0.1 kb

M pen2-4

PCR

product

uncut Col-0

sid2

pen2-4 M Col-0 coi1-16

0.2 kb

PEN2

pen2-4

445 195 66

640 66

AciI AciI

AciI

(Westphal et al., 2008)

COI1 122 25

coi1-16 25 78 44

Tsp509I

Tsp509I Tsp509I

Supplemental Figure 1 - Geng, Cheng, Gangadharan and Mackey

A

B

0.2 kb

0.65 kb

0.4 kb

M coi1 Col-0 M sid2

coi1

npr1

coi1 M Col-0

sid2

npr1

coi1 pen2-4

PCR

product

uncut C

D E

Supplemental Figure 1. DNA analysis of mutant bacteria and plants. (A) At left are schematics of the Cfa6 locus (top) and the Tn5-inserted cfa6 locus (bottom). The hatched bar within cfa6 indicates the region of the cfa6 mutant locus found in plasmid pDB29 and integrated by homologous recombination. P1 through P4 indicate the position of primers. At right are the products (or lack thereof) of colony PCRs including P1, P2, and P3 (top gel) or P2, P3, and P4 (bottom gel). (B) Structure of PEN2 and pen2-4. Shown are schematics of the 706-bp PCR products, including the AciI restriction sites. AciI cuts the PCR fragments from wild-type PEN2 into 445 bp, 195 bp and 66 bp and cuts those from pen2-4 into 640 bp and 66 bp. (C) AciI restriction of the 706-bp PCR fragment of PEN2/pen2-4 from Col-0, coi1-16, sid2 coi1-16, npr1 coi1-16, sid2 npr1 coi1-16, and pen2-4 plants. The results indicate that all coi1-16 lines have been “cured” of the pen2-4 mutation. (D) Structure of COI1 and coi1-16. Shown are schematics of the 147-bp PCR products, including the Tsp509I restriction sites. Tsp509I cuts the PCR fragments from wild-type COI1 into 122 bp and 25 bp and cuts those from coi1-16 into 78 bp, 44 bp and 25 bp. (E) Tsp509I restriction of the 147-bp fragment of COI1/coi1-16 from Col-0, pen2-4, sid2 pen2-4, and coi1-16 plants. The results indicate that the pen2-4 and sid2 pen2-4 lines have been “cured” of the coi1-16 mutation.

1.65KbP1+P3P1+P2

P4+P3P4+P21.65Kb

1.65KbP1+P3P1+P2

P4+P3P4+P21.65Kb

P1+P3P1+P2

P4+P3P4+P21.65Kb

1.65 kb

1.65 kb

P1 P3 Tn5

P2

~1350 bp

~1390 bp no product

no product

P4

P1 P3

1698 bp

1738 bp

P4

Cfa6

cfa6

M Pto cor- ∆CEL

∆CEL

cor-

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

1

0

0.5

1

1.5

2

2.5

3

Col-0

PR

1 e

xp

res

sio

n

Pto cor-

△CEL△CEL cor-buffer

* *

**

Supplemental Figure 2 - Geng, Cheng, Gangadharan and Mackey

Pto cor- ∆CEL ∆CEL cor- buffer

48 h

72 h

Pto cor- ∆CEL ∆CEL cor- buffer

Pto cor- ∆CEL ∆CEL cor- buffer

24 h

A

B

Rubisco

Rubisco Rubisco

Supplemental Figure 2. COR inhibits PR-1 expression. (A) qRT-PCR analysis of PR1 expression 24 h after infiltration of Col-0 leaves with Pto, Ptocor-, Pto∆CEL, Pto∆CEL cor-, or buffer. Shown are the mean and standard error of combined data from of four biological repeats with the level of each transcript induced by Pto∆CEL set to 1. Significant differences were determined by two-tailed t test: * p<0.05 between cor- and DCEL cor- relative to Pto and DCEL, respectively, ** p<0.005 between buffer and all other samples. (B) Immunoblot showing the accumulation of PR-1 in Col-0 leaves 24, 48, and 72 h after infiltration with the indicated bacterial strains. The experiment was done four times with similar results. Arrows point to PR-1 and the upper band is a non-specific cross-reacting protein. Ponceau staining of RuBisCo beneath the blots demonstrates equal loading of samples.

