Isolation of Novel MAMP‐like Activities and Identification of Cognate Pattern Recognition Receptors in Arabidopsis thaliana Using Next‐Generation Sequencing (NGS)–Based Mapping

Li Fan1, Eunyoung Chae2, Andrea A. Gust1, Thorsten Nürnberger1

1 Center of Plant Molecular Biology (ZMBP), Eberhard‐Karls‐University Tübingen, Tübingen, 2 Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20056
Online Posting Date:  September, 2017
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Activation of pattern‐triggered plant immunity requires recognition of microbe‐derived molecular patterns (MAMPs) by plant‐encoded pattern recognition receptors (PRRs). Many plant PRRs are found in selected plant genera only. Transfer of single PRRs or of cassettes expressing several PRRs (PRR stacking) across plant genus boundaries offers the potential to boost disease resistance by improving pathogen recognition features in economically important crop plants. The success of such an approach is most dependent on the availability of a large number of plant PRRs. Here, an efficient method for the identification of novel PRRs in the model plant Arabidopsis thaliana (hereafter, Arabidopsis for simplicity) is described. This method takes advantage of natural variation in microbial pattern sensitivity among hundreds of Arabidopsis accessions currently available. Identification of pattern‐sensitive as well as pattern‐insensitive accessions facilitates next‐generation sequencing (NGS)–assisted mapping of PRRs. This approach is potentially applicable to the identification of PRRs that recognize patterns of any chemical nature. © 2017 by John Wiley & Sons, Inc.

Keywords: microbe‐associated molecular pattern (MAMP); pattern recognition receptor (PRR); next generation sequencing (NGS); restriction site‐associated DNA sequencing (RAD‐seq); Arabidopsis thaliana

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of MAMP‐Like Activity and Identification of Its Cognate PRR
  • Support Protocol 1: MAMP Isolation and Partial Purification
  • Support Protocol 2: Ethylene Assay
  • Support Protocol 3: PseI‐MseI Genotyping‐By‐Sequencing (GBS)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation of MAMP‐Like Activity and Identification of Its Cognate PRR

  • Arabidopsis accessions (1001 Genomes Project;
  • CTAB solution (see recipe)
  • 24:1 (v/v) chloroform:isoamylalcohol
  • Isopropanol
  • 75% (v/v) ethanol
  • DNase‐free water containing 10 µg/ml RNase A
  • Arabidopsis T‐DNA insertion alleles (ABRC or NASC seeds stock; SIGnAL;
  • 96‐well‐format auto‐tube racks with vials (Roth, cat. no. EC05.1) and cap strips (Roth, cat. no. EC09.1)
  • 5/32‐in. stainless steel grinding balls (Ops Diagnostics, cat. no. GBSS 156‐5000‐01)
  • HT Homogenizer (Troemner)
  • 65°C oven
  • 96‐well deep‐well plates
  • Adhesive plate seals (Thermo Fisher Scientific, cat. no. AB0580)
  • Centrifuge for 96‐well deep‐well plates/auto‐tube racks
  • Qubit® Fluorimeter (Life Technologies)
  • Software:
    • SHORE (Ossowski et al., )
    • Burrows‐Wheeler Aligner (BWA; Li & Durbin, )
    • R program (http://www.R‐
    • rQTL package (Arends, Prins, Jansen, & Broman, )
  • Additional reagents and equipment for MAMP isolation and partial purification ( protocol 2), ethylene assay ( protocol 3), genotyping by sequencing ( protocol 4), and agarose gel electrophoresis (Voytas, )

Support Protocol 1: MAMP Isolation and Partial Purification

  • Bacterial strain as a glycerol stock (−80°C) or fungal or oomycete spore or mycelium material
  • Appropriate culture medium for given microorganism
  • Ammonium sulfate
  • 50 mM MES buffer, pH 5.2
  • Columns and buffers for chromatography (Table 20.5.6000)
Table 0.5.1   MaterialsColumns and Buffers for Chromatography

Column by GE Healthcare Buffer A Buffer B
Hitrap SP FF (5 ml) 50 mM MES, pH 5.2 50 mM MES/0.5 M KCl, pH 5.2
Hitrap Q FF (5 ml) 50 mM Tris⋅Cl, pH 8.5 50 mM Tris⋅Cl/0.5 M KCl, pH 8.5
Source 15 S 4.6/100 PE 50 mM MES, pH 5.2 50 mM MES/0.5 M KCl, pH 5.2

  • Shaker (for microbial growth)
  • Sorvall RC‐3B Plus High Capacity Centrifuge, H4000A 4 × 1 liter rotor
  • Centrifuge and rotor accommodating 250‐ml tubes
  • Äkta explorer system with fraction collector Frac‐950 (GE Healthcare)
  • Additional reagents and equipment for dialysis (Zumstein, )

Support Protocol 2: Ethylene Assay

  • Arabidopsis seeds
  • 0.1% (w/v) agarose
  • Soil (Gebrüder Patzer GmbH, cat. no. GS90)
  • Known MAMP as a positive control in ethylene production assay: e.g., synthetic peptides such as flagellin peptide flg22 (Felix, Duran, Volko, & Boller, ) or necrosis and ethylene–inducing peptide nlp20 (Albert et al., ).
  • Nursery pots (5 × 10 × 15 cm)
  • Incubator/grow room equipped for 8‐hr photoperiod at 22°C and 40% to 60% humidity with instantaneous light incident on the surface of 150 µmol/m2/sec
  • Petri dishes
  • 6‐ml glass tubes with rubber cap
  • Shaker
  • 1‐ml plastic syringe and needle
  • Ethylene standard (e.g., Sigma‐Aldrich, 00489)
  • Gas chromatograph (Shimadzu GC‐14A with C‐R4AX chromatopac integrator)

Support Protocol 3: PseI‐MseI Genotyping‐By‐Sequencing (GBS)

  MaterialsPrimers (NNNNN is the barcode 5 to 10 bp in length; each DNA sample has a unique one):
  • 1× elution buffer (10 mM Tris·Cl, pH 8.0 to 8.5).
  • 10× adapter buffer (500 mM NaCl/100 mM Tris·Cl, pH 8.0 to 8.5)
  • Qubit® kit (Thermo Fisher Scientific, e.g., cat no. Q32853)
  • 10× NEBuffer 4 (NEB, cat no. B7004S)
  • PstI‐HF (20,000 U/µl, NEB, cat no. R3140L)
  • MseI (10,000 U/µl, NEB, cat no. R0525L)
  • 10 mM ATP (Thermo Fisher Scientific, cat no. PV3227)
  • T4 DNA ligase (400,000 U/µl, NEB, cat no. M0202L)
  • QIAquick PCR Purification Kit (Qiagen, cat. no. 28106)
  • 2 mM dNTP mix (Thermo Fisher Scientific, cat no. N8080261)
  • Phusion Polymerase (Thermo Fisher Scientific, cat no. F530L)
  • 5× DNA loading buffer (Qiagen, cat no. 239901)
  • Low‐molecular‐weight DNA ladder (NEB, cat no. N3233L)
  • 96‐well microtiter plates
  • PCR machine (MJ Research, PTC‐225 DNA Engine Tetrad Peltier Thermal Cycler)
  • Qubit® assay tubes (Thermo Fisher Scientific, cat no. Q32856)
  • Qubit® Fluorimeter (Thermo Fisher Scientific)
  • ABI 3730×I DNA Analyzer (Applied Biosystems)
  • HiSeq2000 (Illumina)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )
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Internet Resources
  Arabidopsis genome sequences.‐bin/tdnaexpress
  T‐DNA insertion mutants.
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