SUPPLEMENTARY INFORMATION 400 500 0 3 6 9 15 24 48 Time post inoculation (h) SNO content (pmole/mg protein) WT atgsnor1-1 atgsnor1-3 nox1 WT (AvrB) atgsnor1-3 (AvrB) Pst/ Pst (avrB)

0

100

200

300

400

500

0 3 6 9 15 24 48

Time post inoculation (h)

SN

O c

on

ten

t (p

mo

le/m

g p

rote

in)

WT

atgsnor1-1

atgsnor1-3

nox1

WT (AvrB)atgsnor1-3 (AvrB)

Pst / Pst (avrB)

Figure S1 | SNO content in atgsnor1 and nox1 plants

following PstDC3000 challenge. Profile of SNO accumulation

over time in the given plant genotypes following infiltration of

either PstDC3000(avrB) or PstDC3000 (containing an empty

vector lacking avrB). Data points represent the mean of 3

samples ± S.E. Strains of PstDC3000 were infiltrated at 1 x 106

cfu/ml.

SUPPLEMENTARY INFORMATIONdoi:10.1038/nature10427

WWW.NATURE.COM/NATURE | 1


0

50

100

150

200

250

0 3 6 9 15 24 48

Nit

rite

(n

mo

l/m

g p

rote

in)

WT

atgsnor1-1

atgsnor1-3

nox1

Pst (avrB)

d

0

50

100

150

200

250

Nit

rite

(n

mo

l/m

g p

rote

in)

WT

atgsnor1-1

atgsnor1-3

nox1

Pst (avrRps4)

0 3 6 9 15 24 48


WT

0 h 9 h 24 h 24 h + PTIO

atgsnor1-1

atgsnor1-3

nox1

c

ba

0

2

4

6

8

10

12

14

16

18

20

WT atgsnor1-1 atgsnor1-3 nox1

Rela

tiv

e D

AF

-2D

A in

ten

sit

y 0 h

9 h

24 h

24 h + PTIO

Pst (avrB)

Figure S2 | NO and nitrite levels in atgsnor1 and nox1 plants in response to avirulent PstDC3000

strains. a, DAF-2DA fluorescence (green) reports NO accumulation in response to PstDC3000(avrB). The

NO scavenger carboxy-2-phenyl-4,4,5,5-tetra-methylimidazolinone-3-oxide-1-oxyl (c-PTIO; 100 µM) blunts

DAF-2DA fluorescence, confirming the specificity of this fluorophore for bioimaging. Chloroplasts exhibited

red autofluorescence. b, Quantification of DAF-2DA signals following pathogen challenge. c, Levels of nitrite,

a reporter for NO accumulation, in the given plant genotypes following inoculation of PstDC3000(avrB). d,

Nitrite accumulation following PstDC3000(avrRps4) challenge in the stated plant lines. Data points represent

the mean of 3 samples ± S.E. Strains of PstDC3000 were infiltrated at 1 x 106 cfu/ml.

SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature10427


d

e

b

c

0

0.1

0.2

0.3

0.4

0.5

0 3 6 9 15 20 24 48

Fre

e S

A (

µg

/gfr

tiss

ue

) WT

atgsnor1-1

atgsnor1-3

nox1


Pst (avrB)


0

1

2

3

4

5

6

7

8

0 3 6 9 15 20 24 48

SA

G (

µg

/gfr

tis

su

e) WT

atgsnor1-1

atgsnor1-3

nox1

Pst (avrB)


0

1

2

3

4

5

6

0 3 6 9 15 20 24 48

SA

G (

µg

/gfr

tis

su

e) WT

atgsnor1-1

atgsnor1-3

nox1

Pst (avrRps4)


a

0

1

2

3

4

5

6

7

8

0 3 6 9 15 20 24 48


To

tal S

A (

µg

/gfr

tissu

e)

WTatgsnor1-1atgsnor1-3nox1

WT (avrB)atgsnor1-1 (avrB)

Pst / Pst (avrB)

