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Title: Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition

Abstract

Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wildtype Arabidopsis FLS2 confers little or no response. A targeted approach generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovoramore » flagellin peptide. Furthermore, these studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.« less

Authors:
; ; ORCiD logo;
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1346606
Alternate Identifier(s):
OSTI ID: 1287175
Grant/Contract Number:  
FG02-02ER15342
Resource Type:
Published Article
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Name: PLoS ONE Journal Volume: 11 Journal Issue: 6; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science (PLoS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; leucine-rich repeat; bacterial elicitor flagellin; disease-resistance gene; plant immune-responses; elongation-factor tu; pseudomonas-syringae; arabidopsis-thaliana; innate immunity; receptor efr; nicotiana-benthamiana

Citation Formats

Helft, Laura, Thompson, Mikayla, Bent, Andrew F., and Vinatzer, ed., Boris Alexander. Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0157155.
Helft, Laura, Thompson, Mikayla, Bent, Andrew F., & Vinatzer, ed., Boris Alexander. Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition. United States. https://doi.org/10.1371/journal.pone.0157155
Helft, Laura, Thompson, Mikayla, Bent, Andrew F., and Vinatzer, ed., Boris Alexander. Mon . "Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition". United States. https://doi.org/10.1371/journal.pone.0157155.
@article{osti_1346606,
title = {Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition},
author = {Helft, Laura and Thompson, Mikayla and Bent, Andrew F. and Vinatzer, ed., Boris Alexander},
abstractNote = {Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wildtype Arabidopsis FLS2 confers little or no response. A targeted approach generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovora flagellin peptide. Furthermore, these studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.},
doi = {10.1371/journal.pone.0157155},
journal = {PLoS ONE},
number = 6,
volume = 11,
place = {United States},
year = {Mon Jun 06 00:00:00 EDT 2016},
month = {Mon Jun 06 00:00:00 EDT 2016}
}

Journal Article:
Free Publicly Available Full Text
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https://doi.org/10.1371/journal.pone.0157155

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Cited by: 11 works
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Works referenced in this record:

Specific ER quality control components required for biogenesis of the plant innate immune receptor EFR
journal, August 2009

  • Li, Jing; Zhao-Hui, Chu; Batoux, Martine
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 37
  • DOI: 10.1073/pnas.0905532106

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants
journal, November 2004


Control of the pattern-recognition receptor EFR by an ER protein complex in plant immunity
journal, September 2009

  • Nekrasov, Vladimir; Li, Jing; Batoux, Martine
  • The EMBO Journal, Vol. 28, Issue 21
  • DOI: 10.1038/emboj.2009.262

The INs and OUTs of pattern recognition receptors at the cell surface
journal, August 2012

  • Beck, Martina; Heard, William; Mbengue, Malick
  • Current Opinion in Plant Biology, Vol. 15, Issue 4
  • DOI: 10.1016/j.pbi.2012.05.004

Innate Immunity: The Virtues of a Nonclonal System of Recognition
journal, October 1997


Of PAMPs and Effectors: The Blurred PTI-ETI Dichotomy
journal, January 2011

  • Thomma, Bart P. H. J.; Nürnberger, Thorsten; Joosten, Matthieu H. A. J.
  • The Plant Cell, Vol. 23, Issue 1
  • DOI: 10.1105/tpc.110.082602

Analysis of Flagellin Perception Mediated by flg22 Receptor OsFLS2 in Rice
journal, December 2008

  • Takai, Ryota; Isogai, Akira; Takayama, Seiji
  • Molecular Plant-Microbe Interactions, Vol. 21, Issue 12
  • DOI: 10.1094/MPMI-21-12-1635

The Arabidopsis Receptor Kinase FLS2 Binds flg22 and Determines the Specificity of Flagellin Perception
journal, December 2005

  • Chinchilla, Delphine; Bauer, Zsuzsa; Regenass, Martin
  • The Plant Cell, Vol. 18, Issue 2
  • DOI: 10.1105/tpc.105.036574

Plant immunity: towards an integrated view of plant–pathogen interactions
journal, June 2010

  • Dodds, Peter N.; Rathjen, John P.
  • Nature Reviews Genetics, Vol. 11, Issue 8
  • DOI: 10.1038/nrg2812

The Plant Pathogen Pseudomonas syringae pv. tomato Is Genetically Monomorphic and under Strong Selection to Evade Tomato Immunity
journal, August 2011


Genetic Dissection of Basal Resistance to Pseudomonas syringae pv. phaseolicola in Accessions of Arabidopsis
journal, December 2010

  • Forsyth, Alec; Mansfield, John W.; Grabov, Nina
  • Molecular Plant-Microbe Interactions, Vol. 23, Issue 12
  • DOI: 10.1094/MPMI-02-10-0047

