A B

EC50=15.07 µM 100 EC50=57.87 µM

Stomatal aperature (µm)

FDA staining rate (%)

3 80

60
2 40

20
1
0
10 0 10 1 10 2 10 1 10 2
(GlcNAc)8 (µM) (GlcN)8 (µM)

Figure S1. EC50 values for (GlcNAc)8-induced stomatal closure and (GlcN)8-
induced reduction of FDA staining rate
The data of stomatal closure and reduction of FDA staining rate are from Figure 1A
and Figure 7A, respectively. The dose response curves and EC50 values were
calculated via non-linear regression using Prism 8.2.1 software (GraphPad software).

www.pnas.org/cgi/doi/10.1073/pnas.1922319117
 Control
110
60 µM (GlcNAc)8
100 10 µM ABA

Relative leaf weight (%)

90

80

70

60

50
0 30 60 90 120
Time (min)

Figure S2. Water loss from detached leaves treated with ABA and (GlcNAc)8
Relative water loss was represented by the weight of the leaves at various time
points divided by the original weight. Data are mean ± SEM (n=3).
A B
4

Stomatal aperture (μm)

8
3
Log2 fold change

0 2

-4
1
-8
0
-12
Mock GN8
60 µM (GlcNAc)
35S CERK1
cerk1-2

cerk1-1
Col-0

Nossen

gCERK1
gCERK1
cerk1-1
8

Col-0 lyk4-2
lyk4-2 lyk5-2
lyk5-2
35S

C D
4 4

Stomatal aperture (μm)

Stomatal aperture (μm)

3
3
*
2

2 1

0
Mock GN8
60 µM(GlcNAc)
1 8
Control

Col-0 pbl27
pbl27-1 slah
slah3-1
Mock

Ca2+

E F Mock 60 µM (GlcNAc)8
4
Stomatal aperture (μm)

a a a a N.S. N.S.
a a 4
Stomatal aperture (μm)

3 b
*
b b b
3
2

1 2

0
Mock 25 µM(GlcNAc)
GN8 1
8
Col-0 abi1-1C abi2-1C
Col-0 slac1-1
slac1-1 slah3
slah3-1
lyk5
lyk5-2 pbl27
pbl27-1
Figure S3. (GlcNAc)8-induced stomatal closure in lyk4, lyk5, pbl27, slah3, abi1-
1C and abi2-1C
(A) Transcript levels of CERK1. The levels of CERK1 transcript with UBQ10 as a
reference in leaves of different genotypes were normalized to that of Col-0. Data are
mean ± SEM (n=3).
(B) Stomatal closure induced by 60 µM (GlcNAc)8 in Col-0, lyk4-2 and lyk5-2.
Averages from three independent experiments (90 total stomata per bar) are shown.
Data are mean ± SEM (n=3).
(C) Effect of 1mM Ca2+ on cerk1-2 stomatal aperture.
(D) Stomatal closure induced by 60 µM (GlcNAc)8 in Col-0, pbl27-1 and slah3-1.
Averages from three independent experiments (90 total stomata per bar) are shown.
Data are mean ± SEM (n=3).
(E) Stomatal closure induced by 25 µM (GlcNAc)8 in Col-0, slac1-1, slah3-1, lyk5-2
and pbl27-1. Averages from four independent experiments (120 total stomata per bar)
are shown. Data are mean ± SEM (n=4). Different letters indicate statistical
significance (P<0.05, ANOVA with Tukey’s test).
(F) Stomatal closure induced by 60 µM (GlcNAc)8 in Col-0, abi1-1C and abi2-1C.
Averages from three independent experiments (90 total stomata per bar) are shown.
Data are mean ± SEM (n=3). Student’s t test: *, P<0.05. N.S., No significant
difference.
A B
YFP fluorescence Bright field Merged

CERK1D441V-YFPc
+ YFPn-CPK6

LYK4-YFPc
+ YFPn-CPK6
-LW

LYK5-YFPc -LWH
+ YFPn-CPK6 -Ade

FLS2-YFPc
+ YFPn-CPK6

CERK1D441V-YFPc -LW
+ YFPn-OST1
-LWH
-Ade
LYK4-YFPc
+ YFPn-OST1

LYK5-YFPc
+ YFPn-OST1

FLS2-YFPc
+ YFPn-OST1

OST1-YFPc
+YFPn-SLAC1

Figure S4. Additional BiFC and yeast-two hybrid experiments

(A) Confocal microscopy of N. benthamiana leaves transiently expressing the indicated split-
YFP constructs. Representative images are shown. Scale bar, 100 µm.
(B) Yeast two-hybrid assays with the indicated expressing combinations.
 Col-0 cerk1-2 Col-0 cerk1-2
10 µM ABA - + - - + - - + - - + -
60 µM (GlcNAc)8 - - + - - + - - + - - +
100 kDa
75 kDa

