Caractérisation structurale et perception par la plante hôte Medicago truncatula des chitosaccharides pariétaux d'Aphanomyces euteiches, parasite de légumineuses

Aphanomyces euteiches est un oomycète parasite racinaire des légumineuses causant des pertes de rendement récurrentes. La paroi d'A.euteiches contient 10% de N-acétylglucosamine (NAG) sous la forme de chitosaccharides non cristallins, associés aux glucanes pariétaux. Afin de pouvoir étudier leur activité biologique, un bioessai d'élicitation du système racinaire de la plante hôte Medicago truncatula a été mis au point en utilisant une préparation de fragments de chitine comme éliciteur témoin. La purification de fractions de parois hydrolysées, enrichies en NAG, a donné des fragments de glycanes composés de glucose et de NAG. Ces hétéropolymères présentent une structure nouvelle jamais décrite à ce jour. Le bioessai d'élicitation racinaire a révélé une activité biologique des fractions de paroi différente de celle des fragments de chitine chez M.truncatula. De façon intéressante, l'une des fractions induit des oscillations calciques nucléaires dans les cellules épidermiques de cultures de racines de M.truncatula, qui sont différentes de la réponse provoquée par des chitotétramères purs.The oomycete Aphanomyces euteiches is a root pathogen infecting legumes which causes important yield losses. The cell wall of A.euteiches contains 10% N-acetylglucosamine (NAG) in the form of non-crystalline chitosaccharides, which are associated to cell wall glucans. To study their biological activity, a root elicitation bioassay on the host plant Medicago truncatula has been set up using a preparation of chitin fragments as a control elicitor. The purification of hydrolysed cell wall fractions enriched in NAG yielded glycan fragments which were composed of glucose and NAG. These heteropolymers possess a novel structure, which has never been described before. The root elicitation bioassay showed a biological activity of the cell wall fractions different from the one of chitin fragments, on M.truncatula. Interestingly, one fraction induced nuclear calcium oscillations in epidermal cells of M.truncatula root cultures, which are different from the response evoked by pure chitotetramers

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”)

Lipo-chitooligosaccharide signalling blocks a rapid pathogen-induced ROS burst without impeding immunity

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Are chitin synthases targets for antimicrobial compounds and sources of MAMPs in oomycetes?

National audienceChitin is a crystalline N-Acetyl-Glucosamine (GlcNAc) polymer that is essential to cell wall function in Fungi, and chitooligosaccharides derived from it are Microbe-Associated Molecular Patterns recognized by LysM-containing receptors. Whereas GlcNAc is usually a minor component in the cell wall of most oomycetes, large scale sequencing shows the presence of chitin synthase (CHS) genes in their genomes. We are interested in determining the biological role of oomycete, and their involvement in the generation of signals perceived by the plant cell. Our microbial models are Aphanomyces euteiches, a legume root parasite, and a Phytophthora parasitica strain pathogenic to tobacco. Whereas chitin has never been detected in P. parasitica, our data suggest that its CHS gene(s) play(s) an essential role. In A. euteiches, we recently showed that amorphous GlcNAc polymers (chitosaccharides) are involved in cell wall function (Badreddine et al 2008). We are now engaged in characterizing these chitosaccharides in order to determine their links to the other cell wall polymers, and to understand how the host plant Medicago truncatula distinguishes chitosaccharide-derived fragments from other microbial signals such as symbiotic Nod factors. Our approaches include 13C-NMR studies of the cell wall polysaccharides, purification of the chitosaccharides after sequential chemical and enzymatic hydrolyses, elicitation bioassays involving the measurement of reactive oxygen species and expression of defense-related genes, and genetic studies targeting candidate genes of the LysM-containing putative receptors family. Last data obtained on both the microbe and plant sides will be presented

Identification of new MAMPs from the Aphanomyces euteiches cell wall.

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

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NFP, a LysM protein controlling Nod factor perception, also intervenes in Medicago truncatula resistance to pathogens

International audiencePlant LysM proteins control the perception of microbial-derived N-acetylglucosamine compounds for the establishment of symbiosis or activation of plant immunity. This raises questions about how plants, and notably legumes, can differentiate friends and foes using similar molecular actors and whether any receptors can intervene in both symbiosis and resistance. To study this question, nfp and lyk3 LysM-receptor like kinase mutants of Medicago truncatula that are affected in the early steps of nodulation, were analysed following inoculation with Aphanomyces euteiches, a root oomycete. The role of NFP in this interaction was further analysed by overexpression of NFP and by transcriptome analyses. nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A.euteiches, whereas NFP overexpression increased resistance. Transcriptome analyses on A.euteiches inoculation showed that mutation in the NFP gene led to significant changes in the expression of c. 500 genes, notably involved in cell dynamic processes previously associated with resistance to pathogen penetration. nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M.truncatula immunity, suggesting an unsuspected role for NFP in the perception of pathogenic signals

Proton and carbon chemical shifts of glucan-chitosaccharide fraction 2B^a.

a<p>Values are expressed in ppm. The hexose residues were labeled A to G in order of increasing chemical shift of their anomeric protons. B and B’ branching point units were differentiated on the basis of their H-6 and C-6 chemical shifts. Values of the free C-6 for units A, C and D are 60.65, 60.54 and 60.12 ppm.</p

Expression of defense-associated genes in <i>Medicago truncatula</i> in response to glucan-chitosaccharide fractions.

<p>Gene expression in the seedling root system was analyzed by qRT-PCR after 4 h treatment with 100 µg.ml⁻¹ fraction 60A, 60B, 2A or 2B. Chitooligosaccharides (COs) of mean degree of polymerization 6.8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075039#pone.0075039-Nars1" target="_blank">[39]</a> and Flg22 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075039#pone.0075039-Felix1" target="_blank">[40]</a> were used as control elicitors at 20 µg.ml⁻¹ and 1 µM, respectively. Defense-associated gene expression was standardized in each sample using three reference genes encoding an histone H3, a translation elongation factor 1-α and a ubiquitin family protein/phosphatidylinositol 3,4-kinase, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075039#pone.0075039-Nars1" target="_blank">[39]</a>. Mean values from three biological replicates (± S.E.) are given as log2 of fold-expression in elicited seedlings with respect to mock-treated seedlings. Asterisks above the bars indicate that gene induction in elicited seedlings was significant (<i>P</i><0.05). PR10.2, pathogenesis-related protein 10; THA, thaumatin; PI, proteinase inhibitor; CHI I, class I chitinase; LOX, lipoxygenase; PAL, phenylalanine ammonia lyase; EPI, NAD dependent epimerase/dehydratase; VR, vestitone reductase.</p

Linkage analysis of glucan-chitosaccharide fractions.

<p>The glycosidic linkages were identified by electron-impact mass spectrometry. Values are relative mole percentages (mol%) means ± S.D. of 4 technical replicates.</p