Progress in phage display: evolution of the technique and its applications
journal, November 2009


Single Amino Acid Mutations in the Potato Immune Receptor R3a Expand Response to Phytophthora Effectors
journal, July 2014

  • Segretin, María Eugenia; Pais, Marina; Franceschetti, Marina
  • Molecular Plant-Microbe Interactions, Vol. 27, Issue 7
  • DOI: 10.1094/MPMI-02-14-0040-R

Detection of the Virulent Form of AVR3a from Phytophthora infestans following Artificial Evolution of Potato Resistance Gene R3a
journal, October 2014


Receptor quality control in the endoplasmic reticulum for plant innate immunity
journal, September 2009


Structure-function Aspects of Extracellular Leucine-rich Repeat-containing Cell Surface Receptors in Plants: eLRR Receptor Structure and Function
journal, September 2013

  • Zhang, Zhao; Thomma, Bart P. H. J.
  • Journal of Integrative Plant Biology, Vol. 55, Issue 12
  • DOI: 10.1111/jipb.12080

Evasion of Toll-like receptor 5 by flagellated bacteria
journal, June 2005

  • Andersen-Nissen, E.; Smith, K. D.; Strobe, K. L.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 26
  • DOI: 10.1073/pnas.0502040102

Probing the Arabidopsis Flagellin Receptor: FLS2-FLS2 Association and the Contributions of Specific Domains to Signaling Function
journal, March 2012

  • Sun, Wenxian; Cao, Yangrong; Jansen Labby, Kristin
  • The Plant Cell, Vol. 24, Issue 3
  • DOI: 10.1105/tpc.112.095919

Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor
journal, July 2006

  • Kaku, H.; Nishizawa, Y.; Ishii-Minami, N.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 29
  • DOI: 10.1073/pnas.0508882103

Bacterial disease resistance in Arabidopsis through flagellin perception
journal, April 2004

  • Zipfel, Cyril; Robatzek, Silke; Navarro, Lionel
  • Nature, Vol. 428, Issue 6984
  • DOI: 10.1038/nature02485

The dsRNA binding site of human Toll-like receptor 3
journal, May 2006

  • Bell, J. K.; Askins, J.; Hall, P. R.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 23
  • DOI: 10.1073/pnas.0603245103

Effector Biology of Plant-Associated Organisms: Concepts and Perspectives
journal, January 2012

  • Win, J.; Chaparro-Garcia, A.; Belhaj, K.
  • Cold Spring Harbor Symposia on Quantitative Biology, Vol. 77, Issue 0
  • DOI: 10.1101/sqb.2012.77.015933

Identification and Mutational Analysis of Arabidopsis FLS2 Leucine-Rich Repeat Domain Residues That Contribute to Flagellin Perception
journal, October 2007

  • Dunning, F. Mark; Sun, Wenxian; Jansen, Kristin L.
  • The Plant Cell, Vol. 19, Issue 10
  • DOI: 10.1105/tpc.106.048801

The Receptor-Like Protein ReMAX of Arabidopsis Detects the Microbe-Associated Molecular Pattern eMax from Xanthomonas
journal, June 2013


Structural insights for engineering binding proteins based on non-antibody scaffolds
journal, August 2012


Structural Insights into the Evolution of the Adaptive Immune System
journal, May 2013


Pseudomonas Evades Immune Recognition of Flagellin in Both Mammals and Plants
journal, August 2011


Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa
journal, November 2004

  • Ramos, Hugo Cruz; Rumbo, Martin; Sirard, Jean-Claude
  • Trends in Microbiology, Vol. 12, Issue 11
  • DOI: 10.1016/j.tim.2004.09.002

Phytopathogen Effectors Subverting Host Immunity: Different Foes, Similar Battleground
journal, October 2012


Stepwise artificial evolution of a plant disease resistance gene
journal, December 2013

  • Harris, C. J.; Slootweg, E. J.; Goverse, A.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 52
  • DOI: 10.1073/pnas.1311134110

News from the frontline: recent insights into PAMP-triggered immunity in plants
journal, August 2008


Understanding Plant Immunity as a Surveillance System to Detect Invasion
journal, August 2015


Substitutions of Two Amino Acids in the Nucleotide-Binding Site Domain of a Resistance Protein Enhance the Hypersensitive Response and Enlarge the PM3F Resistance Spectrum in Wheat
journal, March 2014

  • Stirnweis, Daniel; Milani, Samira Désiré; Jordan, Tina
  • Molecular Plant-Microbe Interactions, Vol. 27, Issue 3
  • DOI: 10.1094/MPMI-10-13-0297-FI