50 kDa
OST1

37 kDa

32P-signal (Autoradiography) Loading (Coomassie)

Figure S5. Kinase activity of OST1 in response to ABA and (GlcNAc)8

In-gel kinase assays with Histone-III as substrate for whole seedling protein. The
experiment has been repeated three times with similar results.
A B
FDA PI
FDA staining rate (%)

100
80 0

60
*
*
40
20 *
2h
0
0 15 30 60 90 120
Treatment time (min)

C D FDA PI
FDA PI 100 100

FDA staining rate (%)

PI staining rate (%)

80 80

60 60
0
40 40

20 20

0 0
2h Mock CSOS
60 µM (GlcN)8

E F
FDA FDA Bright field

Mock
0

60 μM
2h (GlcN)8
Figure S6. (GlcN)8-induced cell death in Col-0
(A) Time course of (GlcN)8-induced guard cell death. Guard cell death was evaluated
by FDA staining. The time starting (GlcN)8 treatment was considered as 0 time point.
Averages from three independent experiments (360 guard cells in total) are shown.
Data are mean ± SEM (n=3). Asterisks indicate statistical significance compared to
that of 0 time point (P<0.05, Student’s t test).
(B-D) FDA and PI double staining of guard cell treated with 60 µM (GlcN)8. Epidermal
tissues were double stained with FDA and PI to find live guard cells. After 2 h (GlcN)8
treatment, the same epidermal tissues were stained with FDA and PI again to
distinguish live and dead guard cells. The same guard cells before and after (GlcN)8
treatment were used for quantification. Representative staining images were shown in
(B) for Mock treatment and (C) for (GlcN)8 treatment. Blue arrowheads, stomatal pores
stained by PI; white arrowheads, nuclei of dead guard cells stained by PI; scale bar, 20
µm. Quantification of staining rates was shown in (D). Averages from three
independent experiments (200 to 250 guard cells per bar) are shown. Data are mean
± SEM (n=3).
(E) Effect of (GlcN)8 on leaf epidermal cell viability. The same cells were imaged
before and after treatment of 60 µM (GlcN)8. Scale bar, 20 µm.
(F) Effect of (GlcN)8 on mesophyll protoplast viability. Mesophyll protoplasts were
stained with FDA after 2 h (GlcN)8 treatment. Scale bar, 100 µm.
A B
a a a
100 a
FDA staining rate (%)

100

FDA staining rate (%)

80
80
60
60
40 b
b 40
b
20 b
20
0 0
Mock CSOS
60 µM (GlcN)8
60 µM 60 µM (GlcN)8
rbohD (GlcN)8 +20 µM DPI
Col-0 lyk4-2
lyk4-2 lyk5-2
lyk5-2 rbohD
rbohF

C D
100 *
DCF fluorescence
FDA staining rate (%)

200 N.S.
*
(% of Mock)
80
150
60
100
40
50
20
0
0 1 2 203µM 2 mM
4
Mock
60 µM 60 µM (GlcN)8 DPI+20SHAM
µM DPI
(GlcN)8 +2 mM SHAM 60 µM (GlcN)8

Figure S7. Effect of DPI, SHAM, lyk4, lyk5 and rbohD rbohF mutations on
(GlcN)8-induced guard cell death
(A) Effect of lyk4, lyk5 and rbohD rbohF mutations on (GlcN)8-induced guard cell
death. Averages from three independent experiments (200 to 250 guard cells per bar)
are shown. Data are mean ± SEM (n=3). Different letters indicate statistical
significance (P<0.05, ANOVA with Tukey’s test).
(B and C) Effect of DPI and SHAM on (GlcN)8-induced guard cell death. Averages
from three independent experiments (200 to 250 guard cells per bar) are shown. Data
are mean ± SEM (n=3).
(D) ROS accumulation induced by (GlcN)8 in guard cells. ROS accumulation was
expressed as the percentage of DCF fluorescence levels to that of Mock treatment.
Averages from three independent experiments (more than 120 total guard cells per
bar) are shown. Data are mean ± SEM (n=3). Student’s t test: *, P<0.05. N.S., No
significant difference.
 Fungus

