Identification of a high‐affinity binding protein for a hepta‐β‐glucoside phytoalexin elicitor in soybean

Abstract

A putative receptor protein for a hepta-B-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified P-glucan-binding proteins with a photolabile '251-labeled 2-(4-azidophenyl)ethylamino 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 1251-labeling of the protein band by underivatized hepta-fi-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 radioligandbinding 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 '251-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.The perception mechanisms that signal the presence of potential pathogens and lead to the activation of defense responses in plant cells are still poorly understood. A representative case is that of the production of pterocarpan phytoalexins by soybean cells when infected by the pathogenic fungus Phytuphthora megasperma f.sp. glycineu. The phytoalexin response in soybean has been the object of detailed study (Ebel, 1986;Ebel and Grisebach, 1988). The accumulated information on the minimal structural requirements for phytoalexin-elicitor activity of P . megasperma mycelial-wall glucans (Sharp et al., 1984a, b; A commonly proposed model for signal recognition in the phytoalexin response involves a receptor located on the surface of plant cells that specifically binds phytoalexin elicitor(s) (Ebel, 1986;Dixon and Lamb, 1990). The best charac- 3) and (1,6)-linked-P-glucoside with high elicitor activity from mycelial-wall hydrolysates provided initial insight into the minimum structural requirements for elicitor activity in soybean. Additional information was obtained with the chemical synthesis of the hepta-fi-glucoside and comparison of its biological activity with that of a number of oligoglucosides of related structure (Sharp et al., 1984b;Ossowski et al., 1984;. Evidence for the existence of high-affinity-binding sites for P. megasperma (1,3) and (1,6)-linked P-glucan fragments in soybean membranes was provided initially by Schmidt and Ebel (1987) Additional work by our group defined optimal conditions for the solubilization, using detergents, of the glucan-binding proteins and for a partial purification of ...

The Hepta-? -Glucoside Elicitor-Binding Proteins from Legumes Represent a Putative Receptor Family

“…Two-week old transgenic tomato cell cultures or soybean roots were extracted and binding assays were performed as described previously (Cosio et al, 1988(Cosio et al, , 1992 with appropriate amounts of protein. All data were corrected for nonspecific binding determined in the presence of a 1000-fold excess of unlabeled ␤-glucan.…”

“…Radiolabeled derivatives of this compound were used to identify and characterize low abundance ␤-glucan-binding protein(s) (GBP) in soybean. The main component of the GBP was identified as a 75 kDa protein (Cosio et al, 1992;Mithöfer et al, 1996) and was recently purified to homogeneity using affinity chromatography (Mithöfer et al, 1996;Umemoto et al, 1997).…”

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

“…CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-lpropane sulfonate; HPTLC, high performance thin layer chromatography; Zwittergent 3-12, N-dodecyl-N,N-dimethyl-3-ammonio-l-propane sulfonate P. sojae mycelial walls and which may function as a receptor mediating plant defense responses [9,10]. In view of this evidence, and the fact that many oomycetes are plant pathogens of significant impact to agriculture, we considered it of interest to proceed with the study and biochemical characterization of Phytophthora cell wall synthesizing enzymes.…”

Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid.

“…Evidence is accumulating that jasmonic acid (JA) and its derivatives are an integral part of a general signal transduction system (1) that must be present between the elicitorreceptor complex (2) and the gene activation process responsible for the induction of enzyme synthesis (3) that leads to the formation of low molecular weight defense compounds (phytoalexins). Jasmonates have been shown to induce the synthesis of proteinase inhibitor proteins in response to wounding and pathogen attack (1,4) and to trigger the formation of phytoalexins such as flavonoids, alkaloids, terpenoids (5) and their biosynthetic enzymes (5,6), and thionins, polypeptides with antifungal activity, in barley (7).…”