RESUMEN
Pattern-triggered immunity (PTI) is activated in plants upon recognition by pattern recognition receptors (PRRs) of damage- and microbe-associated molecular patterns (DAMPs and MAMPs) derived from plants or microorganisms, respectively. To understand better the plant mechanisms involved in the perception of carbohydrate-based structures recognized as DAMPs/MAMPs, we have studied the ability of mixed-linked ß-1,3/1,4-glucans (MLGs), present in some plant and microbial cell walls, to trigger immune responses and disease resistance in plants. A range of MLG structures were tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca2+ elevations, reactive oxygen species production, phosphorylation of mitogen-activated protein kinases and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, ß-d-cellobiosyl-(1,3)-ß-d-glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. These MLG43-mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, as they were weaker in cerk1 and lyk4 lyk5 mutants than in wild-type plants. Cross-elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [ß-1,4-d-(GlcNAc)6 ], which is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, which is fully impaired in cerk1 and lyk4 lyk5 plants. MLG43 treatment confers enhanced disease resistance in A. thaliana to the oomycete Hyaloperonospora arabidopsidis and in tomato and pepper to different bacterial and fungal pathogens. Our data support the classification of MLGs as a group of carbohydrate-based molecular patterns that are perceived by plants and trigger immune responses and disease resistance.
Asunto(s)
Pared Celular/metabolismo , Resistencia a la Enfermedad , Inmunidad de la Planta , beta-Glucanos/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Calcio/metabolismo , Capsicum/inmunología , Capsicum/metabolismo , Solanum lycopersicum/inmunología , Solanum lycopersicum/metabolismo , Oomicetos/inmunología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , TrisacáridosRESUMEN
Automated chemical synthesis has revolutionized synthetic access to biopolymers in terms of simplicity and speed. While automated oligosaccharide synthesis has become faster and more versatile, the parallel synthesis of oligosaccharides is not yet possible. Here, a chemical vapor glycosylation strategy (VaporSPOT) is described that enables the simultaneous synthesis of oligosaccharides on a cellulose membrane solid support. Different linkers allow for flexible and straightforward cleavage, purification, and characterization of the target oligosaccharides. This method is the basis for the development of parallel automated glycan synthesis platforms.
Asunto(s)
Oligosacáridos , Oligosacáridos/química , GlicosilaciónRESUMEN
We report the automated glycan assembly (AGA) of different oligosaccharide fragments of the bacterial peptidoglycan (PGN) backbone. Iterative addition on a solid support of an acetyl glucosamine and a new muramic acid building block is followed by cleavage from the solid support and final deprotection providing 10 oligosaccharides up to six units.
Asunto(s)
Peptidoglicano/química , Polisacáridos/química , Automatización , Secuencia de CarbohidratosRESUMEN
Considerable research efforts have been devoted to surface-enhanced Raman spectroscopy (SERS), due to its excellent performance in biosensing and imaging. Here, a novel and facile strategy for the fabrication of well-defined and uniform nanodimers as SERS substrates is presented. By the assistance of ultrasound, the violent polyol process for particle generation becomes controllable, enabling the self-assembly of nanostars to nanodimers. Moreover, the aggregation of nanodimers can be easily tuned by post-ultrasonic treatment, which gives a sensitive substrate for SERS.
RESUMEN
The plant cell wall is a cellular exoskeleton consisting predominantly of a complex polysaccharide network that defines the shape of cells. During growth, this network can be loosened through the action of xyloglucan endotransglycosylases (XETs), glycoside hydrolases that "cut and paste" xyloglucan polysaccharides through a transglycosylation process. We have analyzed cohorts of XETs in different plant species to evaluate the substrate specificities of xyloglucan acceptors by using a set of synthetic oligosaccharides obtained by automated glycan assembly. The ability of XETs to incorporate the oligosaccharides into polysaccharides printed as microarrays and into stem sections of Arabidopsis thaliana, beans, and peas was assessed. We found that single xylose substitutions are sufficient for transfer, and xylosylation of the terminal glucose residue is not required by XETs, independent of plant species. To obtain information on the potential xylosylation pattern of the natural acceptor of XETs, that is, the nonreducing end of xyloglucan, we further tested the activity of xyloglucan xylosyl transferase (XXT) 2 on the synthetic xyloglucan oligosaccharides. These data shed light on inconsistencies between previous studies towards determining the acceptor substrate specificities of XETs and have important implications for further understanding plant cell wall polysaccharide synthesis and remodeling.
