Chapter 6

Assays to Investigate the N-Glycosylation State

and Function of Plant Pattern Recognition Receptors
Stacey A. Lawrence, Teresa Ceserani, and Nicole K. Clay

Abstract
The biogenesis and functionality of pattern recognition receptors (PRRs) are critical for robust plant
immune responses. Here, we present methods to determine the N-glycosylation state and ligand-induced
activity of these receptors for comparative quantitative analysis. These techniques can be used to identify
mutants and chemical inhibitors affecting PRR biogenesis and functionality. When combined, these tech-
niques can provide useful insights on biological processes necessary to synthesize a properly membrane-­
localized and ligand-responsive PRR.

Key words Asparagine-linked glycosylation, Protein quality control, Endoglycosidase H, Tunicamycin,

MAPK activation, Seedling growth arrest, Anthocyanin inhibition

1  Introduction

1.1  N-Glycosylation The addition of glycans to asparagine residues (N-glycosylation) is

of FLS2 and EFR an essential, highly conserved co-translational modification. It tar-
gets newly synthesized membrane-localized and secreted proteins in
the endoplasmic reticulum (ER) lumen in eukaryotic cells. The role
of N-glycans in the ER-resident protein quality control (ERQC)
network is to monitor the accurate folding of glycoproteins and
mediate the distribution of correctly folded proteins within the
secretory system (e.g., to the plasma membrane or vacuole) [1, 2].
Some of the best characterized proteins to undergo N-glycosylation
are the Arabidopsis pattern recognition receptors (PRRs) FLS2 and
EFR, which recognize microbe-associated molecular pattern mole-
cules (MAMPs) bacterial flagellin and EF-Tu (as well as their bioac-
tive epitopes flg22 and elf18/elf26), respectively [3, 4].
FLS2 and EFR require protein N-glycosylation for their matu-
ration and transport to their final plasma membrane destination to
mediate antibacterial immunity in response to their ligands [5].
EFR protein abundance and function are compromised when
­single N-glycosylation sites in the EFR protein, N-glycosylation

Libo Shan and Ping He (eds.), Plant Pattern Recognition Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 1578,
DOI 10.1007/978-1-4939-6859-6_6, © Springer Science+Business Media LLC 2017

61
62 Stacey A. Lawrence et al.

enzymes, or single associated ERQC components are mutated [6–

12]. Although individual N-glycan site mutants of FLS2 do not
have the same effect, a significant portion of the mutated FLS2
protein is retained in the ER, reducing its ligand sensitivity and
suggesting that it too is a client of the N-glycan-mediated ERQC
mechanism [11, 12].

1.2  Quantitative FLS2 and EFR have 21 and 16 conserved N-glycosylation consen-
Readouts of FLS2 sus sites—NX(S/T) motifs—on their respective LRR-containing
and EFR ectodomains [11, 12]. In addition, the detected molecular masses
Glycosylation State of FLS2 and EFR are much larger than their predicted masses:
~175 vs. ~130 kDa for FLS2 and ~145 vs. ~110 kDa for EFR [3,
4, 13]. Two tools can be used to deglycosylate the FLS2 and EFR
proteins: the N-glycosylation inhibitor tunicamycin and the degly-
cosylation enzyme endoglycosidase H (Endo H).
Tunicamycin treatment inhibits N-glycosylation and induces
the unfolded protein response in the ER, a cellular stress response
that initiates programmed cell death in plants [14–16]. In wild-­
type seedlings treated with tunicamycin, the FLS2 protein mass is
reduced by ~40 kDa, whereas the EFR protein mass is reduced by
~5 kDa (Fig. 1a). Similar tunicamycin treatments were reported
to produce two differently sized FLS2 proteins at ~175 and
~130 kDa and a single sized EFR protein at ~150 kDa [11].
Because plant N-glycans have an average mass of ~2 kDa, a mass
shift of ~40 kDa for the FLS2 protein suggests that nearly all 21
potential N-glycan attachment sites are occupied, whereas a mass
shift of ~5 kDa for the EFR protein indicates that only a handful
of N-glycan sites may be occupied. The absence of an unglycosyl-
ated EFR protein at ~110 kDa suggests that EFR contains other
substantial post-translational modifications or is more resistant to
tunicamycin treatments.
Endo H has been used to functionally characterize ERQC
components required for the biogenesis of the FLS2 and EFR
proteins [8, 11, 12]. It cleaves N-glycans that are in the early
stages of maturation before processing through the Golgi, and
thus are mainly associated with ER-localized, immature glycosyl-
ated proteins. In contrast, mature N-glycans, which are only asso-
ciated with membrane proteins that process through the Golgi,
are largely insensitive to Endo H cleavage. Therefore, Endo H
treatment combined with functional data allows the differentia-
tion of plasma membrane-localized glycoproteins from their ER-
localized counterparts. In wild-type seedling extracts treated with
Endo H, FLS2, and EFR protein masses are reduced by ~5 kDa.
This suggests that FLS2 and EFR carry mainly mature N-glycans
and are mostly localized at the plasma membrane (Fig. 1b). These
results are consistent with previous reports of similar treatments
[8, 11, 12].
 Receptor N-Glycosylation and Function 63

a b
WT efr WT efr
+ + tunicamycin + + + Endo H

175 kD -FLS2 175 kD

-FLS2 130
130

175
-EFR
130
-EFR 130

95
Loading Loading
control control

c d
6.0
+ + mock
+ + elf26 5.0

Relative signal
tunicamycin + mock
+ + + tunicamycin 4.0

intensity
MPK6 3.0
-p42/44 MAPK
MPK3 2.0
MPK4 mock + elf26
-MPK3 1.0
tunicamycin + elf26
Lane 1 2 3 4 5 6 0.0
1 2 3 4 5 6
Lane

