Abstract
We have previously isolated glycopeptides derived from yeast invertase that acted as highly potent elicitors in suspension-cultured tomato cells, inducing ethylene biosynthesis and phenylalanine ammonia-lyase activity, and we have found that the high mannose oligosaccharides released from the pure glycopeptide elicitors by endo-beta-N-acetylglucosaminidase H acted as suppressors of elicitor activity (Basse, C. W., Bock, K., and Boller, T. (1992) J. Biol. Chem. 267, 10258-10265). One of the elicitor-active glycopeptides (gp 8c) was labeled with t-butoxycarbonyl-L-[35S]methionine and purified by reversed phase high performance liquid chromatography resulting in a specific radioactivity of the derivative of about 900 Ci/mmol. This radiolabeled glycopeptide showed specific, saturable, and reversible binding to whole tomato cells under conditions in which cells are responsive to elicitors as well as to microsomal membranes derived from these cells. Ligand saturation experiments, performed with microsomal membranes, gave a dissociation constant (Kd) of 3.3 nM as determined by Scatchard analysis. Various glycopeptide elicitors and preparations from yeast invertase were compared with respect to their abilities to compete for binding of 35S-labeled gp 8c to tomato membranes and to induce ethylene biosynthesis in tomato cells. These studies revealed a high degree of correlation between elicitor activities in vivo and displacement activities in vitro. In both tests, a high activity depended on the presence of glycan side chains consisting of more than 8 mannosyl residues. The high mannose oligosaccharides that acted as suppressors of elicitor activity in vivo competed for binding of the labeled elicitor also. The suppressor-active glycan Man11GlcNAc and the elicitor-active gp 8c exhibited very similar displacement activities, and the inhibitory constant (Ki) of the glycan Man11GlcNAc was very similar to the Kd value calculated for 35S-labeled gp 8c, indicating that the glycopeptide elicitors and the glycan suppressors derived from these elicitors competed with similar affinities for the same binding site. The suppressor-inactive glycan Man8GlcNAc had a 200-fold lower capacity to compete for binding of 35S-labeled gp 8c to tomato membranes compared with the suppressor-active glycan Man11GlcNAc. Our results demonstrate the existence of a specific elicitor binding site in tomato cell membranes and suggest that glycopeptides and glycans act as agonists and antagonists for induction of the stress response, respectively, by competing for this binding site.
Highlights
Ylene biosynthesis and phenylalanine ammonia-lyase activity, and we have found that the highmannose oligosaccharides released from the pure glycopeptide elicitors by endo-8-N-acetylglucosaminidaseH acted Plants have a capacity to perceive specific components of as suppressors of elicitor activity
This radi- the various classes of elicitors, which have mainly been isoolabeled glycopeptide showed specific, saturable, and reversible binding to whole tomato cells under conditions in which cells are responsive to elicitors as well as tomicrosomal membranes derived from thesecells
ManllGlcNAc and the elicitor-active g p 8c exhibited for elicitor activity (Basse et al, 1992).The peptide moieties very similar displacement activities, and the inhibitoofrtyhe elicitor-active glycopeptidescontained only a few amino constant ( K i )of the glycan ManllGlcNAc was very acids; gp 8, the most active glycopeptide, had the sequence similar to thKed value calculatedfor 3sS-labeled gp 8c, Asn-Gly-Thr-His-Phe (Basse et al, 1992).Cleavage of glycoindicating that theglycopeptide elicitors and thegly- peptide elicitors by Endo H1 or N-glycanase completely incan suppressors derived from these elicitocrsompeted activated elicitor activity and generated molecules that acted with similar affinities for the same binding site
Summary
Plant Cells-Tomato cells (line Msk 8) weregrown as described (Felix et al, 1991a) and used 9-14 days after transferinto fresh growth medium for assays of elicitor and suppressor activities (Basse and Boller, 1992).ACC synthase activity in tomato cells was measured as described by Spanu etal. (1990). Suspension-cultured tomato cells (7-10 days after transfer into fresh growth medium) .were harvested on a sieve andthe fresh weight determined. The cells were suspended in an equal volume of ice-cold homogenization buffer (50 mM Tris-MES, 3 mM MgCl,, 5 mM dithiothreitol, pH 7.5). Binding Ass~y-~~S-Labelegdp 8c (- 900 Ci/mmol, usually 3 X lo cpm/O.l ml corresponding to 1.5 nM or at the concentrations indicated) was incubated with membrane fractions containing 200 pg of protein either alone (total binding) or with appropriate concentrations of competitors in 100 pl of binding buffer (25 mM Tris-MES, pH 7.0, 3 mMMgC12, 0.1 M NaC1, 10 mM methionine) on ice for 1.5 h. The washed filters were transferred into plastic vials, scintillation fluid (3.5ml) was added, and radioactivity (total binding) was determined (Packard 1900TR, Downers Grove, IL).
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