A LysM Receptor-Like Kinase Plays a Critical Role in Chitin Signaling and Fungal Resistance in Arabidopsis
journal, February 2008


Glycosylation Regulates Specific Induction of Rice Immune Responses by Acidovorax avenae Flagellin
journal, May 2011

  • Hirai, Hiroyuki; Takai, Ryota; Iwano, Megumi
  • Journal of Biological Chemistry, Vol. 286, Issue 29
  • DOI: 10.1074/jbc.M111.254029

Pseudomonas syringae Evades Host Immunity by Degrading Flagellin Monomers with Alkaline Protease AprA
journal, July 2014

  • Pel, Michiel J. C.; van Dijken, Anja J. H.; Bardoel, Bart W.
  • Molecular Plant-Microbe Interactions, Vol. 27, Issue 7
  • DOI: 10.1094/MPMI-02-14-0032-R

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors
journal, April 2010

  • Albert, Markus; Jehle, Anna K.; Mueller, Katharina
  • Journal of Biological Chemistry, Vol. 285, Issue 25
  • DOI: 10.1074/jbc.M110.124800

A Decade of Yeast Surface Display Technology: Where Are We Now?
journal, February 2008

  • Shusta, Eric; Pepper, Lauren; Cho, Yong
  • Combinatorial Chemistry & High Throughput Screening, Vol. 11, Issue 2
  • DOI: 10.2174/138620708783744516

The leucine-rich repeat as a protein recognition motif
journal, December 2001


Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models
journal, February 2010

  • Albert, Markus; K. Jehle, Anna; Lipschis, Martin
  • European Journal of Cell Biology, Vol. 89, Issue 2-3
  • DOI: 10.1016/j.ejcb.2009.11.015

Pathogen subversion of cell-intrinsic innate immunity
journal, October 2007

  • Roy, Craig R.; Mocarski, Edward S.
  • Nature Immunology, Vol. 8, Issue 11
  • DOI: 10.1038/ni1528

Plants and animals: a different taste for microbes?
journal, August 2005


Artificial evolution extends the spectrum of viruses that are targeted by a disease-resistance gene from potato
journal, October 2006

  • Farnham, G.; Baulcombe, D. C.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 49
  • DOI: 10.1073/pnas.0605777103

Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7
journal, February 2013

  • Nicaise, Valerie; Joe, Anna; Jeong, Byeong-ryool
  • The EMBO Journal, Vol. 32, Issue 5
  • DOI: 10.1038/emboj.2013.15

Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility
journal, July 2013

  • Clarke, Christopher R.; Chinchilla, Delphine; Hind, Sarah R.
  • New Phytologist, Vol. 200, Issue 3
  • DOI: 10.1111/nph.12408

Chimeric FLS2 Receptors Reveal the Basis for Differential Flagellin Perception in Arabidopsis and Tomato
journal, May 2012

  • Mueller, Katharina; Bittel, Pascal; Chinchilla, Delphine
  • The Plant Cell, Vol. 24, Issue 5
  • DOI: 10.1105/tpc.112.096073

Sensitivity of Different Ecotypes and Mutants of Arabidopsis thaliana toward the Bacterial Elicitor Flagellin Correlates with the Presence of Receptor-binding Sites
journal, September 2001

  • Bauer, Zsuzsa; Gómez-Gómez, Lourdes; Boller, Thomas
  • Journal of Biological Chemistry, Vol. 276, Issue 49
  • DOI: 10.1074/jbc.M102390200

A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana: Sensitivity of A. thaliana to bacterial flagellin
journal, May 1999


Fls2
journal, June 2000


Engineered protein function by selective amino acid diversification
journal, April 2004


LYM2-dependent chitin perception limits molecular flux via plasmodesmata
journal, May 2013

  • Faulkner, C.; Petutschnig, E.; Benitez-Alfonso, Y.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 22
  • DOI: 10.1073/pnas.1203458110

Perception of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated Transformation
journal, May 2006


Disruption of Poly(ADP-ribosyl)ation Mechanisms Alters Responses of Arabidopsis to Biotic Stress
journal, November 2009

  • Adams-Phillips, Lori; Briggs, Amy G.; Bent, Andrew F.
  • Plant Physiology, Vol. 152, Issue 1
  • DOI: 10.1104/pp.109.148049

Pattern Recognition Receptors Require N -Glycosylation to Mediate Plant Immunity
journal, December 2009

  • Häweker, Heidrun; Rips, Stephan; Koiwa, Hisashi
  • Journal of Biological Chemistry, Vol. 285, Issue 7
  • DOI: 10.1074/jbc.M109.063073

