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Plant Cell Reports

, Volume 32, Issue 4, pp 489–502 | Cite as

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors

  • A. Nars
  • T. Rey
  • C. Lafitte
  • S. Vergnes
  • S. Amatya
  • C. Jacquet
  • B. Dumas
  • C. Thibaudeau
  • L. Heux
  • A. BottinEmail author
  • J. FliegmannEmail author
Original Paper

Abstract

Key message

A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay.

Abstract

The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora β-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.

Keywords

Chitooligosaccharide Nuclear magnetic resonance Reactive oxygen species Defense gene activation Aphanomyces 

Notes

Acknowledgments

We thank Dr. S. Bensmihen (LIPM, UMR CNRS-INRA 2594/441, Toulouse) for supplying seeds of the A. thaliana cerk1 GABI-KAT mutant 096F09, and Dr. H. Chanzy (CERMAV-CNRS, Grenoble) and J.J. Bono (LIPM, UMR CNRS-INRA 2594/441, Toulouse) for continuous support. This work was funded by the Région Midi-Pyrénées, the CNRS (PhD Grant INEE 36 to A.N.), and the French Agence Nationale de la Recherche (ANR-08-BLAN-0208-01 “Sympasignal”).

Supplementary material

299_2012_1380_MOESM1_ESM.xls (30 kb)
Online Resource 1 (XLS 30 kb)
299_2012_1380_MOESM2_ESM.xls (31 kb)
Online Resource 2 (XLS 31 kb)
299_2012_1380_MOESM3_ESM.ppt (211 kb)
Online Resource 3 (PPT 211 kb)
299_2012_1380_MOESM4_ESM.xls (35 kb)
Online Resource 4 (XLS 35 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • A. Nars
    • 1
    • 2
  • T. Rey
    • 1
    • 2
  • C. Lafitte
    • 1
    • 2
  • S. Vergnes
    • 1
    • 2
  • S. Amatya
    • 1
    • 2
  • C. Jacquet
    • 1
    • 2
  • B. Dumas
    • 1
    • 2
  • C. Thibaudeau
    • 3
  • L. Heux
    • 3
  • A. Bottin
    • 1
    • 2
    Email author
  • J. Fliegmann
    • 1
    • 2
    • 4
    • 5
    Email author
  1. 1.Université de Toulouse, UPS, UMR5546, Laboratoire de Recherche en Sciences Végétales (LRSV)Castanet-TolosanFrance
  2. 2.CNRS, UMR5546, Laboratoire de Recherche en Sciences Végétales (LRSV)Castanet-TolosanFrance
  3. 3.CERMAV-CNRSGrenoble Cedex 9France
  4. 4.INRA, UMR441, Laboratoire des Interactions Plantes-Microorganismes (LIPM)Castanet-TolosanFrance
  5. 5.CNRS, UMR2594, Laboratoire des Interactions Plantes-Microorganismes (LIPM)Castanet-TolosanFrance

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