AFFINITY PURIFICATION AND CHARACTERIZATION OF A BINDING-PROTEIN FOR AHEPTA-BETA-GLUCOSIDE PHYTOALEXIN ELICITOR IN SOYBEAN

A hepta-beta-glucoside elicitor of phytoalexin accumulation binds with high affinity to the plasma membrane of soybean (Glycine max) cells. One component of the elicitor-binding sites is a protein with an appar ent M(r) of 70 000 in SDS-PAGE as recently identified by photoaffinity labelling using a photosensitive I-125-labelled 2-(4-azidophenyl) eth ylamine conjugate of the heptaglucoside. Heptaglucoside-binding activi ty was solubilized using the zwitterionic detergent, Zwittergent 3-12, and purified by ion-exchange chromatography on Q Sepharose and by aff inity chromatography. The affinity adsorbent consisted of a Phytophtho ra megasperma branched (1 --> 3, 1 --> 6)-beta-glucan fraction conjuga ted by reductive amination to controlled-pore glass beads containing a minopropyl groups. The purified fraction contained a protein of appare nt M(r) of 70 000 which was radiolabelled by photoaffinity labelling a long with two additional proteins of 100 000 and 170 000 labelled to a lesser extent. The 70 000 protein represented also the major protein as visualized by silver staining after SDS-PAGE of the purified fracti on. Ligand-saturation studies and the kinetics of ligand interaction d emonstrated that the hepta-beta-glucoside-binding characteristics of t he solubilized and enriched protein fractions were very similar to tho se of the membrane-associated binding sites. The results provide, thus , a clear identification of a membrane protein of 70 000 with high aff inity and specificity for a fungal signal molecule capable of triggeri ng plant defence. The results also provide a simple method for the iso lation of this protein for further studies at the level of signal perc eption.