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”)

Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures

Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C(4) of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group

Quadruple-phase MDCT of the liver in patients with suspected hepatocellular carcinoma: Effect of contrast material flow rate

OBJECTIVE. The purposes of this study were to evaluate the effect of contrast material flow rate (3 mL/sec vs 5 mL/sec) on the detection and visualization of hepatocellular carcinoma (HCC) with MDCT and the safety profile of iodixanol at different injection rates. SUBJECTS AND METHODS. In a prospective, randomized multicenter trial, 97 patients (83 men and 14 women, with a mean age of 64 years) suspected of having HCC underwent quadruple-phase (double arterial, portal venous, delayed phase) 4- 16-MDCT. Patients were randomized to receive iodixanol, 320 mg I/mL (1.5 mL/kg body weight), at a flow rate of 3 mL/sec (48 patients) or 5 mL/sec (49 patients). Qualitative (lesion detection, image quality) and quantitative (liver and aortic enhancement, tumor-liver contrast) analyses and safety assessment were performed. RESULTS. Overall, 145 HCCs were detected in the 5 mL/sec group and 100 HCCs in the 3 mL/sec group (p < 0.05). More lesions equal to or less than I cm were detected at 5 mL/sec (33 vs 16 lesions). The late arterial phase showed significantly more lesions than the early, arterial phase (133 vs 100 and 96 vs 67 lesions, respectively, p < 0.0001). Hyperattenuating HCCs were better visualized in the late arterial phase at 5 mL/sec (excellent visualization: 54% vs 27%). Using a flow of 5 mL/sec did not increase the rate of patient discomfort or contrast media-related adverse events. Most discomfort in both groups was of mild intensity and there was no severe discomfort. CONCLUSION. For detection of HCC with MDCT, a higher flow rate of 5 mL/sec is recommended. Visualization of hyperattenuating HCC is improved with no greater discomfort or adverse events

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