Carbohydrate–protein interactions trigger a wide range of biological signaling pathways, the mainstays of physiological and pathological processes. However, there are an incredible number of ...carbohydrate-binding proteins (CBPs) that remain to be identified and characterized. This study reports for the first time the covalent labeling of CBPs by triazinyl glycosides, a new and promising class of affinity-based glycoprobes. Mono- and bis-clickable triazinyl glycosides were efficiently synthesized from unprotected oligosaccharides (chitinpentaose and 2′-fucosyl-lactose) in a single step. These molecules allow the specific covalent labeling of chitin-oligosaccharide-binding proteins (wheat germ agglutinin WGA and Bc ChiA1 D202A, an inactivated chitinase) and fucosyl-binding lectin (UEA-I), respectively.
Functionalized oligosaccharides are useful intermediates to prepare products for biological research or for the development of advanced functional materials. Here, we report the unprecedented use of ...aniline as an efficient organocatalyst reaction with “clickable” (azide or alkyne) amine for the transimination-mediated reductive amination of a chitooligosaccharide. Moreover, we demonstrate that alkyne-bearing aniline constitutes an excellent tool for the easy derivatization of chitosan oligosaccharides. Evidence for such improvement has been illustrated by the straightforward design of a FRET substrate to probe chitinase activity and of amphiphilic polycaprolactone-grafted-chitosan. This efficient methodology paves the way to the preparation of novel chitosan oligosaccharide-based advanced materials.
Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling the nodulation of legumes by rhizobia. More recently, LCOs were also found in ...symbiotic fungi and, more surprisingly, very widely in the kingdom Fungi, including in saprophytic and pathogenic fungi. The LCO-V(C18:1, fucosylated/methyl fucosylated), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants such as Medicago truncatula can discriminate between Nod-LCOs and Fung-LCOs. To address this question, we performed a genome-wide association study on 173 natural accessions of M. truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-LCOs and Fung-LCOs stimulated root branching in most accessions, but the root responses to these two types of LCO molecules were not correlated. In addition, the heritability of the root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, of which only one was common. This suggests that Nod-LCOs and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.
Lipo-chitinoligosaccharides (LCOs) are key molecules for the establishment of plant-microorganisms symbiosis. Interactions of leguminous crops with nitrogen-fixing rhizobial bacteria involve Nod ...factors, while Myc-LCOs improve the association of most plants with arbuscular mycorrhizal fungi. Both Nod factors and Myc-LCOs are composed of a chitinoligosaccharide fatty acylated at the non-reducing end accompanied with various substituting groups. One straightforward way to access LCOs is starting from chitin hydrolysate, an abundant polysaccharide found in crustacean shells, followed by regioselective enzymatic cleavage of an acetyl group from the non-reducing end of chitin tetra- or pentaose, and subsequent chemical introduction of N-acyl group. In the present work, we describe the in vitro synthesis of LCO precursors on preparative scale. To this end, Sinorhizobium meliloti chitin deacetylase NodB was produced in high yield in E. coli as a thioredoxin fusion protein. The recombinant enzyme was expressed in soluble and catalytically active form and used as an efficient biocatalyst for N-deacetylation of chitin tetra- and pentaose.
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•Rhizobium NodB deacetylase is expressed and purified in active form in E. coli.•Yield optimization gives up to 100 mg of purified deacetylase from 1 L of culture medium.•In vitro synthesis of lipo-chitinoligosaccharides precursors is performed on preparative scale.
Peptidoglycan oligomers constitute precious tools for the biochemical and structural studies of enzymes involved in the bacterial cell wall metabolism. In this study we show that an unprecedented ...selective Zemplén de‐O‐acetylation of benzyl chitinbioside peracetate leads to a fast and efficient route to N‐acetylmuramyl β(1→4) N‐acetylglucosaminide disaccharide, a central building block for the synthesis of peptidoglycan oligomers. Starting from known benzyl chitinbioside, NAG‐NAM disaccharide pentapeptide is prepared in seven steps with an overall yield of 12%.
A NAG‐NAM disaccharide pentapeptide precursor of peptidoglycan oligomers is prepared in 6 steps from β‐benzyl chitinbioside with 12% overall yield.
The plant hormone strigolactone promotes infection thread formation but does not appear to influence other stages of nodulation, including nitrogen fixation.
Strigolactones (
SL
s) influence the ...ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which
SL
s influence nodulation. We show that
SL
s promote infection thread formation, as a null
SL
-deficient pea (
Pisum sativum
) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic
SL
GR24. We found no evidence that
SL
s influence physical events in the plant before or after infection thread formation, since
SL
-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (
LCO
s) and
SL
-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an
SL
receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that
SL
deficiency may influence the bacterial partner. We found that this influence of
SL
deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of
SL
deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly,
SL
synthesis is down-regulated by mutations in genes of the Nod
LCO
signaling pathway, and this requires the downstream transcription factor
NSP2
but not
NIN
. This, together with the fact that the expression of certain
SL
biosynthesis genes can be elevated in response to rhizobia/Nod
LCO
s, suggests that Nod
LCO
s may induce
SL
biosynthesis.
SL
s appear to influence nodulation independently of ethylene action, as
SL
-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that
SL
s influence ethylene synthesis or vice versa.
Arbuscular mycorrhizal (AM) fungi greatly improve mineral uptake by host plants in nutrient-depleted soil and can intracellularly colonize root cortex cells in the vast majority of higher plants. ...However, AM fungi possess common fungal cell wall components such as chitin that can be recognized by plant chitin receptors to trigger immune responses, raising the question as to how AM fungi effectively evade chitin-triggered immune responses during symbiosis. In this study, we characterize a secreted lysin motif (LysM) effector identified from the model AM fungal species Rhizophagus irregularis, called RiSLM. RiSLM is one of the highest expressed effector proteins in intraradical mycelium during the symbiosis. In vitro binding assays show that RiSLM binds chitin-oligosaccharides and can protect fungal cell walls from chitinases. Moreover, RiSLM efficiently interferes with chitin-triggered immune responses, such as defence gene induction and reactive oxygen species production in Medicago truncatula. Although RiSLM also binds to symbiotic (lipo)chitooligosaccharides it does not interfere significantly with symbiotic signalling in Medicago. Host-induced gene silencing of RiSLM greatly reduces fungal colonization levels. Taken together, our results reveal a key role for AM fungal LysM effectors to subvert chitin-triggered immunity in symbiosis, pointing to a common role for LysM effectors in both symbiotic and pathogenic fungi.
A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety ...(EDANS) as a fluorescent energy donor at the reducing end and a 4‐(4′‐dimethylaminobenzeneazo)‐benzene derivative as an acceptor chromophore at the non‐reducing end. The key steps for the preparation of the target molecule involved transglycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.
The designed hexasaccharide substrate 1, with donor and acceptor chromophores for fluorescence resonance energy transfer, was obtained by an efficient chemoenzymatic synthesis involving condensations catalysed by the Glu‐197‐Ala nucleophile mutant of the retaining cellulase endo‐glucanase (Cel7B) from Humicola insolens.
xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with D-xylosyl residues. Three xyloglucan ...xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher K
m and 9-fold lower k
cat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.
Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of ...Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.
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