0

0.1

0.2

0.3

0.4

0.5

0.6

0 3 6 9 15 20 24 48

Fre

e S

A (

µg

/gfr

tiss

ue

) WT

atgsnor1-1

atgsnor1-3

nox1

Pst (avrRps4)0.7

Figure S3 | Total and free SA and also SAG accumulation over time during attempted pathogen

infection. a, Profile of total SA accumulation over time in the given plant genotypes following infiltration

of PstDC3000 strains. b, Free SA accumulation in the stated Arabidopsis lines over time in response to

attempted PstDC3000(avrB) infection. c, Free SA levels in the given plant genotypes over time in

response to PstDC3000(avrRps4) challenge. d, SAG accumulation in the stated plant lines in response

to attempted PstDC3000(avrB) ingress. e, SAG levels following PstDC3000(avrRps4) infiltration in the

stated Arabidopsis plants. Data points represent the mean of 3 samples ± S.E. All strains of PstDC3000

were infiltrated at 1 x 106 cfu/ml.



0 9 15 24 48 h 0 9 15 24 48 h

a bPst (avrB) Pst (avrRps4)

WT

atgsnor1-1

atgsnor1-3

nox1

Figure S4 | SNOs accelerate the kinetics of HR-cell death. a, Kinetics of HR-cell death

development in the leaves of stated Arabidopsis genotypes over time post PstDC3000(avrB)

challenge. b, Timing of HR formation in the given plant lines following PstDC3000(avrRps4)

infiltration. Avirulent strains of PstDC3000 were infiltrated at 5 x 107 cfu/ml. All experiments

were repeated at least three times with similar results.



0

10

20

30

40

50

60

70

80

0 2 4 6 8 10 12 24


Ele

ctr

oly

te leakag

e (

µs/c

m)

WT

atgsnor1-1

atgsnor1-3

nox1

WT (avrB)

atgsnor1-3 (avrB)

Pst / Pst (avrB)

Figure S5 | Cell death development in atgsnor1 and nox1 plants. Extent of cell death

development established by electrolyte leakage in the given Arabidopsis genotypes

following challenge by PstDC3000 strains. All strains of PstDC3000 were infiltrated at 1 x

108 cfu/ml.



0

20

40

60

80

100

120

140

WT atgsnor1-3 atgsnor1-3 sid2

Cell d

eath

(arb

itra

ry u

nit

) Pst

Pst (avrB)

Pst (avrRps4)

Figure S6 | Cell death development in atgsnor1-3 plants relative to the atgsnor1-3 sid2

double mutant. Wild-type, atgsnor1-3 and atgsnor1-3 sid2 plants were inoculated with the

stated strains of PstDC3000 and cell death development determined by quantification of TB

staining at 48 hpi. All strains of PstDC3000 were infiltrated at 1 x 106 cfu/ml.



0

10

20

30

40

50

60

70

80

WT atgsnor1-3 sid2 atgsnor1-3 sid2

Cell d

eath

(arb

itra

ry u

nit

)

Figure S7 | Extent of cell death in atgsnor1 single and double mutant plants in

response to Emwa1. Wild-type, atgsnor1-3, sid2, and atgsnor1-3 sid2 plants were

challenged with Emwa1 and cell death determined by quantification of TB staining at 5

dpi.



0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

WT atgsnor1-3 sid1 sid2

Fre

e S

A (

µg

/gfr

tissu

e)

0

1

2

3

4

5

6

7

8

9


SA

G (

µg

/gfr

tissu

e)

a

b

c

0

2

4

6

8

10

12


To

tal

SA

(µ

g/g

frti

ss

ue

)

MgCl2Pst

Pst (avrB)

1.6

MgCl2Pst

Pst (avrB)

MgCl2Pst

Pst (avrB)

*

*

Figure S8 | Total SA, free SA and SAG accumulation in sid1 and

sid2 mutants compared to atgsnor1-3 plants. a, Total SA levels in the indicated plant

genotypes following the stated pathogen infiltrations at 48 hpi. b, Free SA concentrations in the

listed plant lines following challenge with the given PstDC3000 strains. c, SAG levels in the

indicated plant genotypes following the stated pathogen infiltrations. Data points represent the

mean of 3 samples ± S.E. When comparing total SA, free SA and SAG levels between

atgsnor1-3 and sid mutant plants, only free SA accumulation between atgsnor1-3 and sid1 in

response to PstDC3000 challenge, exhibits a statistically significant difference in a student’s

t-test (p =0.05). The associated data bars are marked by asterisks (panel b). All strains of