Fig. 1 (a) Immuno-detection of FLS2 and EFR proteins in total protein extracts from Col-0 (WT), fls2, and efr
seedlings treated with 5 μg/mL tunicamycin and separated on an 8.5% SDS-PAGE gel. (b) Immuno-detection
of FLS2 and EFR proteins from Col-0 (WT), fls2, and efr seedlings digested with Endo H and separated on a 6%
SDS-PAGE gel. Black arrows indicate FLS2- or EFR-specific bands before treatment. White arrows indicate
FLS2- or EFR-specific bands after treatment. Molecular mass sizes are indicated on the right. (c) Immuno-­
detection of phosphorylated MPK6, MPK3, and MPK4 proteins from flg22- or elf26-elicited WT total protein
extracts separated on a 10% SDS-PAGE gel. (d) Data represents the combined signal intensities of phosphory-
lated MAPKs relative to that of MPK3 protein (loading control) in (c)

1.3  Quantitative Two MAMP-induced immune responses have been used as high-­
Readouts of FLS2 throughput readouts in forward-genetic screens to identify new
and EFR Function regulators of FLS2 and EFR function: the seedling growth arrest
in Genetic Studies response and the anthocyanin repression response. Both responses
require long-term exposure to elicitors (i.e., in days) and sustained
activation of FLS2/EFR-mediated signaling. In addition, these
assays were successfully used to identify ERQC components
required for the biogenesis of the FLS2 and EFR proteins [6–9,
11, 12]. Here, we describe methods that adapt both MAMP-­
sensitive responses as robust quantitative readouts in small-scale
assays.
Different MAMPs arrest seedling growth in Arabidopsis [3, 4].
With our method, flg22 and elf26 induce a seedling growth arrest
response with different linear dynamic ranges: between 1 nM and
1 μM for flg22 (Fig. 2a) and between 0.1 and 100 nM for elf26
(Fig. 3a). Similar flg22/elf18-induced seedling growth arrest
 64 Stacey A. Lawrence et al.

responses were reported to have a linear dynamic range between 1

and 100 nM, with 100 nM representing the saturating concentra-
tion and concentrations below 1 nM being untested [8]. Responses
in the linear range are directly proportional to the ligand concen-
tration, and thus can be used in comparative quantitative analysis.
Crosstalk between MAMP-induced signaling and abiotic stress-
induced flavonoid accumulation has been described in a wide range
of plant-microbe interactions [9, 17–20]. For example, 1 μM con-
centrations of flg22 and elf18 repress sucrose stress-­induced antho-
cyanin accumulation in Arabidopsis seedlings [9, 20]. With our
method, the linear dynamic range of anthocyanin repression
response starts from 1 nM for flg22 and from 0.1 nM for elf26, and
ends at 100 nM for both elicitors (Fig. 2b to Fig. 3b).
A third MAMP-activated immune response, the MAPK acti-
vation response (i.e., the defense-specific phosphorylation of
MAPKs MPK6, MPK3, and MPK4), is typically used to confirm
findings from the seedling growth arrest and anthocyanin repres-
sion assays [6–9], since a separation between the initial and sus-
tained activation of PRR-mediated signaling has been reported in
Arabidopsis [7, 21, 22]. In contrast to seedling growth and

(a) (b)
140 120 120
A A
120 100 100
Fresh weight (% control)

Fresh weight (% control)

A A
100
80 80
80
60 B 60
60
B
40 C C 40
40

20 20 20

0 0 0
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0.1 1 10
nM µM nM µM
(c) (d)
140 200 140
Relative (A 530-0.25*A 657)/ DW (mg)

Relative (A530 -0.25*A 657)/ DW (mg)

180
120 A 120
160
100 140 100
AB
B 120
80 A 80
100
60 80 60

40 C 60 40
B B B
40
20 20
20
0 0 0
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0.1 1 10
nM µM nM µM

Fig. 2 Flg22-induced, dose-dependent seedling growth arrest (a, b) and anthocyanin repression (c, d) immune
responses in Col-0 (WT) and fls2 seedlings. The response to water is set to 100. (a, c) Data represent the mean
±1 standard deviation of n = 5 biological replicates in (a), n = 10 in (c, left graph), and n = 16 in (c, right graph).
Black bars, WT; white bars, fls2. Different letters indicate statistically significant differences (p ≤ 0.01, two-­
tailed t-test). (b, d) Three independent experiments, including those shown in (a) and (c), illustrate the vari-
ability inherent in the two MAMP immune response assays. DW, dry weight
 Receptor N-Glycosylation and Function 65

(a) (b)
120 140 120
A
120 A
100 A 100

Fresh weight (% control)

B
Fresh weight (% control)

100
80 80
80 B
C
60 B 60
60
B
40 40
40

20 20 20

0 0 0
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0.1 1 10
nM M nM µM
(c) (d)

120 140 120

A A
A

Relative (A530-0.25*A657)/ DW (mg)

120
Relative (A530-0.25*A657)/ DW (mg)

100 100

100
80 B 80

80
60 60
B
C 60
B
40
B 40
40

20 20
20

0 0 0
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0.1 1 10
nM µM nM µM

Fig. 3 Elf26-induced, dose-dependent seedling growth arrest (a, b) and anthocyanin repression (c, d) immune
responses in Col-0 (WT) and efr seedlings. The response to water is set to 100. (a, c) Data represent the mean
±1 standard deviation of n = 5 biological replicates in (a), n = 10 in (c, left graph), and n = 16 in (c, right graph).
Black bars, WT; white bars, fls2. Different letters indicate statistically significant differences (p ≤ 0.01, two-
tailed t-test). (b, d) Data from three independent experiments, including those shown in (a) and (c), illustrate
the variability inherent in the two MAMP immune response assays. DW, dry weight

anthocyanin repression assays, whose MAMP exposures are in the

order of days, the MAPK activation response occurs early in the
FLS2/EFR-mediated signaling and thus requires a much shorter
term of elicitor exposure (in the order of minutes) to flg22 and
elf26. It also has two short linear dynamic ranges: the first
between 1 and 10 nM and the second between 100 nM and 1 μM
(Fig. 4). However, only the second linear range of the flg22-
induced MAPK activation response appears to be significant, with
a near-fold increase in signal intensity between 100 nM and 1 μM
of flg22 (Fig. 4).

1.4  Qualitative The MAPK activation response is useful in pharmacological studies

Readout of FLS2 of FLS2 and EFR function, especially in the presence of toxic chemi-
and EFR Function cals such as tunicamycin. In tunicamycin-treated seedlings, the
in Chemical Studies deglycosylated FLS2 and underglycosylated EFR proteins exhibit
reduced phosphorylation of MPK6, MPK3, and MPK4, in response
to their respective MAMP ligands, demonstrating their decreased
functionality (Fig. 1c, d). This result is consistent with the reported
interference of tunicamycin treatment with FLS2 and EFR’s ligand-
binding ability and plasma membrane-localization [11].
66 Stacey A. Lawrence et al.

(a) (b)
0 10-1 100 101 102 103 104 nM 3 Rep 1
MPK6
Rep 2
-p42/44 MAPK MPK3 2.5

Relative signal intensity

Rep 1 Rep 3
MPK4
-MPK3 2
MPK6 1.5
-p42/44 MAPK MPK3
Rep 2
MPK4 1
-MPK3
MPK6 0.5
-p42/44 MPK MPK3
Rep 3 0
MPK4
-MPK3 0 10-1 100 101 102 103 104
nM

(c) (d)

0 10-1 100 101 102 103 104 nM elf26 2.5 Rep 1

MPK6 Rep 2

Relative signal intensity

-p42/44 MAPK MPK3 2.0
Rep 1 Rep 3
MPK4
-MPK3
1.5
MPK6
-p42/44 MAPK MPK3
Rep 2 1.0
MPK4
-MPK3
0.5
MPK6
-p42/44 MAPK MPK3
Rep 3 0.0
MPK4
-MPK3 0 10-1 100 101 102 103 104
nM elf26

Fig. 4 Dose-dependent MAPK activation response in Col-0 (WT) seedlings elicited with increasing concentra-
tions of flg22 (a, b) or elf26 (c, d). (a, c) Immuno-detection of phosphorylated MPK6, MPK3, and MPK4 (indi-
cated on the right) from three technical replicates (Rep1–3) of WT total protein extracts separated on a 10%
SDS-PAGE gel. (b, d) Data of the combined signal intensities of phosphorylated MAPKs in (a) and (c) relative to
that of MPK3 protein (loading control) illustrate the variability inherent in the MAPK activation assay

2  Materials

2.1  Plant Growth 1. Arabidopsis thaliana seeds Col-0 (wild-type), fls2

(SALK_062054C), and efr (SALK_044334) from Arabidopsis
Biological Resource Center (Columbus, Ohio).
2. Seed sterilization solution: 5% (v/v) hypochlorite, 0.1% (v/v)
Triton X-100.
3. Biosafety cabinet.
4. Autoclaved distilled water (dH20).
5. MS media 1 (pH 5.7–5.8): 0.5× MS basal salts with vitamins
(Phytotechnology labs, M519), 0.05% (w/v) MES, 0.5%
(w/v) sucrose, filter-sterilized (0.45 μm).
6. MS media 2 (pH 5.7–5.8): 1× MS basal salts with vitamins,
0.05% (w/v) MES, 0.5% (w/v) sucrose, filter-sterilized
(0.45 μm).
7. MS media 3 (pH 5.7–5.8): 0.5× MS basal salts with vitamins,
0.05% (w/v) MES, filter-sterilized (0.45 μm).
8. MS media 4 (pH 5.7–5.8): 0.5× MS basal salts with vitamins,
0.05% (w/v) MES, 3.42% sucrose, filter-sterilized (0.45 μm).
9. Sterile 12-well and 24-well tissue culture plates.
 Receptor N-Glycosylation and Function 67

10. Light cart with adjustable light cycle and intensity in a 21 °C
room and water trays to provide humidity.
11. Bacterial MAMP elicitors: 1 mM flg22 (Genscript, ≥85.1%
purity) and 1 mM elf26 (Genscript, ≥96.6% purity) aqueous
solutions.
12. Sterile 2-mL round-bottom and 1.7-mL microcentrifuge
tubes.
13. Sterile 1-mL filter tips and 200 μL tips for pipetmen.
14. Sterile 15-mL conical tubes.

2.2  Tunicamycin 1. 0.5 mg/mL tunicamycin (Sigma, T7765) stock solution in

Treatment methanol.

2.3  Total Protein 1. Total protein extraction buffer: 50 mM Tris-Cl (pH 7.5),
Extraction 50 mM DTT, 4% (w/v) SDS, 10% (v/v) glycerol; add DTT
prior to use.
2. 2-mL round-bottom and 1.7-mL microcentrifuge tubes.
3. Liquid nitrogen.
4. TissueLyzer homogenizer (Qiagen) and 5-mm stainless steel
beads (Thomson, 440CSS).
5. Room-temperature benchtop microcentrifuge.
6. 95 °C heat block.

2.4  SDS-PAGE 1. Distilled H2O (dH2O).

2. Mini PROTEAN tetra cell and 1.5-mm plates (Bio-Rad).
3. 6% separating gel: 375 mM Tris-Cl (pH 8.8), 6% (w/v) 37.5:1
acrylamide to bisacrylamide, 0.1% (w/v) SDS, 0.05% (w/v)
APS, 10% (v/v) TEMED.
4. 8.5% separating gel: 375 mM Tris-Cl (pH 8.8), 8.5% (w/v)
37.5:1 acrylamide to bisacrylamide, 0.1% (w/v) SDS, 0.05%
(w/v) APS, 10% (v/v) TEMED.
5. 10% separating gel: 375 mM Tris-Cl (pH 8.8), 10% (w/v)
37.5:1 acrylamide to bisacrylamide, 0.1% (w/v) SDS, 0.05%
(w/v) APS, 10% (v/v) TEMED.
6. 2-propanol or 0.1% SDS solution for gel overlay.
7. 4% stacking gel: 125 mM Tris-Cl (pH 6.8), 4% (w/v) 37.5:1
acrylamide to bisacrylamide, 0.1% (w/v) SDS, 0.05% (w/v)
APS, 10% (v/v) TEMED.
8. Lint-free tissues (Kimwipes).
9. Prestained 10–175 kDa molecular-weight markers (Abcam,
ab115832).
10. Running buffer: 25 mM Tris, 192 mM glycine, 0.1% (w/v)
SDS.
68 Stacey A. Lawrence et al.

2.5  Western Blotting 1. Two-gel tetra blotter or Criterion blotter (Bio-Rad).

2. Transfer buffer: 25 mM Tris, 192 mM glycine, freshly pre-
pared and stored at 4 °C.
3. PVDF membrane, 0.45 μm pore size (Millipore, IPVH00010).
4. Methanol.
5. Precut chromatography paper.
6. 1% (w/v) Ponceau S (IBI Scientific, IB01100) in 3% (w/v)
trichloroacetic acid.
7. 5% (w/v) acetic acid.
8. Sterile 10× phosphate-buffered saline (PBS) (pH 7.4): 1.369 M
NaCl, 26.8 mM KCl, 1 mM Na2HPO4, 17.9 mM KH2PO4.
9. 5% (w/v) nonfat dry milk in 1× PBS.
10. Primary antibodies in 5% (w/v) nonfat dry milk in 1× PBS:
anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Cell
Signaling Technology, 9101), 1:2000 dilution; anti-AtMPK3
(Sigma, A6979), 1:2000 dilution; anti-FLS2 (custom-made
polyclonal antibody against the peptide CTKQRPTSLNDEDSQ,
A. thaliana gene ID: At5g46330), 1:1000 dilution; anti-EFR
(custom-made polyclonal antibody against the peptide
CITESPRDAPQSSPQ, A. thaliana gene ID: At5g20480),
1:250 dilution.
11. Secondary antibody: Goat anti-rabbit IgG-HRP (Jackson
Laboratories, 111-035-003), 1:20,000 dilution in 5% (w/v)
nonfat dry milk in 1× PBS.
12. Enhanced chemiluminescence (ECL) prime western blotting
detection reagent (GE Healthcare, RPN2232).
13. National Institutes of Health (NIH) ImageJ software (http://
imagej.nih.gov/ij/).

2.6  Membrane 1. Stripping solution: 50 mM Tris-Cl (pH 7.5), 2% (w/v) SDS,
Stripping 0.16% (v/v) β-mercaptoethanol.
2. 60 °C oven.
3. 1× PBS.

2.7  Endo H 1. Endoglycosidase H (Endo H; 1,000,000 units/mL; New

Treatment England Biolabs, P0703L).
2. 10× citrate buffer (pH 5.4): 127.5 mM citric acid, 372.5 mM
sodium citrate.
3. 37 °C incubator.
4. 80 °C heat block.

2.8  Seedling Growth 1. 18–25 G1½ hypodermal needle.

Inhibition and 2. Bunsen burner.
Anthocyanin Assays
3. Liquid nitrogen.
 Receptor N-Glycosylation and Function 69

4. Lyophilizer (Labconco).
5. Balance (≥1 mg).
6. TissueLyzer homogenizer (Qiagen) and 5-mm stainless steel
beads (Thomson, 440CSS).
7. Acidic methanol: 99% (v/v) methanol, 1% (v/v) 12.1 M HCl.
8. 70 °C incubator.
9. Visible light spectrophotometer (Beckman).

