Solubilization of functional plasma membrane-localized hepta-beta-glucoside elicitor-binding proteins from soybean.

Total membranes prepared from roots of soybean (Glycine max L.) seedlings have previously been shown to contain proteinaceous binding site(s) for a hepta-beta-glucoside elicitor of phytoalexin accumulation. The hepta-beta-glucoside elicitor-binding proteins have now been shown to co-migrate with a plasma membrane marker enzyme (vanadate-sensitive H(+)-ATPase) on linear sucrose density gradients. With the use of detergents, the elicitor-binding proteins have been solubilized in functional form from soybean root membranes. The nonionic detergents n-dodecylsucrose, n-dodecylmaltoside, and Triton X-114, at concentrations of 5 to 10 mg/mL, each solubilizes between 50 and 60% of the elicitor-binding activity in a single extraction of the membranes. A zwitterionic detergent, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZW 3-12), also solubilizes about 40% of the total binding activity at detergent concentrations between 1 and 2 mg/mL, but the total binding activity recovered is only approximately 50% of that recovered with the nonionic detergents. The elicitor-binding proteins solubilized with either n-dodecylsucrose or ZW 3-12 retain the high affinity for radiolabeled hepta-beta-glucoside elicitor (apparent dissociation constant [Kd] = 1.8 nM and 1.4 nM, respectively) that was observed with the membrane-localized binding proteins (apparent Kd = 1 nM). Competitive ligand-binding experiments with several structurally related synthetic oligoglucosides demonstrate that the solubilized binding proteins retain specificity for elicitor-active oligosaccharides, irrespective of the detergent used for solubilization. Moreover, the binding affinities of the oligoglucosides for the solubilized binding proteins correlate well with their abilities to induce phytoalexin accumulation in soybean cotyledon tissue. Gel-permeation chromatography of n-dodecylsucrose-solubilized elicitor-binding proteins demonstrate that the bulk of the elicitor-binding activity is associated with large detergent-protein micelles (relative molecular weight > 400,000). Our results suggest that n-dodecylsucrose is a suitable detergent for solubilizing elicitor-binding proteins from soybean root membranes with minimal losses of binding activity. More importantly, we demonstrate that solubilization does not significantly after the binding properties of the proteins for elicitor-active oligoglucosides.     

Structure-activity relationships of oligo-beta-glucoside elicitors of phytoalexin accumulation in soybean.

The abilities of a family of chemically synthesized oligo-beta-glucosides, ranging in size from hexamer to decamer, to induce phytoalexin accumulation in soybean cotyledons were investigated to determine which structural elements of the oligoglucosides are important for their biological activity. The results of the biological assays established that the following structural motif is necessary for the oligo-beta-glucosides to have high elicitor activity: [formula; see text] The branched trisaccharide at the nonreducing end of the oligoglucosides was found to be essential for maximum elicitor activity. Substitution of either the nonreducing terminal backbone glucosyl residue or the side-chain glucosyl residue closest to the nonreducing end with glucosaminyl or N-acetylglucosaminyl residues reduced the elicitor activity of the oligoglucosides between 10-fold and 10,000-fold. Elicitor activity was also reduced 1000-fold if the two side-chain glucosyl residues were attached to adjacent backbone glucosyl residues rather than to glucosyl residues separated by an unbranched residue. In contrast, modifications of the reducing terminal glucosyl residue of an elicitor-active hepta-beta-glucoside by conjugation with tyramine and subsequent iodination had no significant effect on the elicitor activity of the hepta-beta-glucoside. These results demonstrate that oligo-beta-glucosides must have a specific structure to trigger the signal transduction pathway, which ultimately leads to the de novo synthesis of phytoalexins in soybean.     

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

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-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-beta-glucoside conjugate (Kd = 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 (Kd = 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.     

Characterization of binding proteins that recognize oligoglucoside elicitors of phytoalexin synthesis in soybean

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H⁺-ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K_d? 1 nM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K_d= 1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.     

Host-Pathogen Interactions : XVII. HYDROLYSIS OF BIOLOGICALLY ACTIVE FUNGAL GLUCANS BY ENZYMES ISOLATED FROM SOYBEAN CELLS

