The hepta-β-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to β-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound β-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-β-glucoside conjugate (K-d = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (K-d = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex

Aphanomyces euteiches cell wall fractions containing novel glucan-chitosaccharides induce defense genes and nuclear calcium oscillations in the plant host Medicago truncatula

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.Amaury Nars, Claude Lafitte, Mireille Chabaud, Sophie Drouillard, Hugo Mélida, Saïda Danoun, Tinaig Le Costaouëc, Thomas Rey, Julie Benedetti, Vincent Bulone, David George Barker, Jean-Jacques Bono, Bernard Dumas, Christophe Jacquet, Laurent Heux, Judith Fliegmann, Arnaud Botti

Ginkgo biloba Responds to Herbivory by Activating Early Signaling and Direct Defenses

Background: Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses. Methodology/Principal Findings: Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca 2+]cyt) and H2O2 production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca 2+] cyt and H 2O 2. Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides

Isolation of a French bean (Phaseolus vulgaris L.) homolog to the β-glucan elicitor-binding protein of soybean (Glycine max L.)

A high-affinity membrane-bound β-glucan elicitor-binding protein has been purified from microsomal preparations of French bean (Phaseolus vulgaris L.) roots. A 5900-fold purification was achieved by affinity chromatography of functionally solubilized membrane proteins. The β-glucan-binding protein had an apparent molecular mass of 78 kDa when subjected to SDS-PAGE. Western blot analysis showed specific crossreactivity of this French bean protein with an antiserum raised against a synthetic peptide representing an internal 15 amino acid fragment of the β-glucan-binding protein from soybean. Northern blot analysis with a cDNA probe of the soybean β-glucan-binding protein gene revealed a crosshybridizing transcript of 2.4 kb in French bean. These results indicate that the β-glucan-binding proteins of French bean and soybean are conserved homologs involved in β-glucan elicitor recognition

LuBiA (Luciferase-Based Binding Assay): Glowing Peptides as Sensitive Probes to Study Ligand-Receptor Interactions.

The quantitative and qualitative biochemical description of molecular interactions is fundamental to the study of ligand/receptor pairs and their structure/function relationships. Bioactive peptides often are active at (sub-)nanomolar concentrations, indicating they have a high affinity for their sites of action, notably binding sites on receptors. Since such receptor proteins are commonly of low abundance, highly sensitive detection methods are required to study these ligand/receptor interactions. We present a protocol for an inexpensive luminescence-based detection setup in which the peptide ligand of interest is extended with the 11-amino acid HiBiT tag. This tag can be quantified easily down to fmol amounts by its ability to reconstitute the enzymatic activity of LgBiT, a truncated version of the Oplophorus gracilirostris luciferase

An ancient enzyme domain hidden in the putative beta-glucan elicitor receptor of soybean may play an active part in the perception of pathogen-associated molecular patterns during broad host resistance

A successful defense against potential pathogens requires that a host organism is able to discriminate between self and nonself structures. Soybean (Glycine max L.) exploits a specific molecular pattern, a 1,6-beta-linked and 1,3-beta-branched heptaglu

Signal perception and transduction in the activation of plant defense by beta-glucan elicitors

The best characterized elicitors of the oomycete Phytophthora sojae for activating a multicomponent defense response, including the production of phytoalexins, in soybean (Glycine max L.) are the branched 1,3- and 1,6-linked beta-glucans that are structural polysaccharides of the hyphal walls of the pathogen. The soybean microsymbiont Bradyrhizobium japonicum synthesizes cyclic 1,3-1,6-beta-glucans that suppressed the fungal beta-glucan-induced phytoalexin response in soybean, indicating a novel mechanism by which the symbiotic bacteria might avoid a defense response that is normally activated in pathogenic interactions. A putative receptor (M-r = 75 kDa) for the P. sojae beta-glucans was isolated from soybean membranes and partially characterized. Partial amino acid sequences were used to raise an anti-peptide antiserum and to generate two oligonucleotides which served as primers for PCR using soybean cDNA as template. Northern blot experiments with a PCR product indicated that the glucan-binding protein mRNA has a size of about 2.4 kb. Since several species of the plant family Fabaceae were shown to possess related high-affinity beta-glucan-binding sites, glucan-based perception mechanisms may be a more common feature of this plant family. Following plasma membrane binding of the beta-glucan elicitor, the subsequent signal transduction might involve a rapid, transient increase in the cytosolic Ca2+ level and in permeability changes of the plasma membrane to Ca2+, H+, and Cl-. Production of the isoflavonoid phytoalexins, glyceollins, in soybean involves a multi-step biosynthetic pathway. Among the pathway enzymes are several cytochrome P450s. A gene family-specific differential display approach was used in the identification of several elicitor-inducible P450 cDNAs

Eur. J. Biochem.

4-Coumarate:CoA ligase (4CL) is involved in the formation of coenzyme A thioesters of hydroxycinnamic acids that are central substrates for subsequent condensation, reduction, and transfer reactions in the biosynthesis of plant phenylpropanoids. Previous studies of 4CL appear to suggest that many isoenzymes are functionally equivalent in supplying substrates to various subsequent branches of phenylpropanoid biosyntheses. In contrast, divergent members of a 4CL gene family were identified in soybean (Glycine max L.). We isolated three structurally and functionally distinct 4CL cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene Gm4CL3. A fourth cDNA encoding 4CL4 had high similarity with 4CL3. The recombinant proteins expressed in Escherichia coli possessed highly divergent catalytic efficiency with various hydroxycinnamic acids. Remarkably, one isoenzyme (4CL I) was able to convert sinapate; thus the first cDNA encoding a 4CL that accepts highly substituted cinnamic acids is available for further studies on branches of phenylpropanoid metabolism that probably lead to the precursors of lignin. Surprisingly, the activity levels of the four isoenzymes and steady-state levels of their transcripts were differently affected after elicitor treatment of soybean cell cultures with a beta-glucan elicitor of Phytophthora sojae, revealing the down-regulation of 4CL1 vs. up-regulation of 4CL3/4. A similar regulation of the transcript levels of the different 4CL isoforms was observed in soybean seedlings after infection with Phytophthora sojae zoospores. Thus, partitioning of cinnamic acid building units between phenylpropanoid branch pathways in soybean could be regulated at the level of catalytic specificity and the level of expression of the 4CL isoenzymes