2.9  MAPK 1. MAPK extraction buffer: 25 mM β-glycerophosphate, 10%

Activation Assay (v/v) glycerol, 50 mM Tris-Cl (pH 7.5), 200 mM NaCl, 0.1%
(v/v) Tween-20, 5 mM NaF, 0.5 mM DTT, 1 mM EDTA (pH
8.0), 2 mM NaVO3, 1 mini-complete protease inhibitor tablet
(Roche, 4693159001), and 1 phosphatase inhibitor tablet
(Roche, 4906845001), freshly prepared on ice (see Note 1).
2. 2× Laemmli buffer: 125 mM Tris-Cl (pH 6.8), 4% (w/v) SDS,
10% (v/v) β-mercaptoethanol, 20% (v/v) glycerol [23].
3. 2-mL round-bottom and 1.7-mL microcentrifuge tubes.
4. Liquid nitrogen.
5. TissueLyzer homogenizer (Qiagen) and 5-mm stainless steel
beads (Thomson, 440CSS).
6. 4 °C benchtop microcentrifuge.
7. 95 °C heat block.
8. Room-temperature benchtop microcentrifuge.

3  Methods

3.1  Tunicamycin 1. Sterilize Col-0 Arabidopsis seeds in an excess of seed steriliza-

Treatment tion solution for 10 min. In biosafety cabinet, under sterile
conditions, rinse seeds with autoclaved dH20. Repeat three to
five times.
2. Add 1 mL of MS media 2 and 12 seeds per well in a 12-well
tissue culture plate.
3. Place sample-containing plate on a light cart, under a 16-h light
cycle at 21 °C, 70–80 μE intensity, and 50–75% humidity.
4. On day 7, in biosafety cabinet, exchange media with 1 mL of
MS media 2 (see Note 2).
5. On day 8, in biosafety cabinet, add 10 μL of tunicamycin to a
final concentration of 5 μg/mL. Incubate the sample-­containing
plate under the same conditions as in step 3 for 24 h.
6. On day 9, air-dry seedlings from each well on paper towels for
5 min, transfer seedlings to 2-mL round-bottom microcentri-
fuge tubes, and freeze samples in liquid nitrogen. Samples can
be stored at −80 °C until processing (see Note 3).
70 Stacey A. Lawrence et al.

3.2  Total Protein 1. Cool the homogenizer plates by pouring liquid nitrogen on
Extraction them. Quickly transfer the sample tubes to the homogenizer
plate and open the lids. Pour liquid nitrogen on top of the
samples to keep them frozen. Add 80 μL of total protein
extraction buffer and a liquid nitrogen-cooled 5-mm stainless
steel bead to each sample.
2. Homogenize the samples for 2 min at 25 Hz.
3. Centrifuge at 3000 × g for 5 s to collect homogenate at the
bottom of the tube.
4. Incubate samples at 95 °C for 10 min.
5. Centrifuge at 11,000 × g for 8 min at room temperature to
precipitate insoluble material.

3.3  SDS-PAGE 1. Cast an 8.5% SDS polyacrylamide separating gel of 1.5-mm

thickness (see Note 4).
2. Overlay gel with 0.1% SDS or 2-propanol and allow gel to
polymerize for 20–30 min.
3. Gently rinse off the 0.1% SDS or 2-propanol layer with dH20 and
use a lint-free tissue to remove excess liquid on top of the gel.
4. Cast a 4% stacking gel on top of the separating gel, insert
comb, and allow gel to polymerize for at least 10 min.
5. Prerun gel at 100 V for 10 min (see Note 5).
6. Load 35 μL of each sample per well of the gel. Load 7.5 μL of
prestained molecular-weight markers in the first well.
7. Separate proteins at 150 V until the 62-kDa marker band
reaches the bottom of the gel.

3.4  Western Blotting 1. Once the run is complete, incubate gel in dH20 for 5 min with
gentle agitation.
2. Equilibrate gel and sponges in cold transfer buffer for 20 min.
3. Incubate PVDF membrane in methanol for 1–5 min and equil-
ibrate in ice-cold transfer buffer for 20 min.
4.
Assemble blotting sandwich following manufacturer’s
instructions.
5. In cold-room, transfer proteins onto membrane at 100 V for
1 h or until current reaches 0.7 mA for a Tetra blotter appara-
tus, or at 100 V for 30 min or until current reaches 1 mA for a
Criterion blotter apparatus.
6. Allow membrane to air dry completely (10–30 min).
7. Stain with Ponceau S for 5 min.
8. Destain using 5% acetic acid for 10 min and rinse three times
in dH20.
 Receptor N-Glycosylation and Function 71

9. Image and rinse with 1× PBS on a shaker until Ponceau S stain-

ing has faded.
10. Block with 5% nonfat dry milk for 20–30 min at room tem-
perature with gentle rocking.
11. Incubate with primary antibody anti-FLS2 overnight at 4 °C
with gentle rocking.
12. Wash blot with 1× PBS for 5 min with gentle agitation. Repeat
two times.
13. Incubate with secondary antibody at room temperature for 1 h
with gentle agitation.
14. Wash blot with 1× PBS for 5 min with gentle agitation. Repeat
two times.
15. Incubate with ECL solution per manufacturer’s instructions.
16. Perform detection using digital imaging system or film.

3.5  Membrane 1. After detection, rinse membrane with 1× PBS and incubate in
Stripping stripping solution at 60 °C for 30 min.
2. Rinse membrane with 1× PBS several times to remove all SDS
and β-mercaptoethanol.
3. Proceed to step 10 of Subheading 3.4, except incubate in pri-
mary antibody anti-EFR.

