HIGH-AFFINITY BINDING OF GLYCOPEPTIDE ELICITOR TO TOMATO CELLS AND MICROSOMAL-MEMBRANES AND DISPLACEMENT BY SPECIFIC GLYCAN SUPPRESSORS

We have previously isolated glycopeptides derived from yeast invertase that acted as highly potent elicitors in suspension-cultured tomato c ells, inducing ethylene biosynthesis and phenylalanine ammonia-lyase a ctivity, and we have found that the high mannose oligosaccharides rele ased from the pure glycopeptide elicitors by endo-beta-N-acetylglucosa minidase H acted as suppressors of elicitor activity (Basse, C. W., Bo ck, K., and Boller, T. (1992) J. Biol. Chem. 267, 10258-10265). One of the elicitor-active glycopeptides (gp 8c) was labeled with t-butoxyca rbonyl-L-[S-35]methionine and purified by reversed phase high performa nce liquid chromatography resulting in a specific radioactivity of the derivative of about 900 Ci/mmol. This radiolabeled glycopeptide showe d specific, saturable, and reversible binding to whole tomato cells un der conditions in which cells are responsive to elicitors as well as t o microsomal membranes derived from these cells. Ligand saturation exp eriments, performed with microsomal membranes, gave a dissociation con stant (K(d)) of 3.3 nm as determined by Scatchard analysis. Various gl ycopeptide elicitors and preparations from yeast invertase were compar ed with respect to their abilities to compete for binding of S-35-labe led gp 8c to tomato membranes and to induce ethylene biosynthesis in t omato cells. These studies revealed a high degree of correlation betwe en elicitor activities in vivo and displacement activities in vitro. I n both tests, a high activity depended on the presence of glycan side chains consisting of more than 8 mannosyl residues. The high mannose o ligosaccharides that acted as suppressors of elicitor activity in vivo competed for binding of the labeled elicitor also. The suppressor-act ive glycan Man11GlcNAc and the elicitor-active gp 8c exhibited very si milar displacement activities, and the inhibitory constant (K(i)) of t he glycan Man11GlcNAc was very similar to the K(d) value calculated fo r S-35-labeled gp 8c, indicating that the glycopeptide elicitors and t he glycan suppressors derived from these elicitors competed with simil ar affinities for the same binding site. The suppressor-inactive glyca n Man8GlcNAc had a 200-fold lower capacity to compete for binding of S -35-labeled gp 8c to tomato membranes compared with the suppressor-act ive glycan Man11GlcNAc. Our results demonstrate the existence of a spe cific elicitor binding site in tomato cell membranes and suggest that glycopeptides and glycans act as agonists and antagonists for inductio n of the stress response, respectively, by competing for this binding site.