The ability of β-glucosylase I, a soybean cell wall β-glucosyl hydrolase, to degrade elicitors of phytoalexin accumulation was studied. Extensive β-glucosylase I treatment of the glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae results in hydrolysis of 77% of the glucosidic bonds of the elicitor and destruction of 94% of its activity. Soybean cell walls contain some additional factor, probably one or more additional enzymes, which can assist β-glucosylase I in hydrolyzing the glucan elicitor. This was demonstrated by the more rapid hydrolysis of the glucan elicitor by a mixture of soybean cell wall enzymes (containing β-glucosylase I). In a single treatment, the mixture of cell wall enzymes hydrolyzed 91% of the glucosidic bonds and destroyed 85% of the activity of the elicitor. The enzymes from soybean cell walls will also hydrolyze elicitor-active oligoglucosides prepared from the mycelial walls of Phytophthora megasperma var. sojae. The active oligoglucosides are more susceptible than the glucan elicitor to hydrolysis by these enzymes. The mixture of cell wall enzymes or β-glucosylase I, by itself, hydrolyzes more than 96% of the glucosidic bonds and destroys more than 99% of the activity of the oligoglucoside elicitor. Two possible advantages for the existence of these enzymes in the walls of soybean cells are discussed.     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in theplasma membrane from rice leaf and root cells

Presence of a high-affinity binding protein for N-acetylchitooligosaccharide (fragments of chitin) elicitor in the plasma membrane from rice leaf and root cells was shown by affinity labeling experiments with an ¹²⁵I-labeled N-acetylchitooligosaccharide derivative. Binding studies also showed that binding site in the leaf cells has a high affinity to highly elicitor-active, larger chitin fragments but much lower or no affinity to less elicitor-active or elicitor-inactive oligosaccharides. The amount of the binding protein in the leaf cells was slightly smaller than that in the suspension-cultured cells but much larger compared to that in the root cells. These results indicate the possible- involvement of the elicitor binding protein in the perception of the elicitor signal in intact rice plant.     

Sensitivity of different ecotypes and mutants of Arabidopsis thaliana toward the bacterial elicitor flagellin correlates with the presence of receptor-binding sites

Flagellin, the main building block of the bacterial flagellum, acts as potent elicitor of defense responses in different plant species. Genetic analysis in Arabidopsis thaliana identified two distinct loci, termed FLS1 and FLS2, that are essential for perception of flagellin-derived elicitors. FLS2 was found to encode a leucine-rich repeat transmembrane receptor-like kinase with similarities to Toll-like receptors involved in the innate immune system of mammals and insects. Here we used a radiolabeled derivative of flg22, a synthetic peptide representing the elicitor-active domain of flagellin, to probe the interaction of flagellin with its receptor in A. thaliana. The high affinity binding site detected in intact cells and membrane preparations exhibited specificity for flagellin-derived peptides with biological activity as agonists or antagonists of the elicitor responses. Specific binding activity was measurable in all ecotypes of A. thaliana that show sensitivity to flagellin but was barely detectable in the flagellin-insensitive ecotype Ws-0 affected in FLS1. A strongly impaired binding of flagellin was observed also in several independent flagellin-insensitive mutants isolated from the flagellin-sensitive ecotype La-er. In particular, no binding was found in plants carrying a mutation in the LRR domain of FLS2. These data indicate that the formation of functional receptor-binding sites depends on genes encoded by both loci, FLS1 and FLS2. The tight correlation between the presence of the binding site and elicitor response provides strong evidence that this binding site acts as the physiological receptor of flagellin.     

Host-Pathogen Interactions : XIX. THE ENDOGENOUS ELICITOR, A FRAGMENT OF A PLANT CELL WALL POLYSACCHARIDE THAT ELICITS PHYTOALEXIN ACCUMULATION IN SOYBEANS

An elicitor of phytoalexin accumulation (endogenous elicitor) is solubilized from purified cell walls of soybean (Glycine max [L.] Merr., cv. Wayne) by extracting the walls with hot water or by subjecting the walls to partial acid hydrolysis. The endogenous elicitor obtained from soybean cell walls binds to an anion exchange resin. The elicitor-active material released from the resin contains oligosaccharides rich in galacturonic acid; small amounts of rhamnose and xylose are also present. The preponderance of galacturonic acid in the elicitor-active fragments suggests that the elicitor is, in fact, a fragment of a pectic polysaccharide. This possibility is supported by the observation that treatment of the wall fragments with a highly purified endopolygalacturonase destroys their ability to elicit phytoalexin accumulation. This observation, together with other evidence presented in this paper, suggests that galacturonic acid is an essential constituent of the elicitor-active wall fragments. Endogenous elicitors were also solubilized by partial hydrolysis from cell walls of suspension-cultured tobacco, sycamore, and wheat cells.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [¹²⁵I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([¹²⁵I]-GN₈-AzPEA) and a [¹²⁵I]-labeled 2- (4-aminophenyl)ethylamino conjugate ([¹²⁵]-GN₈-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)₈ showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)₈, cellopentaose nor α-1,4 linked N-acetylgalactosamine octamer (GalNAc)₈ at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high-affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.     

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.     

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl₂ increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.     

Functional reconstitution of beta-glucan elicitor-binding activity upon incorporation into lipid vesicles.