3.6  Endo H 1. Sterilize Col-0 Arabidopsis seeds in an excess of seed steriliza-

Treatment tion solution for 10 min. In biosafety cabinet, under sterile
conditions, rinse seeds with autoclaved dH20. Repeat three to
five times.
2. Add 1 mL of MS media 2 and 12 seeds per well in a 12-well
tissue culture plate.
3. Place sample-containing plate on a light cart, under a 16-h light
cycle at 21 °C, 70–80 μE intensity, and 50–75% humidity.
4. On day 7, in biosafety cabinet, exchange media with 1 mL of
MS media 2 (see Note 2).
5. On day 9, air-dry seedlings from each well on paper towels for
5 min, transfer seedlings to 2-mL round-bottom microcentri-
fuge tubes, and freeze samples in liquid nitrogen. Samples can
be stored at −80 °C until processing (see Note 3).
6. Proceed to Subheading 3.2.
7. Set up two sample reactions:
Reaction H: 34 μL of protein extract and 1000 U of Endo
H in 1× citrate buffer.
Reaction O: 34 μL of protein extract in 1× citrate buffer.
8. Incubate at 37 °C for 1–3 h (see Note 6).
72 Stacey A. Lawrence et al.

9. Incubate the reaction at 80 °C for 10 min to heat-inactivate

the enzyme.
10. Centrifuge samples at max speed for 1 min at room temperature.
11. Proceed to Subheading 3.3, except casting a 6% SDS-PAGE
gel and separate proteins at 150 V until 95 kDa band reaches
the bottom of the gel.
12. Proceed to Subheading 3.4.

3.7  Seedling Growth 1. Sterilize Col-0 and fls2 (or efr) Arabidopsis seeds in an excess
Inhibition Assay of seed sterilization solution for 10 min. In biosafety cabinet,
under sterile conditions, rinse seeds with autoclaved dH20.
Repeat three to five times.
2. Label the first 12-well tissue culture plate with “Col-0” and
the second plate with “fls2” (or “efr”). Add 1 mL of MS media
1 and five seeds of the appropriate genotype to each well. Each
12-well plate can accommodate the treatment of one genotype
with a mock dilution and three tenfold serial dilutions of the
MAMP elicitor flg22 (or elf26) in triplicate (see Note 7).
3. Synchronize seeds at 4 °C in the dark for a minimum of 48 h.
4. Place the sample-containing plate on a light cart, under a 16-h
light cycle at 21 °C, 70–80 μE light intensity, and 50–75%
humidity.
5. On day 3, in biosafety cabinet, prepare 100× working solution
for each tenfold serial dilution (e.g., 1 mM, 100 μM, 10 μM)
of the MAMP elicitor flg22 (or elf26) for a total of three con-
centrations and add 60 μL of each solution to a 15-mL conical
tube containing 5.94 mL of MS media 1 for a total of three
final concentrations (e.g., 10 μM, 1 μM, 100 nM).
6. Insert a 1-mL filter tip into a 200-μL tip and use it to remove
the liquid media from the first column of wells of both plates
using a pipetman.
7. Pipette 1 mL of MS media 1 (mock dilution) into each well in
the first column of both plates. Repeat steps 6 and 7 for the
remaining columns of wells using the first serial dilution instead
of the mock dilution. Repeat steps 6 and 7 for the third and
fourth columns of wells using the second and third serial dilu-
tions, respectively (see Note 8).
8. Incubate the sample-containing plates under the same condi-
tions as in step 4 for 2 days, and then count the number of
seedlings per well (see Note 9).
9. After an additional 3 days, air-dry the seedlings from each well
on paper towels for 10 min and measure their collective fresh
weight (FW) using a balance. Each 12-well plate generates 12
samples, three biological replicates per elicitor concentration
for a given genotype (see Note 10).
 Receptor N-Glycosylation and Function 73

10. Create a data sheet of the number of seedlings and their FW

from each sample to calculate the FW per seedling. Set the
mock treated value to 100% and calculate the percentage of
inhibition of seedling growth induced by each MAMP elicitor
concentration with respect to mock-treated samples. Repeat
for each genotype.

3.8  Anthocyanin 1. Sterilize Col-0 and fls2 (or efr) Arabidopsis seeds in an excess
Accumulation Assay of seed sterilization solution for 10 min. In biosafety cabinet,
under sterile conditions, rinse seeds with autoclaved dH20.
Repeat three to five times.
2. Label the first 24-well tissue culture plate with “Col-0” and
the second with “fls2” (or “efr”). Add 0.5 mL of MS media 3
and five (or eight) seeds of the appropriate genotype to each
well (see Note 11). Each 24-well plate can accommodate the
treatment of one genotype with a mock dilution and three ten-
fold serial dilutions of the MAMP elicitor flg22 (or elf26) in
triplicate (see Note 12).
3. Synchronize seeds at 4 °C in the dark for a minimum of 48 h.
4. Place the sample-containing plate on a light cart, under a 24-h
light cycle at 21 °C, 100 μE light intensity, and 50–75%
humidity.
5. On day 3, in biosafety cabinet, prepare 100× working solution
for each tenfold serial dilution (e.g., 1 mM, 100 μM, 10 μM)
of the MAMP elicitor flg22 (or elf26) for a total of three con-
centrations and add 60 μL of each solution to a 15-mL conical
tube containing 5.94 mL of MS media 4 for a total of three
final concentrations (e.g., 10 μM, 1 μM, 100 nM).
6. Insert a 1-mL filter tip into a 200-μL tip and use it to remove
the liquid media from the first row of wells of both plates using
a pipetman.
7. Pipette 0.5 mL of MS media 3 (mock dilution) into each well
in the first row of both plates. Repeat steps 6 and 7 for the
second row of wells of both plates using the first serial dilution
instead of the mock dilution. Repeat steps 6 and 7 for the
third and fourth rows of wells using the second and third serial
dilutions, respectively (see Note 13).
8. Incubate plates under the same conditions as in step 4 for 4
days (see Note 14).
9. Heat a needle using a Bunsen burner and poke two air holes in
the lids of each 1.7-mL microcentrifuge tube for ventilation.
Label tubes with an alcohol-resistant marker.
10. Combine seedlings from two wells for each row and air-dry
them on paper towels for 2–5 min. Transfer the seedlings to
the ventilated and labeled microcentrifuge tubes. Each 24-well
74 Stacey A. Lawrence et al.