Asunto(s)
Pared Celular/metabolismo , Glicosiltransferasas/metabolismo , Oligosacáridos/metabolismo , Cromatografía Líquida de Alta Presión , Plantas/clasificación , Plantas/metabolismo , Especificidad de la EspecieRESUMEN
The mixed-linkage (1â3),(1â4)-d-glucan (MLG) specific glycosyl hydrolase lichenase is an important biochemical tool for the structural characterization of MLGs. It holds potential for application in the brewery, animal feed, and biofuel industries. Several defined MLG oligosaccharides obtained by automated glycan assembly are used to analyze the substrate specificities of Bacillus subtilis lichenase. Two glucose building blocks (BBs), equipped with a temporary fluorenylmethyloxycarbonyl chloride (Fmoc) protecting group in the C-3 or C-4 position, served to assemble different oligosaccharides by using an automated oligosaccharide synthesizer. Light-induced cleavage of the glycan products from the solid support followed by global deprotection provided seven MLG oligosaccharides of different length and connectivity. After incubation of the MLG oligosaccharides with lichenase, the digestion products were analyzed by HPLC-MS. These digestion experiments provided insights into the enzyme's active site that is in line with other recent evidence suggesting that the substrate specificity of lichenases has to be reconsidered. These results demonstrate that synthetic MLG oligosaccharides are useful tools to analyze mixed-linkage ß-glucanases.
Asunto(s)
Bacillus subtilis/enzimología , Glucanos/metabolismo , Glicósido Hidrolasas/metabolismo , Oligosacáridos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Glucanos/química , Glicósido Hidrolasas/química , Espectrometría de Masas , Oligosacáridos/química , Especificidad por SustratoRESUMEN
We report the automated glycan assembly of oligosaccharides related to the plant cell wall hemicellulosic polysaccharide xyloglucan. The synthesis of galactosylated xyloglucan oligosaccharides was enabled by introducing p-methoxybenzyl (PMB) as a temporary protecting group for automated glycan assembly. The generated oligosaccharides were printed as microarrays, and the binding of a collection of xyloglucan-directed monoclonal antibodies (mAbs) to the oligosaccharides was assessed. We also demonstrated that the printed glycans can be further enzymatically modified while appended to the microarray surface by Arabidopsis thaliana xyloglucan xylosyltransferase 2 (AtXXT2).
Asunto(s)
Anticuerpos Monoclonales/química , Arabidopsis/química , Automatización , Pared Celular/química , Oligosacáridos/síntesis química , Polisacáridos/química , Arabidopsis/enzimología , Pared Celular/enzimología , Análisis por Micromatrices , Oligosacáridos/química , Oligosacáridos/metabolismo , Pentosiltransferasa/metabolismo , Polisacáridos/metabolismoRESUMEN
We report the automated glycan assembly of oligosaccharide fragments related to the hemicellulose xyloglucan (XG). Iterative addition of monosaccharide and disaccharide building blocks to a solid support provided seven cellulose and xyloglucan fragments including XXGG- and XXXG-type oligosaccharides.
Asunto(s)
Automatización , Glucanos/síntesis química , Oligosacáridos/síntesis química , Xilanos/síntesis química , Glucanos/química , Estructura Molecular , Oligosacáridos/química , Xilanos/químicaRESUMEN
Organic semiconductors, such as carbon nitride, when employed as powders, show attractive photocatalytic properties, but their photoelectrochemical performance suffers from low charge transport capability, charge carrier recombination, and self-oxidation. High film-substrate affinity and well-designed heterojunction structures may address these issues, achieved through advanced film generation techniques. Here, we introduce a spin coating pretreatment of a conductive substrate with a multipurpose polymer and a supramolecular precursor, followed by chemical vapor deposition for the synthesis of dual-layer carbon nitride photoelectrodes. These photoelectrodes are composed of a porous microtubular top layer and an interlayer between the porous film and the conductive substrate. The polymer improves the polymerization degree of carbon nitride and introduces C-C bonds to increase its electrical conductivity. These carbon nitride photoelectrodes exhibit state-of-the-art photoelectrochemical performance and achieve high yield in C-H functionalization. This carbon nitride photoelectrode synthesis strategy may be readily adapted to other reported processes to optimize their performance.
RESUMEN
Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10 000 individual reactions cm- 2 . An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening.