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

Abstract

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. 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.

Reactive oxygen species and plant resistance to fungal pathogens

“…The chitin-elicited production of ROS and of H 2 O 2 in particular has been demonstrated in roots of M. truncatula and Arabidopsis (Kim et al, 2006;Nars et al, 2013;Plancot et al, 2013). Using an elicitor from Fusarium oxysporum, increased levels of superoxide and H 2 O 2 were also detected in the border-like cells of Arabidopsis and flax roots (Plancot et al, 2013).…”

Aphanomyces euteiches Cell Wall Fractions Containing Novel Glucan-Chitosaccharides Induce Defense Genes and Nuclear Calcium Oscillations in the Plant Host Medicago truncatula

“…The facts that the majority of GlcNAc residues are 1,4-linked in the A. euteiches cell wall [19] and that we used a glucanase mixture which was able to release all the chitosaccharides from the cell wall, indicate that most chitosaccharides containing 1,4-linked GlcNAc residues were lost during the purification of the fractions. We have recently shown that β-1,4-linked GlcNAc oligomers of DP above 8, which are poorly soluble in water, can occur in a metastable soluble state before irreversible precipitation [39]. We thus conclude that 1,4-linked GlcNAc in A. euteiches essentially occurs in poorly soluble carbohydrates which precipitated out during the purification, whereas the soluble glucan-chitosaccharides characterized here contain a high proportion of unusual 1,6-linked GlcNAc residues.…”

“…The capacity of fractions 60A, 60B, 2A and 2B to activate the expression of defense-associated genes in wild type (WT) and nfp mutant seedlings of M. truncatula was investigated following a previously established experimental setup [39]. The root systems of hydroponically-grown seedlings were incubated for 4 h in the presence of each fraction (100 µg.ml −1 ).…”

Foliar treatments with Gaultheria procumbens essential oil induce defense responses and resistance against a fungal pathogen in Arabidopsis

“…The categories selected included genes induced by phytohormones (SA, JA, ethylene, and absicic acid), by microbial PAMPs (FLG22, chitosaccharides, harpin, and lipopolysaccharides), pathogen infection and housekeeping genes. To validate the selection of marker genes, Arabidopsis plants (Col-0) grown in microtiter plates were treated with solutions containing mehyljasmonate (MeJA), FLG22 peptide, BTH (BION® formulation), and chitosaccharidic fragments obtain from crab shell chitin (DP 6-9) (Nars et al, 2013). RNAs were extracted 1, 6, and 24 h after treatments and gene expression was analyzed by real-time quantitative-RT PCR using the dynamic array (Figure 2).…”

“…Flg22 peptide was synthesized by Proteogenix (Strasbourg, France) with purity greater than 90%. Chitin oligomers obtained from Crab shell chitin (CSC, Sigma-Aldrich) were prepared as described by Nars et al (2013). BTH formulated as BION® was obtained from Syngenta.…”