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The syringolides, specified by avirulence gene D from Psendomonas syringae. elicit a disease reaction only in soybean cultivars carrying the Rpg4 disease resistance gene. In order to identify structural features important for clicilor activity and construct highly labeled elicitor-active probes, several syringolide derivatives were synthesized (Tsurushima et al. 1996). The synthesis of syringolidc derivatives were started from changing the hemikclal group of syringolide 1 to ketal group (Fig. I). Syringolidc I was reacted with the corresponding alcohol in the presence of dry Dowex/HCI. All ketal derivatives had no clicilor activity in soybean leaves. Esterification of the C-4’ hydroxyl group on syringolide 1 was performed using N.N’-dicyclohcxylcarbodiimide(DCC), l-(3-dimcthylaminopropyl)-3-clhylcarhodiimidc methiodide (EDCI) or 4-(dimethylamino) pyridine (DMAP). However the esterification reaction destroyed the syringolidc ring system. Since syringolidc ketals were stable to esterification using base catalysis, (C-3)-O-bcnzyl syringolidc I was esterified with 3.4.5-trimethoxyphenylacctic acid. 3-(4-mcthoxyphenyl)propionic acid or succimc anhydride. After the reaction the protecting group was removed by hydrogenation. Hemikelal esters of syringolidel elicited the HR only in soybean cotyledon carrying Rpg4. whereas the ketal esters were inactive. Hemikelal esters were also active elicitors of glyceollin in soybean coiyledons. while ketals had no elicitor activity. 4-(3.4.5-Trinicthoxyphenylacetyl)syringolidc I was unlimited by adding AgOCOCF3 and iodine in anhydrous CH2Cl2to produce 4’-(2-indo-3,4,5-trimcthoxyphcnylacctyl) syringolide 1 in 95 % yield.

How Bradyrhizobium sp. avoid elicitation of a host defense response during symbiosis is not known. Periplasmid cyclic beta-(1–3),(1–6)-glucans of B. japonicum have been shown to suppress a fungal (Phytopthora sojae) beta-glucan-induced host defense response in soybean cotyledons. B. japonicum ndvB mutant strain AB-14 which did not synthesize beta-glucan and strain AB-1 (ndvC) which synthesized a cyclic beta-(1–3)-linked molecular (cyclodecalaminarinose), were both defective in nodule morphogenesis and had elevated levels of glyceollin in the nodule tissue. Consistent with the proposed suppressor function, cyclodecalaminarinose from strain AB-1 did not suppress a fungal beta-glucan-induced plant defense response in soybean cotyledons. Data will be presented on the glucan and phytoalexin levels in several fix mutants of B. japonicum.

Pectin was irradiated with electron beam (0 – 2000 kGy) in air to obtain the various types of pectic fragments. Low molecular weight pectic fragments (< M.W. 5000) was collected. Pectin fragments prepared by irradiation with 1000 and 2000 kGy had the activity for induction of glyceollins (phytoalexin of soybean). The same pectic fragments also showed inhibition of IAA-induced pea stem growth.

The defence of plants against microbial pathogens is a multistep process characterized by a bidirectional flow of signals. In the interaction between soybean (Glycine max L.) and Phytophthora megasperma f. sp. glycinea, a hepta-(1→3, 1→6)-s-glucoside derived from fungal cell walls is regarded as a highly active elicitor of phytoalexin production. Until now, the biological activity of this compound and its synthetic analogue was mainly monitored by a bioassay using wounded cotyledons. Recent studies in our group showed that the application of the synthetic hepta-(1→3, 1→6)-s-glucoside or a partially purified s-glucan fraction from fungal cell walls to both unwounded roots of soybean seedlings (up to 1 g/1) and to soybean cell-suspension cultures (up to 0.1 g/1) neither led to a significant accumulation of the pterocarpan phytoalexin glyceollin nor to the induction of chalcone synthase, a key enzyme of phytoalexin biosynthesis, respectively. However, when applied simultaneously with the protein kinase inhibitor K-252a, the hepta-(1→3, 1→6)-s-glucoside and the partially purified s-glucan fraction showed a profound induction of chalcone synthase activity in soybean cell cultures. Accumulation of glyceollin in roots of soybean seedlings was increased by coincubation with s-glucan and K-252a to a level comparable to that induced by crude elicitor preparations or observed after fungal infection. These results indicate that the hepta-(1→3, 1→6)-s-glucoside and purified s-glucans derived from fungal cell walls are indeed able to elicit defence responses in soybean tissues other than wounded cotyledons when combined with an additional signal molecule.