Expression of a bacterial flagellin gene triggers plant immune responses and confers disease resistance in transgenic rice plants
journal, June 2008


The growth–defense pivot: crisis management in plants mediated by LRR-RK surface receptors
journal, October 2014

  • Belkhadir, Youssef; Yang, Li; Hetzel, Jonathan
  • Trends in Biochemical Sciences, Vol. 39, Issue 10
  • DOI: 10.1016/j.tibs.2014.06.006

Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy
journal, August 2003

  • Yonekura, Koji; Maki-Yonekura, Saori; Namba, Keiichi
  • Nature, Vol. 424, Issue 6949
  • DOI: 10.1038/nature01830

FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation
journal, October 2014


Flagellin Perception Varies Quantitatively in Arabidopsis thaliana and Its Relatives
journal, January 2012

  • Vetter, M. M.; Kronholm, I.; He, F.
  • Molecular Biology and Evolution, Vol. 29, Issue 6
  • DOI: 10.1093/molbev/mss011

A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides
journal, May 2010

  • Brutus, A.; Sicilia, F.; Macone, A.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 20
  • DOI: 10.1073/pnas.1000675107

Flagellin Glycosylation Island in Pseudomonas syringae pv. glycinea and Its Role in Host Specificity
journal, October 2003


Hierarchy and Roles of Pathogen-Associated Molecular Pattern-Induced Responses in Nicotiana benthamiana
journal, April 2011

  • Segonzac, Cécile; Feike, Doreen; Gimenez-Ibanez, Selena
  • Plant Physiology, Vol. 156, Issue 2
  • DOI: 10.1104/pp.110.171249

LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains
journal, July 2011


Direct Regulation of the NADPH Oxidase RBOHD by the PRR-Associated Kinase BIK1 during Plant Immunity
journal, April 2014


Structural Basis for flg22-Induced Activation of the Arabidopsis FLS2-BAK1 Immune Complex
journal, October 2013


The leucine-rich repeat domain in plant innate immunity: a wealth of possibilities
journal, February 2009


Advances in the directed evolution of proteins
journal, October 2014


Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses
journal, May 2009


Early molecular events in PAMP-triggered immunity
journal, August 2009


The Bacterial Elicitor Flagellin Activates Its Receptor in Tomato Cells According to the Address–Message Concept
journal, September 2000

  • Meindl, Thomas; Boller, Thomas; Felix, Georg
  • The Plant Cell, Vol. 12, Issue 9
  • DOI: 10.1105/tpc.12.9.1783

Callose Deposition: A Multifaceted Plant Defense Response
journal, February 2011

  • Luna, Estrella; Pastor, Victoria; Robert, Jérôme
  • Molecular Plant-Microbe Interactions, Vol. 24, Issue 2
  • DOI: 10.1094/MPMI-07-10-0149

Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes
journal, April 2007

  • Reetz, Manfred T.; Carballeira, José Daniel
  • Nature Protocols, Vol. 2, Issue 4
  • DOI: 10.1038/nprot.2007.72

Tomato I2 Immune Receptor Can Be Engineered to Confer Partial Resistance to the Oomycete Phytophthora infestans in Addition to the Fungus Fusarium oxysporum
journal, December 2015

  • Giannakopoulou, Artemis; Steele, John F. C.; Segretin, Maria Eugenia
  • Molecular Plant-Microbe Interactions, Vol. 28, Issue 12
  • DOI: 10.1094/MPMI-07-15-0147-R

Layered pattern receptor signaling via ethylene and endogenous elicitor peptides during Arabidopsis immunity to bacterial infection
journal, February 2013

  • Tintor, N.; Ross, A.; Kanehara, K.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 15
  • DOI: 10.1073/pnas.1216780110

Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance
journal, March 2010

  • Lacombe, Séverine; Rougon-Cardoso, Alejandra; Sherwood, Emma
  • Nature Biotechnology, Vol. 28, Issue 4
  • DOI: 10.1038/nbt.1613

Plant pattern-recognition receptors
journal, July 2014


Flagellin Is Not a Major Defense Elicitor in Ralstonia solanacearum Cells or Extracts Applied to Arabidopsis thaliana
journal, June 2004

  • Pfund, Christine; Tans-Kersten, Julie; Dunning, F. Mark
  • Molecular Plant-Microbe Interactions, Vol. 17, Issue 6
  • DOI: 10.1094/MPMI.2004.17.6.696

Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility
journal, November 2003

  • Smith, Kelly D.; Andersen-Nissen, Erica; Hayashi, Fumitaka
  • Nature Immunology, Vol. 4, Issue 12
  • DOI: 10.1038/ni1011

Discovery and Directed Evolution of a Glyphosate Tolerance Gene
journal, May 2004