PstDC3000 were infiltrated at 1 x 106 cfu/ml.



a b

0

50

100

150

200

250


SN

O (

pm

ole

/mg

pro

tein

)

Mock

H.a (Emwa1)

0

50

100

150

200

250


Nit

rite

(n

mo

le/m

g p

rote

in)

Mock

H.a (Emwa1)

Figure S9 | Determination of SNO and nitrite levels in Arabidopsis sid mutants. a,

SNO levels were measured in the given plant genotypes at 5dpi following H. Arabidopsidis

Emwa1 inoculation. b, Nitrite levels were measured in the given plant genotypes following

H. Arabidopsidis Emwa1 inoculation. Data points represent the mean of 3 samples ± S.E.



0 3 6 15 h 3 6 15 h

a bPst (avrB) Pst (avrRps4)

WT

atgsnor1-1

atgsnor1-3

nox1

Figure S10 | ROI accumulation in atgsnor1 and nox1 plants following pathogen challenge.

a, Wild-type, atgsnor1 and nox1 plants were infiltrated with PstDC3000(avrB) and subsequently

stained with DAB to detect ROI accumulation at the times indicated. b, Wild-type, atgsnor1 and

nox1 plants were challenged with PstDC3000(avrRps4) and then stained with DAB to score for

ROI accumulation at the stated times. Avirulent strains of PstDC3000 were infiltrated at 1 x 106

cfu/ml.



WT atgsnor1-3 atrbohD atrbohF atrbohD atgsnor1-3 atgsnor1-3 atgsnor1-3

atrbohF atrbohD atrbohF atrbohD atrbohF

Figure S11 | Cell death development in atgsnor1-3 and atrboh single, double and

triple mutants. Plants of the indicated genotypes were challenged with 1 x 106 cfu / ml

PstDC3000(avrB) and then stained with TB at 48 hpi.



0

10

20

30

40

50

60

70

WT atgsnor1-3 atrbohD atgsnor1-3

atrbohD

atrbohD atrbohF

atgsnor1-3

Cell d

eath

(arb

itra

ry u

nit

)

MgCl2

Pst

Pst (avrB)

0

20

40

60

80

100

120

Inte

nsit

y o

f D

AB

sta

in

(arb

itra

ry u

nit

)

MgCl2

Pst

Pst (avrB)

WT atgsnor1-3 atrbohD atgsnor1-3

atrbohD

atrbohD atrbohF

atgsnor1-3

a

b

Figure S12 | Cell death formation and ROI accumulation in atgsnor1-3 and atrboh single,

double and triple mutants. a, Extent of cell death development in the stated plant lines

determined by quantification of TB staining following infiltration with either PstDC3000(avrB) or

PstDC3000 (containing an empty vector lacking avrB). PstDC3000 strains were inoculated at

1 x 106 cfu / ml and leaves harvested at 48 hpi. b, atgsnor1-3 and atrboh single, double and

triple mutants were challenged with either PstDC3000(avrB) or PstDC3000 and then stained

with DAB to score for ROI accumulation. PstDC3000 strains were inoculated at

1 x 106 cfu / ml and leaves harvested at 6 hpi. Data points represent the mean of 3 samples ±

S.E.



AtRBOH D::Myc

anti-c-Myc

Loading

control

0h 6h 12h

WT

atrb

ohD

atgs

nor1

-1

atrb

ohD

atgs

nor1

-3

atrb

ohD

atrb

ohD

atgs

nor1

-1

atrb

ohD

atgs

nor1

-3

atrb

ohD

atrb

ohD

atgs

nor1

-1

atrb

ohD

atgs

nor1

-3

atrb

ohD

Figure S13 | SNO levels do not impact AtRBOHD protein abundance. Levels of

endogenous AtRBOHD in the given plant genotypes over time following infiltration of

PstDC3000(avrB) at 1 x 107 cfu/ml. The experiment was repeated twice with similar

results.