plate should generate 12 samples, three biological replicates

per elicitor concentration (see Note 15).
11. Freeze samples in liquid nitrogen and lyophilize them over-
night. Cover container with aluminum foil to prevent light-­
induced degradation of anthocyanins.
12. Calibrate the balance with the weight of an empty 2-mL
round-bottom microcentrifuge tube, transfer the lyophilized
contents from the 1.7-mL tube to the 2-mL tube, and record
the sample dry weight (DW). Repeat for all samples.
13. Add one 5-mm stainless steel bead to each sample-containing
2-mL microcentrifuge tube and homogenize the sample to a
fine powder for 1 min at 25 Hz.
14. Carefully open the microcentrifuge tubes so not to lose any
homogenized contents and add 500 μL of acidic methanol to
each sample.
15. Cap samples tightly and vortex until sample is completely dis-
solved in solution.
16. Incubate samples at 70 °C for 15 min to extract anthocyanin.
Caution: this is the boiling point of methanol and can cause
tubes to pop open when handled.
17. Centrifuge extracts at max speed for 5 min.
18. Transfer 200 μL of supernatant to a new 1.7-mL microcentri-
fuge tube containing 300 μL of acidic methanol.
19. Measure the absorbance (Abs) value of each sample with the
spectrophotometer set at 530 and 657 nm.
20. Create a data sheet with DW, Abs530, and Abs657 of each sample.
21. Determine the anthocyanin content using the following equa-
tion: (Abs530–0.25×Abs657)/DW (mg) [24]. Set the anthocy-
anin content of the water-treated samples to 100% and calculate
the relative anthocyanin repression elicited by each MAMP
concentration (see Note 16).

3.9  MAPK 1. Sterilize Col-0 Arabidopsis seeds in an excess of seed steriliza-

Activation Assay tion solution for 10 min. In biosafety cabinet, under sterile
conditions, rinse seeds with autoclaved dH20. Repeat three to
five times.
2. Add 1 mL of MS media 2 and 12 seeds to each well of a 12-well
tissue culture plate for a total of seven wells. Each 12-well plate
can accommodate the treatment of a single genotype with a
mock dilution and 3 serial dilutions of the MAMP elicitor
flg22 (or elf26) in triplicate.
3. Place the sample-containing plate on a light cart, under a 16-h
light cycle at 21 °C, 70–80 μE light intensity, and 50–75%
humidity.
 Receptor N-Glycosylation and Function 75

4. On day 7, in biosafety cabinet, exchange liquid media with

1 mL of MS media 2 (see Note 2). Place the sample-­containing
plate under the same conditions as in step 3 (see Note 17).
5. On day 9, prepare 100× working solution for each tenfold
serial dilution (e.g., 1 mM, 100 μM, 10 μM, 1 μM, 100 nM,
10 nM) of the MAMP elicitor flg22 (or elf26) for a total of six
concentrations. Add 10 μL of mock dilution to the first well
and repeat for each point of the serial dilution.
6. After 5 min, gently transfer seedlings to a paper towel to dry
excess media (see Note 18).
7. After another 5 min, transfer seedlings to a 2 mL round-­
bottom sample tube (see Note 19).
8. Freeze samples in liquid nitrogen. Samples can be stored at
−80 °C until processing.
9. Cool the homogenizer plates by pouring liquid nitrogen on
them. Quickly transfer the sample tubes to the homogenizer
plate and open the lids. Pour liquid nitrogen on top of the
samples to keep them frozen. Add 100 μL of ice-cold MAPK
extraction buffer and a liquid nitrogen-cooled 5-mm stainless
steel bead to each sample (see Note 1).
10. Homogenize the samples for 2 min at 25 Hz.
11. Centrifuge at 4 °C for 3 min.
12. Transfer 90 μL of supernatant to a precooled 1.7-mL tube and
add 90 μL of freshly prepared 2× Laemmli buffer. Repeat for
each sample.
13. Incubate samples at 95 °C for 3 min.
14. Centrifuge for 1 min at room temperature at maximum speed.
15. Load 25 μL onto a 10% SDS-PAGE gel.
16. Continue from Subheading 3.3, except casting three 10% SDS
polyacrylamide separating gels of 1.5-mm thickness, loading
25  μL of samples per well on triplicate gels, and separating
proteins at 150 V until the 29-kDa marker band reaches the
bottom of the gel.
17. Continue from Subheading 3.4, except incubating in primary
antibody anti-phospho-p44/42 MAPK (see Notes 20 and 21).
18. After detection, quantify phosphorylated MPK6, MPK3, and
MPK4 signal intensities en bloc using NIH ImageJ, following
software instruction.
19. Continue from Subheading 3.5, except using primary anti-
body anti-AtMPK3 (see Note 20).
20. After detection, quantify MPK3 signal intensity using NIH
ImageJ, following software instruction.
76 Stacey A. Lawrence et al.

