HIGH-AFFINITY BINDING OF FUNGAL BETA-GLUCAN ELICITORS TO CELL-MEMBRANES OF SPECIES OF THE PLANT FAMILY FABACEAE

Microsomal preparations of six species of the plant family Fabaceae ii ;ere screened for high-affinity binding of branched (1 --> 3), (1 --> 6)-beta-glucaas. Oligoglucosides of this type are specific elicitors o f phytoalexin accumulation in soybean (Glycine max L.): a member of th is family. The species studied were alfalfa (Medicago sativa L.), broa dbean (Vicia faba L.), chickpea (Cicer arietinum L.), french bean (Pha seolus vulgaris L.), pea (Pisum sativum L.), and white lupin (Lupinus albus L.). A I-125-labeled 4-(2-aminophenyl)ethylamine conjugate of a (1 --> 3), (1 --> 6)-beta-glucan fraction with an average degree of po lymerization (DP) of 18, obtained from mycelial walls of Phytophthora sojae, was used as radioligand for initial screening. The structural c omplexity of this fraction allowed the identification of binding sites with affinities for isomeric structures other than the (1 --> 3), (1 --> 6)-hepta- beta-glucoside for which soybean binding sites display h ighest affinity. Radioligand competition experiments against unlabeled fungal beta-glucan resulted in the identification of high-affinity bi nding in alfalfa, bean, lupin, and pea. Half-maximal competition conce ntrations (ICS,) for fungal beta-glucan in these species were between 5 and 30 nM. Pseudoheterologous radioligand competition by unlabeled h epta-beta-glucoside showed that for alfalfa, lupin and pea the IC50 va lues for this structure (4 to 16 nM) were similar to those of soybean (7.7 nM). Bean microsomes, however, displayed an IC50 significantly hi gher than soybean (68 nM) suggesting that the structural motif recogni zed by its binding sites is not identical to that of soybean or the ot her three species. Radioligand saturation assays with alfalfa, lupin a nd pea microsomes using an I-125-Iabeled aminophenylethylamine hepta-b eta-glucoside conjugate gave dissociation constants (K-d) of 5.3, 3.7, and 1.8 nM; respectively. The affinity of these sites for hepta-beta- glucoside was in the same range as that of soybean (K-d 1-3 nM), where as the affinity of the binding sites of bean for the same ligand was s ignificantly lower (K-d = 33 nM). Good correlation was found between t he presence of high-affinity binding and the accumulation of isoflavon oid phytoalexins in roots of alfalfa, bean, chickpea and pea seedlings after exposure to fungal beta-glucan. Lupin displayed a strong wound- induced accumulation of prenylated isoflavone which was independent of the presence of beta-glucan, making it impossible to determine phytoa lexin induction in response to elicitor. No specific binding or phytoa lexin accumulation in response to beta-glucans was observed in broadbe an. This is the first report on the existence of possibly homologous e licitor-binding sites within a plant taxonomic family and may provide preliminary evidence for putative evolutionary relationships in pathog en perception mechanisms in plants.