chitinase

chitin deacetylase

chitin
oligosaccharide

Ca2+ ?
chitosan
SLAC1 activation ? oligosaccharide

Stomatal closure Cell death

Guard cell

Figure S8. Proposed model of guard cell-fungus interaction

During fungal penetration through stomata, chitin in the cell wall of fungi is digested by
chitinases preexisted in the apoplast or secreted by plant cells, leading to the release of
CTOS. Guard cells then perceive the CTOS through its receptor, CERK1, which induces
SLAC1 activation through a Ca2+-dependent pathway and consequently stomatal
closure to prevent fungal invasion. On the other hand, fungi secrete chitin deacetylases,
converting CTOS to CSOS, to evade CTOS-triggered immune response. When CSOS
accumulates over a threshold, it is sensed by yet-unknown receptors in the plasma
membrane, which triggers cell death also in a Ca2+-dependent manner to inhibit fungal
infection. PM, plasma membrane; CW, cell wall. For simplicity, plant cell wall is not
illustrated.
Table S1. Primers for vector construction
Assay Constructs Primer name Primer sequence (5’→3’)
BiFC pXY106-CPK6 CPK6-BclI-F CGTGATCAATGGGCAATTCATG
TCGTGG
CPK6-SalI-R CGCGTCGACCTACACATCTCTC
ATGCTGAT
pXY106-OST1 OST1-BamHI-F CGGGATCCATGGATCGACCAG
CAGTGA
OST1-SalI-R CGCGTCGACTCACATTGCGTAC
ACAATCTC
pXY106-SLAC1 SLAC1-BamHI-F CGGGATCCATGGAGAGGAAAC
AGTCAAAT
SLAC1-SalI-R CGCGTCGACTCAGTGATGCGA
CTCTTCCTC
pXY104-LYK4 LYK4-Sal1-F GGGATCCTCTAGAGTCGACAT
GATCTCGTTTTCATTTC
LYK4-Sal1-R CGCTGCCACCGCCGTCGACGT
ACGACGATTCTTCCCAG
pXY104-LYK5 LYK5-Sal1-F GGGATCCTCTAGAGTCGACAT
GGCTGCGTGTACACTC
LYK5-Sal1-R CGCTGCCACCGCCGTCGACGT
TGCCAAGAGAGCCGGAAC
pXY104-FLS2 FLS2-Sal1-F GGGATCCTCTAGAGTCGACAT
GAAGTTACTCTCAAAG
FLS2-Sal1-R CGCTGCCACCGCCGTCGACAA
CTTCTCGATCCTCGTTAC
pXY104-CERK1D441V CERK1-BamHI-F1 CGGGATCCATGAAGCTAAAGAT
TTCTCTAAT
CRRK1-SalI-R1 CGCGTCGACCCGGCCGGACAT
AAGACTGAC
CERK1-F2 GTTTATGTCCATAGGGtCATTAA
ATCTGCCAAT
CERK1-R2 ATTGGCAGATTTAATGaCCCTAT
GGACATAAAC
Yeast two-hybrid pGADT7-CREK1KD CREK1KD-EcoRI-F CATGGAGGCCAGTGAATTCAAT
TTGTCTTTTAAGATTG
CREK1KD-EcoRI-R GCCCACCCGGGTGGAATTCCC
GGCCGGACATAAGACTG
pGBKT7-CPK6 CPK6-EcoRI-F GCCATGGAGGCCGAATTCATG
GGCAATTCATGTCGTG
CPK6-SalI-R TGCGGCCGCTGCAGGTCGACG
CACATCTCTCATGCTGATG
pGBKT7-OST1 OST1-EcoRI-F GCCATGGAGGCCGAATTCATG
GATCGACCAGCAGTGA
OST1-SalI-R TGCGGCCGCTGCAGGTCGACG
CATTGCGTACACAATCTC
pGBKT7-SLAC1 NT SLAC1 NT-EcoRI-F GCCATGGAGGCCGAATTCATG
GAGAGGAAACAGTCAAATG
SLAC1 NT-SalI-R TGCGGCCGCTGCAGGTCGACG
TAGGAGAAACGGCCATTG
pGBKT7-SLAC1 CT SLAC1 CT-EcoRI-F GCCATGGAGGCCGAATTCTTTG
TCTGGCAAACGTTG
SLAC1 CT-SalI-R TGCGGCCGCTGCAGGTCGACG
GTGATGCGACTCTTCCTC