4  Notes

1. Due to the labile nature of the phosphate bond, short process-

ing times and ice-cold temperature are a must for this tech-
nique. Therefore, keep all reagents, tubes, and samples on ice.
We recommend processing no more than 12 samples at a time,
although a maximum of 24 samples can be processed. The
MAPK extraction buffer is stable on ice for two consecutive
rounds of sample extractions.
2. Due to the evaporation, absorption, and alteration of the
media by growing seedlings over the course of a week, the
media in each well must be replenished with fresh media. To
ensure enough time for the seedlings to recover from the
mechanical stress caused by the media exchange, we recom-
mend the media exchange to occur on day 7.
3. If performing a chemical treatment followed by MAPK activa-
tion assay after step 6 proceed with step 5 of Subheading 3.9.
4. We use different percentage (5–13.5%) SDS-PAGE separating
gels to allow for differential separation of specific and nonspe-
cific proteins for immuno-detection by a given primary anti-
body, especially for proteins whose molecular masses may be
altered by chemical treatments.
5. Prerunning gels eliminates contaminants that may be present
in the separating gel and thus can interfere with protein
migration.
6. Although the incubation time can vary from 1 to 16 h, with
3 h as the ideal, longer incubation times will result in more
protein degradation.
7. This plating scheme is used to test the elicitation strength of
three serial MAMP dilutions. When comparing wild-type to
mutant seedlings, label the first column of a 12-well tissue cul-
ture plate with “Col-0,” the second with “m1” (for mutant
number 1), the third with “m2” (for mutant number 2), and
the last with “fls2” (or “efr”). Repeat this for two other 12-well
plates to obtain three biological replicates per genotype.
8. The described exchange of media is valid when testing a dilu-
tion series of an elicitor. When comparing immune responses
between wild-type, 1–2 mutants, and fls2 (or efr) for a total of
two serial MAMP dilutions, each column of a triplicate plate
contains a different genotype. Exchange the media for each
row of wells, so that the first row will receive the mock treat-
ment; the second, the first serial dilution of elicitor; and the
third, the second dilution of elicitor. Repeat for the second and
third plates.
 Receptor N-Glycosylation and Function 77

9. Total seedling count may be less than the total seed count due
to reduced seed germination rates and seedling losses from
media exchanges.
10. After 8 days of growth, we observed the growth rate of mock-­
treated seedlings to slow due to space limitations in the wells.
11. To ensure that all seedlings in a well are equally exposed to the
MAMP elicitor, we recommend plating five seeds per well for
MAMP treatments <100 nM, and plating eight seeds per well
for MAMP treatments ≥100 nM.
12. The plating scheme provided is valid when testing an elicitor
dilution series. When comparing immune responses between
wild-type, 1–2 mutants, and fls2 (or efr) for one concentration
of elicitor, label the first row of a 24-well tissue culture plate
with “Col-0,” the second with “m1” (for mutant number 1),
the third with “m2” (for mutant number 2), and the last with
“fls2” (or “efr”). Repeat this for two other 24-well plates.
­Proceed by adding the media and five seeds of the appropriate
genotype to each well.
13. The described exchange of media is valid when testing a dilu-
tion series of an elicitor. When comparing immune responses
among wild-type, 1–2 mutants, and fls2 (or efr) for one MAMP
concentration, draw a vertical line on the lid for all three plates,
between columns 3 and 4 and through all four rows. Exchange
the media in the wells to the left of the line with media con-
taining the mock dilution and the media in the wells to the
right of the line with media containing the MAMP concentra-
tion to be tested.
14. To ensure that seedlings have dry weights of more than 1 mg,
which is the lower limit of the balance, we recommend extend-
ing the length of elicitation to 5 days for MAMP elicitor treat-
ments >100 nM. We also observed that increasing the length
of elicitation can enhance differences in growth arrest or
anthocyanin repression responses between wild-type and
mutant seedlings.
15. When comparing immune responses among wild-type, 1–2
mutants, and fls2 (or efr), combine seedlings from three wells per
genotype. Each 24-well plate should generate one biological rep-
licate of the mock-treated sample and one of the MAMP-elicited
samples per genotype for a total of eight samples. Three plates are
needed to generate the three biological replicates per elicitor
concentration per genotype to test statistical significance.
16. Due to pipetting errors, plate-to-plate variability, and impre-
cise treatment-lengths, we highly recommend growing wild-­
type and fls2 (or efr) seedlings on each 24-well plate to serve as
normalization factors for comparative analysis.
78 Stacey A. Lawrence et al.

17. When performing a tunicamycin treatment, add the desired

amount of chemical to every other well on day 8 and place the
sample-containing plate under the same conditions as in step 3.
18. Mechanical wounding can also activate MAPKs; therefore,
gently handle the samples.
19. MAMP-induced MAPK activation is fast and dynamic, with a
maximum signal intensity between 10 and 15 min.
20. Incubations with strong antibodies, like the anti-phospho-
­p44/42 MAPK and anti-MAPK3, can be carried out at room
temperature for 1.5–2 h, with gentle rocking.
21. To minimize the technical variability due to transfer, it is pos-
sible to cut each of the 10% replicate gels between the 37 and
50 kDa molecular weight markers and transfer the three strips
to a single PVDF membrane.

Acknowledgments

This work was supported by T32 GM007499 (to S.A.L.) and Yale
University Elizabeth Brown Postdoctoral Fellowship (to T.C.).

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