Figure S14

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

m/z

561.37

1088.44

925.38

720.91

944.29

618.37 765.35

880.501358.561235.48

708.33

497.30 861.47 1245.501024.46417.13

310.95 1275.40269.97 1214.66953.39779.50 1108.75 1368.79 1498.28

885IGVFYC890GMPGMIK897

b12y10

y9

y5

y1

0+

2

y8

b8y7

y4

b4 y6

b5752.0 753.0 754.0 755.0

752.8537

753.3549

753.8574

m/z

a

*

Rela

tive A

bu

nd

an

ce

c

200 300 400 500 600 700 800 900 1000 1100 1200

964.42

311.08

1127.47

564.28

946.34

628.71778.29

865.26515.25 677.20598.37473.71352.59 747.28409.52 834.32 1010.51883.46293.11202.25 1109.56 1143.34687.89 1237.39

821LHNYC825TSVYE830

b9

b8

b8

-H2O

b7b6

y6

b5

MH-H2O+2

y2

b9

+2

y3

b9

-H2O

+2

636.0 637.0 638.0 639.0 640.0

m/z

637.7625

638.2641

638.7665

*

Rela

tive A

bu

nd

an

ce

0.0E+00

2.0E+03

4.0E+03

6.0E+03

8.0E+03

1.0E+04

1.2E+04

1.4E+04

1.6E+04

1.8E+04

Control CysNO

rep 1 rep 2 rep 1 rep 2

Inte

nsit

y

b

m/z



Figure S14 | Mass spectrometric analysis identifies C890 as a site of cysteine S-

nitrosylation. a, MSMS spectra of C890 (y8) from tryptic fragment 885IGVFYC890GMPGMIK897 (peptide MSMS spectra shown with two methionine oxidised and

cysteine blocked with carbamidomethyl). This analysis is consistent with C890 S-

nitrosylation. The S-nitrosothiol formed at C890, following NO donor exposure, reacted with

iodoacetamide to form a carbamidomethyl ion. LC-MS spectra of the corresponding peak

(752.8537 amu) of this peptide fragment is shown in inset. b, Changes of MS peak intensity

of the tryptic peptide in (a), 885IGVFYC890GMPGMIK897 containing C890, following NO donor

treatment. As shown in two independent runs, no carbamidomethyl ion formation was

detected at C890 in the absence of NO donor. In contrast, the formation of this ion at C890

was striking following NO donor treatment. c, MSMS spectrum of C825 from tryptic fragment 821LHNYC825TSVYE830. In contrast to C890, no S-nitrosylation of C825 was detected. Rather,

only methylthiol formation was found at C825 following treatment with methyl

methanethiosulfonate (MMTS), suggesting C825 was not S-nitrosylated following NO donor

exposure. LC-MS spectra of the corresponding peak (637.7625 amu) of this peptide

fragment blocked with MMTS is shown in inset.



AtRbohD

HNOX2

DNOX

AtRbohD

HNOX2

DNOX

HNOX2

DNOX

HNOX2

DNOX

AtRbohD

AtRbohD

Figure S15 | Cys890 of AtRBOHD is evolutionary conserved across kingdoms.

Arrow head indicates conserved cysteine residue in AtRBOHD(C890), HNOX2(C537)

and DNOX(C1315) from a C-terminus amino acid sequence comparison. Sequences

were aligned by the CLUSTAL method using the ClustalW programme.



a

b

c

0 50 100 250 500 500 500 500 500 µMDTT

GSNO CysNO GSNO CysNO

HNOX2 C537A HNOX2 WT

0 50 100 250 500 500 GSH 500 500 µMDTT DTT

GSNO CysNO

anti-Biotin

HNOX2

anti-Biotin

HNOX2

0 50 100 250 500 500 GSH 500 500 µMDTT DTT

GSNO CysNO

0 50 100 250 500 500 500 500 500 µMDTT

GSNO CysNO GSNO CysNO

DNOX C1315A DNOX WT

anti-Biotin

anti-Biotin

DNOX

DNOX

d

Figure S16 | S-nitrosylation of human and fly NADPH oxidase.

a, Recombinant HNOX2 is S-nitrosylated in a concentration dependent fashion by GSNO

and this modification can be reversed by DTT. CysNO can also drive HNOX2-SNO

formation and this redox-based modification can be reversed by DTT. b, Drosophila DNOX

is S-nitrosylated by either GSNO or CysNO and in each case this modification is reversible

by DTT. c, The C537A mutant of HNOX2 is not S-nitrosylated by GSNO. d, The C1315A

mutant of DNOX is not S-nitrosylated by GSNO. All experiments were repeated at least

twice with similar results.



