A

B

C

mock HopM10.2

0.25

0.3

0.35

0.4Controlflg22

mock HopM10.2

0.25

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0.35

ControlABA

mock HopM10.2

0.25

0.3

0.35

ControlH2O2

*****

Stom

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/len

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mock HopM1

0

2000

4000

6000

mock HopM1

0

50

100

150

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250flg22 chitin

D

*

*

WT mock

WT DEX 2h

HopM1 mock

HopM1 DEX 2h0

5

10

15

Rela

tive

Hop

M1

expr

essi

on

E

Tota

l pho

ton

coun

t

H2O2

Figure S1. Suppression of the PAMP-triggered oxidative burst and stomatal closure by HopM1 in N. benthamiana and characterization of HopM1 expression. A. Flg22- and chitin-induced oxidative burst in the absence and presence of in planta expressed HopM1. ROS generation (indicated as total photon count) was monitored over time for 30 minutes. Each experiment was repeated at least three times; results from a representative experiment are shown. Error bars represent standard error based on n=28 samples. B. Flg22-, ABA- or H2O2-mediated stomatal closure in the absence or presence of in planta expressed HopM1 in N. benthamiana. Each experiment was repeated at least three times; results from a representative experiment are presented. Error bars represent standard error based on n≥30. C. RT-PCR to detect HopM1 in N. benthamiana leaves non-agroinfiltrated (Naïve), agroinfiltrated with the empty Agrobacterium (Control), or agroinfiltrated with Agrobacterium to express HopM1 (HopM1), two days post-infiltration. Amplification of Actin 2 (ACT2) is shown as a control. D. Tissue collapse in HopM1-expressing N. benthamiana leaves five days post-infiltration. E. Relative HopM1 expression in mock- or DEX-treated Col-0 or transgenic HopM1 Arabidopsis plants. UBQ10 was used as the internal control. The expression in the mock-treated HopM1 transgenics was set to 1. 

mock HopM10.1

0.2

0.3

0.4

Controlflg22

+MG132

**

N. benthamiana

Stom

atal

ape

rtur

e (w

idth

/len

gth)

Figure S2. The proteasome inhibitor MG132 reverts the HopM1-mediated inhibition of PAMP-triggered stomatal closure in N. benthamiana. Flg22-mediated stomatal closure in the absence or presence of in planta expressed HopM1 upon treatment with the proteasome inhibitor MG132 in N. benthamiana. This experiment was repeated at least three times; results from a representative experiment are presented. Error bars represent standard error based on n≥30. Asterisks indicate a statistically significant difference compared to controls according to Student t-test, p<0.05.

Arabidopsis thaliana

WT

HopM1

atmin7

Nicotiana benthamiana

WT

HopM1

A

B

flg22

flg22

ABA

ABA

Control

Control

Figure S3. Confocal imaging of stomata. Micrographs of Arabidopsis leaves of the indicated lines (A) and in N. benthamiana leaves (B) show merged z-stacks of stomatal apertures under control, ABA- and flg22-treated conditions. The grey lines represent measurements of the width and length of the stomatal pore. Scale bar = 10m.

A

B

Figure S4. HopM1 does not affect the accumulation of the receptor FLS2 or the NADPH oxidase RBOHD. A. FLS2 accumulation in wild type (WT) or transgenic HopM1 Arabidopsis plants upon DEX treatment. Coomassie staining (CBB) is included as loading control. B. FLS2-YFP and RBOHD-YFP accumulation in the absence or presence of HopM1 in transiently co-transformed N. benthamiana plants. Coomassie staining (CBB) is included as loading control.

A

B

CBZR1-YFP mock BZR1-

YFP+AICAR

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nsity

ratio

*

BZR1-YFP+HopM1

BZR1-YFP

BZR1-YFP+AvrE

BZR1-YFP

BZR1-YFP+AvrPtoB

BZR1-YFP

0

2

4

6

8

10

Nuc

lear

/cyt

opla

smic

inte

nsity

ratio

a

bc

b b b

Figure S5. AICAR inhibits 14-3-3 proteins in N. benthamiana, and HopM1, but not other bacterial effectors, exerts a similar effect. A. Confocal micrographs show BZR1-YFP localization in mock- or AICAR-treated transiently transformed N. benthamiana leaves. B. Nuclear/cytoplasmic signal ratio in mock- or AICAR-treated transiently transformed N. benthamiana leaves. This experiment was repeated three times with similar results; in each case, at least 15 cells were measured. Results from one representative experiment are shown. Error bars represent standard error. Asterisks indicate a statistically significant difference compared to controls according to Student’s t-test, p<0.05. C. Expression of HopM1, but not other bacterial effectors, triggers strong nuclear accumulation of BZR1-YFP in Nicotiana benthamiana. Nuclear/cytoplasmic intensity ratio in control BZR1-YFP or BZR1-YFP+HopM1, BZR1-YFP+AvrE or BZR1-YFP+AvrPtoB transiently co-transformed N. benthamiana leaves. In each case, co-expression with the bacterial effector and control were performed in two halves of the same leaf. The results are the average of two independent biological replicates; in each case, at least 15 cells were measured. Error bars represent standard deviation. Letters indicate a statistical significant difference according to a one-way ANOVA test with post hoc Tukey analysis, p<0.05. 

PRR

RBOHD/B

ROS

Stomatal closure

GRF8/TFT1

*****

PAMPs

HOPM1

26S Proteasome

Figure S6. Proposed model for the effect of HopM1 on PAMP-triggered ROS burst and stomatal closure. Upon PAMP perception by Pathogen Recognition Receptors (PRRs), the RBOHD/B-mediated production of ROS is activated. RBOHD/B function is required to trigger stomatal closure. The 14-3-3 GRF8/TFT1 acts as a positive regulator of the PAMP-triggered ROS burst. HopM1 binds to and destabilizes GRF8/TFT1, ultimately interfering with both the PAMP-triggered ROS production and stomatal closure.