Chapter 5

Analysis for Protein Glycosylation of Pattern Recognition

Receptors in Plants
Takaakira Inokuchi and Yusuke Saijo

Abstract
Recognition of molecules typical of microbes or aberrant cellular states, termed microbe- or danger-­
associated molecular patterns (MAMPs/DAMPs), respectively, provides an important step in plant and
animal innate immunity. In plants, pattern recognition receptors (PRRs) identified to date are limited to
membrane-associated proteins, of which the majority has an extracellular leucine-rich repeat (LRR) or
lysine-motif (LysM) domain. These PRRs undergo quality control (QC) in the Endoplasmic Reticulum
(ER) that is dependent on Asn (N)-linked glycosylation (Glc3Man9GlcNAc2 conjugation) of their extracel-
lular domain. In Arabidopsis, genetic studies have revealed that a subset of these PRRs require an intact
N-glycosylation pathway in the ER for their biogenesis and function. Here, we describe methods for
immunoblot-based detection of protein glycosylation states in plants.

Key words Plant immunity, Endoplasmic Reticulum quality control (ERQC), Pattern recognition
receptors (PRRs), Immunoblot analysis, N-linked glycosylation, Glycosidase treatment

1  Introduction

The LRR receptor kinases (RKs) EFR and FLS2 recognize the
bacterial MAMPs elf18 and flg22, derived from elongation factor-
Tu (EF-Tu) and flagellin, respectively. Genetic studies have shown
that EFR function, but not FLS2 function, is impaired in the
absence of the following ERQC branches: The first involves the
oligosaccharyltransferase (OST) complex subunits STT3A and
OST3/6, glucosidases I and II (GI and GII), the ER chaperone
calreticulin3 (CRT3), and UDP-glucose:glycoprotein glucosyl-
transferase (UGGT); the second involves the Hsp70 family mem-
ber BiP that acts together with the Hsp40 family members ERdJ
and stromal cell-derived factor 2 (SDF2) [1]. This indicates a
model in which EFR is a client protein that strictly requires ERQC
mediated by these protein folding pathways. However, it cannot be
ruled out that the observed dysfunction of the receptor in these

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_5, © Springer Science+Business Media LLC 2017

55
56 Takaakira Inokuchi and Yusuke Saijo

ERQC-related mutant plants is an indirect consequence of ERQC

defects. A simple protein biochemical analysis can help determine
whether the protein of interest directly undergoes N-glycosylation.
N-glycosylation on the client proteins can be typically detected as
a shift of the mobility on SDS-PAGE. In the case of EFR and FLS2,
protein biochemical analysis revealed an increase of the apparent
size in a manner dependent on the OST complex [2]. This leads to
the view that both receptors undergo N-glycosylation, despite the
aforementioned divergence in the requirement for OST-mediated
ERQC. Here, we describe methods that are useful for the detec-
tion of N-glycosylated proteins from plant protein extracts.

2  Materials

Prepare all solutions using Milli-Q (MILLIPORE Q-guard).

2.1  Plants Ten-day-old Arabidopsis thaliana seedlings grown on 1/2 × MS

medium (pH 5.6) containing 25–34 mM sucrose and 0.8% agar
were treated with or without elf18 at 0.5 μM for 24 h, and then
subjected to immunoblot analysis.

2.2  Lysis Buffer Prepare for a 10× stock solution of Lysis Buffer, containing 50 mM
Tris–HCl (pH 7.5), 150 mM NaCl, 10% Glycerol, 2 mM EDTA,
0.5% (v/v) NP-40, which can be stored at 4 °C. Immediately
before use, dilute it to the working concentration, and then freshly
add 2 mM DTT and 1% (v/v) P9599 protease inhibitor cocktail
(Sigma).

2.3  Immunoblotting 1. PVDF membranes.

Reagents 2. For 1 L of Immunoblot transfer buffer, dissolve 3 g of Tris, 14
g of Glycine and 200 mL of 100% MeOH in water.
3. For 1 L of 10× Tris-buffered saline (TBS), dissolve 87.66 g of
NaCl and 100 mL of 1 M Tris–HCl (pH 7.5) in water.
4. For 1 L of TBS containing 0.1% Tween-20 (TBST), dilute 100
mL of 10× TBS and 5 mL of 20% Tween-20 in water.
5. Blocking solution: dissolve 5% skim milk in 0.1% TBST. Store
at 4 °C.
6. Criterion™ Blotter.
7. Medium binder clips.
8. Plastic container.
9. Whatman 3 MM Chr paper.
10. Anti-FLS2 antibodies.
11. For 10 mL of 6× SDS sample buffer, dissolve 3 g of sucrose,
1.2 g of SDS, and 10 mg of bromophenol blue (BPB) in 6 mL
 Protein Glycolsylation of Plant PRRs 57

of 0.5 M Tris–HCl (pH 6.8), and then top up with water (if
necessary). Store at room temperature before adding a
reducing agent.

