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

[EN] N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.SIThis work is part of the “Laboratoire d’Excellence” (LABEX) entitled TULIP (ANR -10-LABX-41); it was funded by the Région Midi-Pyrénées, the CNRS (PhD grant INEE 36 to AN), and the French Agence Nationale de la Recherche (ANR-08-BLAN-0208-01 “Sympasignal”)

Étude des mécanismes de perception du facteur Nod et rôle de protéines à domaines LysM chez Medicago truncatula

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Recherche de partenaires protéiques de la protéine DMI3/CCaMK chez Medicago truncatula (analyse comparée du phosphoprotéome de plante sauvage et mutante ; Isolement de la protéine nucléaire IPD3 (Interacting Protein of DMI3) par une approche de double-hybride chez la levure)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Coordination entre l'épiderme et le cortex dans l'établissement des endosymbioses racinaires chez Medicago truncatula (rôle du gène DMI3 codant une protéine calcium et calmoduline dépendante)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Evolutionary History of Plant LysM Receptor Proteins Related to Root Endosymbiosis

LysM receptor-like kinases (LysM-RLKs), which are specific to plants, can control establishment of both the arbuscular mycorrhizal (AM) and the rhizobium-legume (RL) symbioses in response to signal molecules produced, respectively, by the fungal and bacterial symbiotic partners. While most studies on these proteins have been performed in legume species, there are also important findings that demonstrate the roles of LysM-RLKs in controlling symbiosis in non-legume plants. Phylogenomic studies, which have revealed the presence or absence of certain LysM-RLKs among different plant species, have provided insight into the evolutionary mechanisms underlying both the acquisition and the loss of symbiotic properties. The role of a key nodulation LysM-RLK, NFP/NFR5, in legume plants has thus probably been co-opted from an ancestral role in the AM symbiosis, and has been lost in most plant species that have lost the ability to establish the AM or the RL symbiosis. Another LysM-RLK, LYK3/NFR1, that controls the RL symbiosis probably became neo-functionalised following two rounds of gene duplication. Evidence suggests that a third LysM-RLK, LYR3/LYS12, is also implicated in perceiving microbial symbiotic signals, and this protein could have roles in symbiosis and/or plant immunity in different plant species. By focusing on these three LysM-RLKs that are widespread in plants we review their evolutionary history and what this can tell us about the evolution of both the RL and the AM symbioses

Signalling during the early stage of the Rhizobium/legume symbiosis.

National audienceThe establishment of the symbiosis between Rhizobia and the plants of the legume family depends on a signal exchange between the two partners, and shows a high degree of specificity determined largely by the bacterial signals called nodulation factors (Nod factors). Nod factors are able to induce in the host plant, at very low concentrations (10-12M) and in a structure dependent manner, many of the responses observed in the presence of the bacterium, suggesting the presence of receptors. By using radiolabelled Nod factors, we have been able to characterise in Medicago truncatula, a high affinity binding site (Kd = 0.45 nM) for the major Nod factor produced by its symbiont Sinorhizobium meliloti. This site termed Nod Factor Binding Site 3 (NFBS3) is associated to a high density fraction prepared from roots. Studies of Nod factor binding in root extracts of early symbiotic mutants of M. truncatula, reveals that NFBS3 is present in a nfp mutant, affected in a putative Nod factor receptor, but is absent in dmi1 and dmi2 mutants affected in genes required for the establishment of symbioses with both rhizobia and arbuscular mycorrhizal fungi. The potential role of NFBS3 in the establishment of root endosymbioses is discussed

Identification and partial characterization of an extracellular manganese-dependent peroxidase in Armillaria ostoyae and <em>Armillaria mellea</em>

National audienceAbstract Laccase and manganese-dependent peroxidase (Mn peroxidase) activities were detected in the culture media of Armillaria ostoyae and A. mellea. Mn peroxidase was produced in significantly higher quantity by the A. ostoyae isolates and was purified by chromatography from one isolate of this species. Some properties of the purified enzyme were examined (absorption spectrum, H2O2 and MnSO4 optimal concentrations, pH optimum and lactate stimulation). Enzymes of potential importance in the lignin degradation (especially Mn peroxidase) by Armillaria sp. are compared to those of other root-rotting fungi. The possible role of Mn peroxidase in modulating the pathogenicity of Armillaria sp. is discussed

Docking of Chitin Oligomers and Nod Factors on Lectin Domains of the LysM-RLK Receptors in the Medicago-Rhizobium Symbiosis

Legume plants establish a symbiotic association with N2-fixing soil bacteria (rhizobia) to benefit from a nitrogen source independent of soil nitrates for their protein syntheses, growth, and development. The infection and subsequent development of functional rhizobia-containing nodules require a first step by the host plant’s roots in which they specifically recognize the signal molecules produced by the rhizobia, known as Nodulation (Nod) factors [1]. Nod factors consist of amphipathic lipo-chitooligosaccharides built up from an oligochitin backbone of 4-5 β1,4-linked GlcNAc residues carrying a fatty acid chain on the nonreducing end of the GlcNAc residue (Fig. 27.1). Diverse substitutions, e.g. acetyl groups, sulfate groups, or O-linked sugars, on the GlcNAc residues of both the reducing and nonreducing ends of the oligochitin backbone define the different host specificities that are necessary for correct rhizobia–legume recognition

The DMI1 and DMI2 early symbiotic genes of Medicago truncatula are required for a high-affinity nodulation factor-binding site associated to a particulate fraction of roots

International audienceThe establishment of the legume-rhizobia symbiosis between Medicago spp. and Sinorhizobium meliloti is dependent on the production of sulfated lipo-chitooligosaccharidic nodulation (Nod) factors by the bacterial partner. In this article, using a biochemical approach to characterize putative Nod factor receptors in the plant host, we describe a high-affinity binding site (Kd = 0.45 nM) for the major Nod factor produced by S. meliloti. This site is termed Nod factor-binding site 3 (NFBS3). NFBS3 is associated to a high-density fraction prepared from roots of Medicago truncatula and shows binding specificity for lipo-chitooligosaccharidic structures. As for the previously characterized binding sites (NFBS1 and NFBS2), NFBS3 does not recognize the sulfate group on the S. meliloti Nod factor. Studies of Nod factor binding in root extracts of early symbiotic mutants of M. truncatula reveals that the new site is present in Nod factor perception and does not make infections 3 (dmi3) mutants but is absent in dmi1 and dmi2 mutants. Roots and cell cultures of all these mutants still contain sites similar to NFBS1 and NFBS2, respectively. These results suggest that NFBS3 is different from NFBS2 and NFBS1 and is dependent on the common symbiotic genes DMI1 and DMI2 required for establishment of symbioses with both rhizobia and arbuscular mycorrhizal fungi. The potential role of this site in the establishment of root endosymbioses is discussed