Among the vast array of responses of higher plants to pathogens, the synthesis of phytoalexins is believed to play in some cases an important role in the development of resistance. As an example, soybean (Glycine max L.) seedlings produce and accumulate a mixture of structurally related isoflavonoid derivatives (glyceollins) following either inoculation with the fungus Phytophthora megasperma f sp. glycinea (Pmg) or treatment with a s-glucan elicitor isolated from the fungal cell walls. While the different steps leading to the glyceollin synthesis in soybean cells are now well documented (1), the sequence of reactions involved between the recognition and/or the binding of the elicitor and the cellular response remains to be investigated. The external surface of the plasma membrane (PM) of soybean cells has been shown to contain high affinity binding sites for s-glucans (2). The PM also contains the bulk of cellular free sterols as a mixture of sitosterol, stigmasterol and 24-methylcholesterol (3). In order to shed some light on the possible involvement of sterols in some plant defense reactions, especially the elicitation of phytoalexin synthesis, cultured soybean (Glycine max L. cv Harosoy 63) cells were grown in the presence of fenpropimorph, a sterol biosynthesis inhibitor, which triggers the replacement of up to 95 % of the usual Δ5-sterols by 9s,19-cyclopropylsterols, and then tested for their ability to synthesize glyceollins in response to the addition of a Pmg s-glucan elicitor. In such cells, the elicitor was found to induce an earlier synthesis and a five-fold higher accumulation of glyceollins than in elicited control (i.e. with a non modified sterol profile) cells. These isoflavonoids are undetectable in unelicited cultures treated only with the sterol biosynthesis inhibitor, indicating that the overstimulation of their synthesis requires the presence of the elicitor.

Overproduction of reactive oxygen species have been described in plant cells exposed to pathogens. A rapid and transient production of H2O2 as well as of lipoxygenase activity have been shown to occur in plants cells and tissues after infection or biotic elicitation. These observations lead us to assume that accumulation of lipid hydroperoxides occurs during elicitation. Furthermore, our previously published data show that various hydroperoxides (HPX) are powerful inducers of glyceollin synthesis in soybean roots.

Linear a-1,4-oligogalacturonides derived from plant cell walls elicit a variety of changes putatively associated with defense responses of plants to pests (Nothnagel et al. 1983; Robertsen, 1986; Cervone et al. 1987; De Lorenzo et al. 1987; Bruce and West, 1989). Effects not apparently related to defense responses and considered to be controlled by the classical phytohormones are affected by oligogalacturonides (Branca et al. 1988; Hayashi and Yoshida, 1988; Baldwin and Pressey, 1989). A possible connection between the role played by oligogalacturonides and that played by one phytohormone, auxin, has been observed. It has been shown that IAA-induced pea stem elongation is competitively inhibited by oligogalacturonides and that both the early and the late growth responses to IAA are antagonized by oligogalacturonides (Branca et al. 1988). We have now tested the hypothesis that competition of oligogalacturonides with auxin is of general occurrence and that oligogalacturonides interfere with the regulatory effect of auxin in pea stems and other plant tissue systems. We report here that a purified oligogalacturonide preparation, [average degree of polymerization (DP)=131 capable of eliciting glyceollin production in soybean cotyledons, not only inhibits the IAA-induced pea stem elongation but also antagonizes the IAA-induced rhizogenic ability of tobacco leaf explants. Moreover, we report that elicitor-active oligogalacturonides inhibit the induction of auxin-binding protein (ABP) and compete with auxin for auxin-binding sites in carrot cell membrane preparations.