FDR HNOX2 AtRBOH Da

b

Figure S17 | Computational modelling of HNOX2 and

AtRBOHD. a, Computational models of FDR, HNOX2 and AtRBOHD. b, Computational model

of the C-terminus of AtRBOHD showing the site of S-nitrosylation, C890 (yellow), Phe921

(blue) and FAD (green). The model predicts that S-nitrosylation of AtRBOHD at Cys890 may

disrupt the side-chain position of Phe921 impeding FAD binding.



a

b

Figure S18 | Computational modelling reveals the impact of SNO formation at C890 on

AtRBOHD structure. a, Computational model of the C-terminus of AtRBOHD showing the site of

S-nitrosylation, C890 (yellow), Phe921 (magenta) and FAD (green). In its reduced form (-SH)

Cys890 may stabilize Phe921 through a hydrogen bond (dashed line). Consequently, Phe921 and

the flavin group of FAD are coplanar, maximizing π-orbital overlapping. This is thought to be

necessary for high catalytic efficiency, as mutation of the according Phe570 and homologous

Tyr314 in hNOX2 and FDR, respectively, strikingly reduces the activity of these enzymes. b, S-

nitrosylation (-SNO) of Cys890 is expected to disrupt the coplanar localization of Phe921, thereby

destabilizing or sterically ejecting FAD.



NA

DP

H o

xid

as

e a

cti

vit

y

(∆O

D/m

g p

rote

in)

0

5

10

15

20

25

30

0 2 4 6 8 10


a

b

c

0

20

40

60

80

100

120

Ele

ctr

oly

te l

ea

kag

e (

µs

/cm

)

0 2 4 6 8 10


WT

C890A

0 2 4 6 8 10


0

20

40

60

80

100

120

140

160

180

200

SN

O c

on

ten

t (p

mo

le/m

g p

rote

in)

WT

C890A

WT

C890A

Figure S19 | Analysis of the kinetics of SNO formation, NADPH oxidase activity

and HR-cell death following pathogen challenge. a, SNO accumulation in wild-type

and C890A mutant plants following infiltration of PstDC3000(avrB). b, NADPH oxidase

activity in wild-type and C890A mutant plants following infiltration of PstDC3000(avrB).

c, Cell death determined by electrolyte leakage in wild-type and C890A mutant plants

following infiltration of PstDC3000(avrB). All pathogen infiltrations were undertaken with

1 x 108 cfu/ml. Data points represent the mean of 3 samples ± S.E.



R/avr

[SA ↑]

[SNOs ↑]HR

Cell Death

[ROIs ↑]

AtRBOHD

Cys890

AtRBOHD

Cys890

SH

SNO

- +SNO SNO

AtGSNOR1

Figure S20 | Model showing a central role for SNOs in the regulation of cell death

development during attempted infection.

Following R protein-mediated recognition SA and SNO levels rise and AtRBOHD is

activated (red shape) to synthesise ROIs. Collectively, these small molecules help drive cell

death development. As SNO levels rise they prime negative feedback loops, suppressing SA

accumulation and antagonising the activity of AtRBOHD by S-nitrosylation of Cys890 (blue

shape) leading to a decrease in ROI generation. In aggregate, these latter mechanisms help

limit the extent of cell death formation.



Documents

SUPPLEMENTARY INFORMATION 400 500 0 3 6 9 15 24 48 Time post inoculation (h) SNO content (pmole/mg protein) WT atgsnor1-1 atgsnor1-3 nox1 WT (AvrB) atgsnor1-3 (AvrB) Pst/ Pst (avrB)