and 12 h) of WT and ELENA1 OX plant (OX-16). Expression levels (FPKMs) of all detected genes were used for

hierarchical clustering analysis. (B) Venn diagram showing up- and down-regulated protein coding genes (fold change

≥ 2 or ≤ 0.5, P value < 0.05) in WT (top) and OX (bottom). (C) Gene Ontology (GO) enrichment analysis of 535 and

603 co-upregulated protein coding genes. Top 15 (with the lowest P values) enriched GO terms of biological process

category are shown. (D) Scatter plot showing log2-transformed expression levels (FPKMs) of detected genes in WT (x-

axis) compared to OX (y-axis). Up-regulated genes, down-regulated genes and non-differentially expressed genes are

shown in red, blue and black, respectively.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

150

(kD)

100

150

(kD)

75

100

150

75

100

50

37

*

S l t l Fi 5 ELENA1 i t ith M di t b it i it (S t Fi 5)Supplemental Figure 5. ELENA1 associates with Mediator subunits in vitro. (Supports Figure 5)

In vitro binding assay with IVT biotinylated ELENA1 RNA and recombinant mediators (MBP-MEDs).

Proteins were detected with anti-MBP antibody. Asterisk indicates the full-length protein of MBP-MED19a.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

At5g12230(MED19a)

Mediator subunit SALK_034955(med19a-2)

A B

SALK_037435(med19a-1)

At5g05140(MED26b)

TFIISelongation factor

SALK_020870(med26b-1)

**

** **

**

C

** ** ** ** ** ** ** **

Supplemental Figure 6. PR1 expression levels in MED19a and MED26a KO mutants after elf18

treatment. (Supports Figure 5 and 6)

(A) Schematic diagram of T-DNA insertion sites and Salk line numbers in each mediator knock-out mutants

(med19a-1, med19a-2, and med26b). (B) Time course of PR1 expression after elf18 treatment in the

mediator mutants. (C) Time course of MED19a expression after elf18 treatment in the med19a mutants.

Transcript levels were normalized to ACT2 expression levels. Bars represent average ±SD (n = 3

independent seedling pools). Asterisks indicate statistically significant differences compared with the wild

type (Col-0). **P < 0.01 two-tailed t test.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

20%INPUT

ELENA1

A

+ -+

Biotinylated sense RNA

Biotinylated anti sense RNA

B

20%INPUT

ELENA1

+ -+

non-Biotinylated RNA

MBP-MED19a

- + Biotinylated anti-sense RNA

anti-MBP

MBP-MED19b

- + Biotinylated RNA

anti-MBP

MBP MBP

Supplemental Figure 7. ELENA1 associates with both MED19a and MED19b in vitro. (Supports

Fi 5 d 6)Figure 5 and 6)

(A) In vitro binding assay with IVT biotinylated ELENA1 RNA and recombinant MED19b protein tagged with

MBP. (B) In vitro binding between recombinant MBP-MED19a and IVT biotinylated sense or anti-sense

ELENA1 RNA.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

A B

*

****

**

med19a-1med19a-1

Supplemental Figure 8. PR1 expression levels in med19a/med19b double mutant plants. (Supports Figure 6) (A) PR1 expression levels in med19a-1, MED19b RNAi/med19a-1 (19bi/19a) after elf18 treatment. Bars ( ) p , ( )

represent average ±SD (n = 3 independent seedling pools). (B) Relative expression of MED19b in 19bi/19a

transgenic lines (#19). Transcript levels were normalized to ACT2 expression levels. Bars represent

average ±SD (n = 3 independent seedling pools). Asterisks indicate statistically significant difference. *P

< 0.05, **P < 0.01; two-tailed t test.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

A B C

** ** **

** ****

** ****

Supplemental Figure 9. ELENA1 and MED19a expression levels in ELENA1 and MED19a double

mutants. (Supports Figure 6)

(A) ELENA1 i i 35S ELENA1/ d19 1 (E1/ 19 ) l t E1#16 i ELENA1 OX 16 (B)(A) ELENA1 expression in 35S:ELENA1/med19a-1 (E1/m19a) plants. E1#16 is ELENA1 OX-16. (B)

MED19a expression in ELENA1 KD-10/UBQ:MED19a (e1#10/M19a) plants. M19a#1 is UBQ:MED19a-1

(C) MED19a expression in 35S:ELENA1-16/UBQ:MED19a (E1#16/M19a) plants. Bars represent average

±SD (n = 3 independent seedling pools). Asterisks indicate statistically significant difference compared with

WT (Col-0). **P < 0.01; two-tailed t test.

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

A B

Supplemental Figure 10. Time course expression of genes neighboring ELENA1 after elf18

t t ttreatment.

(A) Relative expression of CBL6 after 5 µM elf18 treatment in the ELENA1 mutants (B) Relative expression

of AT4g16360 after elf18 treatment in the ELENA1 mutants. For (A) and (B), transcript levels were

normalized to ACT2 expression levels. Bars represent average ±SD (n = 3 independent seedling pools).

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

Bacteria

M b

EFR

elf18

Membrane

(A)nELENA1

Nucleus

PR1TF

MED19aEnrichment

in PR1 promoter AS-1 like

MED19a

Immune responses

Supplemental Figure 11. A working model of transcriptional regulation of PR1 by ELENA1 in

Arabidopsis.

Elf18 (red star) is recognized by EFR receptor and the signal transduced into the nucleus. ELENA1

transcriptional level was induced by the signal. Association between ELENA1 and MED19a enhances

MED19a enrichment on the PR1 promoter. Finally, PR1 expression is increased and plant shows disease

resistance phenotype

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

Supplemental Data. Seo et al. (2017). Plant Cell 10.1105/tpc.16.00886

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