In temperature-induced Triton X-114 phase separation experiments the beta-glucan elicitor-binding site from soybean (Glycine max L.) root membranes was identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-solubilized beta-glucan-binding proteins were incorporated into lipid vesicles by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-beta-glucoside ligand was saturable, reversible and of high affinity (K(d)=6-7 nM). Competition studies using beta-glucans with different degrees of polymerization (DP 7-15; DP 15-25) showed effective displacement of the radioligand from the binding site whereas beta-glucan fragments with DP <7 were ineffective. The total amount of reconstituted binding activity was dependent on the acyl chain length of the phospholipids used for the reconstitution with a preference for decanoic (C10) and dodecanoic (C12) chains. Restored ligand binding was maximally 37% as compared to the former detergent-solubilized binding activity. The presence of a lipid environment stabilized the purified beta-glucan-binding proteins.     

A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase.

The release of elicitor-active carbohydrates from fungal cell walls by beta-1,3-endoglucanase contained in host tissues has been implicated as one of the earliest processes in the interaction between soybean (Glycine max) and the fungal pathogen Phytophthora megasperma f. sp. glycinea leading to host defense responses such as phytoalexin production. The present study was conducted to evaluate the primary structure of the glucanase-released elicitor (RE). Gel-filtration chromatography of carbohydrates released from mycelial walls by purified soybean beta-1,3-endoglucanase resolved them into the four fractions (elicitor-active RE-I, -II, and -III and elicitor-inactive RE-IV). Sugar composition analysis indicated that all of the fractions were composed almost entirely of glucose. 1H- and 13C-nuclear magnetic resonance analysis indicated the presence of both beta-1,3- and beta-1,6-linkages for the elicitor-active RE-I, -II, and -III fractions and only beta-1,3 linkage for the elicitor-inactive RE-IV fraction. Methylation analysis and degradation studies employing beta-1,3-endo- and beta-1,3-exoglucanase further suggested that the basic structure of elicitor-active RE consists of beta-1,6-linked glucan backbone chains of various lengths with frequent side branches composed of beta-1,3-linked one or two glucose moieties. From these structural analyses of RE, a structural model of how RE is originally present in fungal cell walls and released by host beta-1,3-endoglucanase is also proposed.     

感染叶锈菌的小麦细胞间隙液中激发子的定性及初步分离

感染叶锈菌后小麦叶片细胞间隙液(1wF)中有激发子存在,它(们)能诱导寄主PAL、PO活性的增强及细胞过敏性坏死的产生。这种有诱导活性的物质分别用NaIO4和蛋白酶处理证明含有糖基和蛋白质成分,可能是糖蛋白。IWF经凝胶过滤分离后,各部分诱导不同的防卫反应的活性强度不等。因此IWF中可能含有几种不同成分的激发子,或是同种成分而聚合度不同的激发子。     

A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation

Soybean membrane preparations specifically bound [¹⁴C]mycolaminaran, a branched β-1,3-glucan produced by Phytophthora sp. which elicits production of the phytoalexin glyceollin in soybean tissues. A Scatchard plot of the binding data disclosed the presence of a single affinity class of binding sites with a K_d value of 11.5 micromolar for the glucan. To assess the physiologic importance of mycolaminaran binding in phytoalexin elicitation, several derivatives of mycolaminaran were prepared. Reduced mycolaminaran had slightly greater elicitor activity and binding affinity than the native substance, while periodinated mycolaminaran was virtually devoid of either elicitor activity orbinding capability. Phosphorylated mycolaminaran, on the other hand, gave values for both elicitor activity and membrane binding which were intermediate between the native and periodinated preparations. No other tested carbohydrates competed with the binding of [¹⁴C]mycolaminaran. Soybean membrane preparations contained β-1,3-endoglucanase activity that degraded mycolaminaran and reduced both its efficiency as a phytoalexin elicitor and its membrane binding at temperatures above 0°C. Once [¹⁴C]mycolaminaran bound to membranes, however, it was not appreciably susceptible to glucanase attack and could not be displaced with excess unlabeled ligand. Taken collectively, the observations suggest that the membrane binding sites are mycolaminaran-specific receptors which are physiologically involved in the initiation of phytoalexin production in soybean cotyledons. Because the binding of mycolaminaran to membranes was abolished by heat and proteolytic enzymes, the receptor is probably a protein(s) or glycoprotein(s).     