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

2

Supplemental Figure 3. SA accumulation after bacterial infiltration. Accumulation of free SA was measured by HPLC after infiltrating Col-0, sid2, coi1 and sid2 coi1 plants with the indicated bacterial strains or buffer. Leaves were collected 15 h post infiltration. Shown is the combined data from three biological replicates and error bars indicate standard deviations. No SA was detected in sid2 and sid2 coi1, which, based on our SA standards, corresponds to < 0.02 mg/g fresh weight. Statistical analyses of the indicated samples were by one-way ANOVA and Fisher LSD test with significant differences (p<0.05) indicated by lowercase letters.

Supplemental Figure 3

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Col 0 sid2 coi1 sid2 coi1

Ptocor-∆CEL∆CEL cor-buffer

Col-0 sid2 coi1 sid2 coi1

ab

ab

bc

c c

c

a

c

SA μ

g/g

fre

sh w

eigh

t

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

3

2.5

3.5

4.5

5.5

6.5

7.5

8.5

9.5

Col-0 coi1 sid2 npr1 sid2 npr1coi1

Log

CFU

/cm

2

Ptocor-ΔCELΔCEL cor-

Col-0 coi1 sid2 npr1 sid2 npr1 coi1

A

B

BC C a a a a

A’

B’

C’

BC’ a’

c’ c’ b’

Supplemental Figure 4 - Geng, Cheng, Gangadharan and Mackey A

B

Supplemental Figure 4. COR promotes bacterial virulence in sid2 npr1 plants independent of targeting COI1. (A) Growth of the indicated strains 4 days after inoculation into Col-0, coi1, sid2 npr1 and sid2 npr1 coi1 plants. The dashed line indicates the starting inoculum of bacteria. Shown are the mean and standard error of five biological replicates. Different letter types indicate significant differences (p<0.05) by one-way ANOVA and Tukey HSD test of comparisons between the different bacterial strains on individual plant genotypes. (B) Quantification of callose deposits following infiltration of the indicated strains into Col-0, coi1, sid2 npr1 and sid2 npr1 coi1 leaves. Shown are the mean and standard error of combined data from two independent biological replicates. Statistical analyses of log transformed data of indicated samples were by one-way ANOVA and Tukey HSD test (p<0.05).

0

0.5

1

1.5

2

2.5

3

3.5

Pto cor- ΔCEL ΔCEL/cor- buffer

Log

Cal

lose

dep

osits

/1.1

mm

2 Col-0coi1

sid2 npr1

sid2 npr1 coi1

b bc c

a

Pto cor- ∆CEL ∆CEL cor- buffer

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

4

Supplemental Figure 5 - Geng, Cheng, Gangadharan and Mackey

Supplemental Figure 5. PEN2 is not required for flg22-induced callose deposition in soil-grown plants. (A) Callose deposition 15 hours after infiltration of 30 mM flg22 or buffer into Col-0 and pen2-1 leaves. Shown are representative fluorescence microscopy images of aniline blue-stained leaves (scale bar = 0.2 mm). The experiment was done two times with similar results. (B) Callose deposition in the cotyledons of 10-day-old liquid-grown seedlings of Col-0, sid2, pen2-1, pen2-4 and sid2 pen2-4 after 18 h of exposure to 1 mM flg22 or the control treatment. Shown are representative fluorescence microscopy images of aniline blue-stained leaves (scale bar = 0.2 mm). The experiment was done two times with similar results.

A

B

Col-0 pen2-1

water

flg22

Col-0 sid2 pen2-1 sid2 pen2-4 pen2-4

water

flg22

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

5

0

0.5

1

1.5

2

Col-0 sid2 pen2-1 sid2pen2-4

bufferflg22 (10 μM)ΔCELΔCEL + 3 μM CORΔCEL cor- ΔCEL cor- + 3 μM COR

Supplemental Figure 6 - Geng, Cheng, Gangadharan and Mackey A

B

Supplemental Figure 6. COR inhibits indole glucosinolate accumulation upstream of PEN2. Accumulation of 1MI3G (A) and I3G (B) after infiltration of buffer, flg22, or the indicated bacterial strains, with or without 3 mM COR, into Col-0, sid2, pen2-1 and sid2 pen2-4 leaves. Quantities of 1MI3G and I3G were calculated relative to sinigrin (spiked into each sample) and normalized with the amount elicited by Pto∆CEL in pen2-1 set to 1. Shown are the means and standard error from three biological replicates. Different letter types indicate significant differences (p<0.05) by one-way ANOVA and Tukey HSD test of comparisons between the indicated bacterial strains on individual plant genotypes.