3  Method

3.1  Extraction 1. Grind tissues in liquid nitrogen (Fig. 1a).

of PRRs 2. Suspend tissues in Lysis Buffer (100 mg in 300 μL lysis buffer).
If necessary, keep the lysates on ice until they thaw, and then
mix well.
3. Centrifuge at 13,200 × g 4 °C for 15 min.
4. Recover the supernatant (crude lysates) to a clean tube (see
Note 1).

3.2  Pull Down with
ConA Sepharose
1. Dilute crude lysates with Lysis buffer up to ~1.3 mL in a 1.5 mL
tube (Fig. 1a).
2. Add 50 μL of ConA Sepharose™ 4B (GE Healthcare) after
vortex (see Note 2).
3. Rocking at 4 °C for 1–3 h.
4. Centrifuge at 800 × g- 4 °C for 1–2 min and discard the super-
natant (see Note 3).
5. Wash the Sepharose pellet with Lysis buffer (see Note 4).

a b
ConA Sepharose
Spin down
and
Discard the supernatant
Rocking
Glucose
Mannose
GlcNAc

Add SDS sample buffer Endo H

and Cleavage site
Heat at 95°C
Asn(N) X Ser(S)/Thr(T)
Collect the supernatant Immunoblotting
N-linked Glycosylation
with or without
Endo H treatment

Fig. 1 (a) Schematic diagram for the isolation of ConA Sepharose-binding proteins. (b) Endo H cleavage site in
N-linked glycan
58 Takaakira Inokuchi and Yusuke Saijo

6. Repeat three times the steps 4 and 5.

7. Add 50 μL of 1× SDS sample buffer (with DTT added before
use to a final concentration of 10–20 mM).
8. Heat at 95 °C for 5 min.
9. Centrifuge at 800 × g 4 °C for 1–2 min and collect the
supernatant.

3.3  Cleavage 1. Add 2 μL of GlycoBuffer 3 (New England BioLabs) and 1 μL

of N-Linked of Endo H (New England BioLabs) in 17 μL of the aforemen-
Glycosylation tioned ConA Sepharose-bound protein solution (see Note 5).
of PRRs by Endo H 2. Incubate this mixture for 1 h at 37 °C (Fig. 1b).

3.4  Immunoblot 1. SDS-PAGE on a 6.0–7.5% gel (see Note 6).

Analysis for PRRs 2. Transfer proteins to PVDF membrane for 1 h at 4 °C.
3. Stain the membrane with Ponceau S (see Note 7), to assess
equal loading of the samples.
4. Destain the membrane with 1× TBS.
5. Block the membrane with blocking solution for 1 h at room
temperature (see Note 8).
6. Incubate the membrane overnight at 4 °C with appropriate
dilutions of the first antibodies of choice in Lysis buffer.
7. Wash the membrane with 0.1% TBST for 10 min. Repeat this
step three times.
8. Incubate the membrane with appropriate dilutions of the
second antibodies of choice in Lysis buffer for 1 h at room
temperature.
9. Wash the membrane with 0.1% TBST three times for
10 min each.
10. Develop chemiluminescent signals with ECL reagent and
detect them using a CCD camera-based imager (Fig. 2).

4  Notes

1. Floating debris should be filtered out of the recovered

supernatant.
2. ConA Sepharose should be suspended well before use.
3. Do not exceed 3300 × g in centrifugation to avoid malfunc-
tioning of ConA Sepharose.
4. Suspend gently by pipetting. Do not Vortex.
5. Suspend GlycoBuffer 3 and Endo H by pipetting. Endo H works
in the presence of SDS, which is likely to increase the accessibility
of Endo H to the cleavage sites on N-glycan chains.
 Protein Glycolsylation of Plant PRRs 59

a b
Col-0 stt3a-2

Endo H - + - +
Col-0 stt3a-2 fis2
255 kDa

142 kDa
142 kDa < de-glycosylated FLS2

52 kDa

Fig. 2 Immunoblot analysis for an N-glycosylation-dependent mobility shift of the Arabidopsis pattern recogni-
tion receptor FLS2. (a) The position of the molecular weight marker is shown on the left. Top, immunoblot
probed with anti-FLS2 antibodies; Bottom, immunoblot stained with Ponceau S for equal loading verification.
(b) ConA Sepharose-binding proteins were digested with Endo H, and then subjected to immunoblot analysis
with anti-FLS2 antibodies

6. According to the molecular weight of the proteins of interest,

the gel percentage needs to be determined. Electrophoresis
should be performed until the frontline of BPB reaches the
bottom (Fig. 2).
7. Since the staining of Ponceau S is reversible, this staining does
not affect immunoblotting.
8. When the background signal is high, blocking and washing
should be extensively performed. Blocking and/or washing
even overnight help enhance the signal/background ratio.

Acknowledgments

The researches in the Saijo Lab have been supported in part by JST
PRESTO and Grant-in-Aid for Scientific Research to Y.S.

References

1. Saijo Y (2010) ER quality control of immune S, Schulze-Lefert P (2009) Receptor quality

receptors and regulators in plants. Cell control in the endoplasmic reticulum for
Microbiol 12:716–724 plant innate immunity. EMBO J 28:
2. Saijo Y, Tintor N, Lu X, Rauf P, Pajerowska-­ 3439–3449
Mukhtar K, Haweker H, Dong X, Robatzek