Oligoglucoside elicitor-mediated activation of plant defense

Plants have acquired defense mechanisms to counteract potential pathogens. One such strategy involves inducible defense reactions that are activated by elicitors, signaling compounds of diverse nature. For one class of elicitors, oligoglucosides, recent developments in the characterization and isolation of an oligoclucan-binding protein, a putative elicitor receptor, and isolation of a cDNA that encodes the binding protein are discussed. Furthermore, the discovery of a role for calcium in the elicitation process is described. Finally, the identification of polymerase chain reaction products whose sequences indicate that they encode cytochrome P-450–dependent enzymes with possible roles in the formation of phytoalexins, antimicrobial plant defense compounds, is reported. These advances may lay the foundation for the first characterization of a receptor and subsequent signaling events in oligoglucan elicitor perception by higher plants. BioEssays 20: 569–576, 1998. © 1998 John Wiley & Sons Inc.     

Identification of high-affinity binding sites for the hepta-β-glucoside elicitor in membranes of the model legumes Medicago truncatula and Lotus japonicus

 Previous studies have led to the identification and characterization of specific, high-affinity binding sites for a hepta-β-glucoside elicitor in soybean. A survey of plant species for elicitor-binding activity reveals that among the plants tested, the hepta-β-glucoside elicitor is only recognized by plants belonging to the legume family. We have characterized in detail the glucan elicitor-binding site in the model legume Medicago truncatula Gaertn., and partially characterized the site in Lotus japonicus. These sites have characteristics that are very similar to the one in soybean, with dissociation constants of 4.7 and 8.9 nM respectively. The elicitor-binding sites from both plants are stable during solubilization with non-ionic alkylglycoside detergents. However, differences are observed in the abundance of the binding sites and their selectivity towards structurally related analogues of the hepta-β-glucoside elicitor. Our results suggest that similar, but perhaps not identical, binding sites for the hepta-β-glucoside elicitor exist in diverse legumes, but not in plants outside of the legume family. Received: 15 December 1999 / Accepted: 28 February 2000     

Protein phosphatase inhibitors activate anti-fungal defence responses of soybean cotyledons and cell cultures

The application of a variety of structurally different protein phosphatase inhibitors (okadaic acid, acanthifolicin, microcystins, nodularin, tautomycin, calyculin A, cantharidin and endothall) to cut surfaces of soybean cotyledons (Glycine max L.) resulted in the production of isoflavonoid phytoalexins (plant defence compounds). Daidzein was the predominant isoflavonoid produced by soybean cotyledons in response to protein phosphatase inhibitors. In contrast, several isoflavonoid phytoalexins were seen after application of either an elicitor β-glucan fraction isolated from yeast extract or hepta-(1→3, 1→6)-β-glucoside which is the most potent elicitor-active component isolated from the soybean pathogen Phytophthora megasperma f. sp. glycinea. Isoflavonoid production in response to either protein phosphatase inhibitors or elicitors reached a maximum after 20–24 h. The addition of protein phosphatase inhibitors to a soybean cell suspension culture induced the expression of phenylalanine ammonia-lyase (PAL), the first enzyme in the isoflavonoid biosynthetic pathway. Induction of PAL activity was blocked by protein synthesis inhibitors, cycloheximide or anisomysin, and largely prevented by a protein kinase inhibitor, K252a. Another common response of plant cells to fungal elicitation, alkalinization of the soybean cell culture media, was induced within minutes in response to protein phosphatase inhibitors and was largely prevented by K252a. These studies suggest a direct role for phosphorylation in activation of plasma membrane ion flux(es), whereas the longer-term effects of protein phosphatase inhibitors on isoflavonoid production and PAL expression could be due to either direct effects of increased protein phosphorylation, or the secondary consequences of other phosphorylation-induced cellular changes. They also indicate that protein phosphatase inhibitors are likely to be of general use in investigating mechanisms of plant responses to environmental stimuli.     

The bacterial elicitor flagellin activates its receptor in tomato cells according to the address-message concept

flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, acts as a potent elicitor in plants. Here, we have used an iodinated derivative of flg22 ((125)I-labeled Tyr-flg22) as a molecular probe for the flagellin receptor in tomato cells. This radioligand showed rapid binding to a single class of specific, saturable, high-affinity receptor sites in intact cells and membrane preparations. Binding, although essentially nonreversible under physiological conditions, was not covalent, and chemical cross-linking was required to specifically label a single polypeptide of 115 kD. Intact flagellin and elicitor-active flagellin peptides but not biologically inactive analogs efficiently competed for binding of radioligand. Peptides lacking the C terminus of the conserved domain, previously found to act as competitive antagonists of elicitor action in tomato cells, also competed for binding of radioligand. Thus, this novel, high-affinity binding site exhibited all the characteristics expected of a functional receptor of bacterial flagellin. For a model of receptor activation, we propose a two-step mechanism according to the address-message concept, in which binding of the N terminus (address) is the first step and activation of responses with the C terminus (message) is the second step.