0

0.5

1

1.5

2

2.5

3

3.5

4

Col-0 sid2 pen2-1 sid2pen2-4

I3G

acc

umul

atio

n

buffer

flg22 (10 μM)

ΔCEL

ΔCEL + 3 μM COR

ΔCEL cor-

ΔCEL cor- + 3 μM COR

A

A

A

A

a

a

a a

0

0.5

1

1.5

2bufferflg22 (10 μM)ΔCELΔCEL + 3 μM CORΔCEL/cor- ΔCEL/cor- + 3 μM COR

AA A

A

a

aa

a

Col-0 sid2 pen2-1 sid2 pen2-4

I3G

acc

umul

atio

n

A A A

A

a

a

a

a

1MI3

G a

ccum

ulat

ion

0

0.5

1

1.5

2bufferflg22 (10 μM)ΔCELΔCEL + 3 μM CORΔCEL/cor- ΔCEL/cor- + 3 μM COR

AA A

A

a

aa

a

Col-0 sid2 pen2-1 sid2 pen2-4

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

6

Supplemental Table 1. Phenotype of Arabidopsis types used in this study

Supplemental Tables

Plant type Phenotype

Col-0 Wild-type

sid2 Lacks isochorismate synthase required for the majority of defense-

associated production of SA

npr1 Lacks a key signaling molecule for transducing the SA-signal to a

transcriptional response

coi1 Lacks the only known receptor for active JA-conjugates and

coronatine

pen2 Lacks the myrosinase that hydrolyzes 4-methoxy-indol-

3ylmethylglucosinolate (4MI3G)

sid2 pen2 Deficient in SA-production and hydrolysis of 4MI3G

sid2 coi1 Deficient in SA-production and JA-signaling

npr1 coi1 Deficient in SA-signaling and JA-signaling

sid2 npr1 Deficient in SA-production and SA-signaling

sid2 npr1 coi1 Deficient in SA-production, SA-signaling and JA-signaling

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

7

Allele Forward

primer Reverse primer Comments

sid2-2 5’-

gaaagacgacct

cgagttctcta-3’

5’-cgtaagtctccctgcca

attgag-3’

Deletion mutant: SID2 product

= 470bp, sid2-2 gives no

product

npr1-1 5’-

atgtctcgaatgta

cataaggc-3’

5’-catgagtgcggttctac

cttc-3’

NlaIII digestion: NPR1

products = 200 and 100bp,

npr1-1 product = 300bp

coi1-16

5’-caaagacttggaa

accatagctagaa

attg-3’

5’-ccaatatcctcattcaag

gagccaccacaagat-

3’

Tsp509I digestion: COI1

products = 122 and 25bp, coi1-

16 products = 78, 44, and 25bp

pen2-4 5’-

aaacgttgccgtt

gatttct-3’

5’-cagcaacactagcgcc

atta-3’

AciI digestion: PEN2 products

= 445, 195, and 66bp, pen2-4

products = 640 and 66bp

Cfa6

and

cfa6::Tn5

P1 = 5’-

ctggtccggtcgg

tctaggta-3’

P4 = 5’-

cgagcaactcgtc

gagcatac-3’

P3 = 5’-

cggacctacgtcgagg

taaca-3’

P2 = 5’-

agatctgatcaagaga

cag-3’

See Supplemental Figure 1 for

details on the position of these

primers within the Cfa6 and

cfa6::Tn5 loci.

Supplemental Table 2. Primers used for screening mutant plants and bacteria

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

8

Supplemental Table 3. Primers used for qRT-PCR

Gene Primers

ACTIN2 (AT3G18780) 5’-CTAAGCTCTCAAGATCAAAGGCTTA-3’ 5’-TTAACATTGCAAAGAGTTTCAAGGT-3’

MYB51 (AT1G18570) 5’-ACAAATGGTCTGCTATAGCT-3’ 5’-CTTGTGTGTAACTGGATCAA-3’

CYP79B2 (AT4G39950) 5’-GTAACTTCGGAGCATTCGT-3’

5’-TCGCCGGATATCACATCC-3’

CYP79B3 (AT2G22330) 5’- AGTCACTTCCGAACACTCA-3’

5’-TCGCAGGTTACCATATTCC-3’

CYP83B1 (AT4G31500) 5’-TCACGCCATATCTACCAGC-3’

5’-TGGACGTCATGACTGGAC-3’

CYP81F2 (AT5G57220) 5’-CTCATGCTCAGTATGATGC-3’ 5’-CTCCAATCTTCTCGTCTATC-3’

PR-1 (AT2G14610) 5’-CTACGCAGAACAACTAAGAGGCAAC-3’

5’-TTGGCACATCCGAGTCTCACTG-3’

Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312

9