The hepta-β-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to β-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound β-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-β-glucoside conjugate (K-d = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (K-d = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex

Tissue-Specific Expression of as-1 in Transgenic Tobacco.

When integrated as a transgene in one or a few copies, the -90 35S promoter of cauliflower mosaic virus confers expression in roots with little or no expression in cotyledons and leaves. The responsible cis element, activation sequence-1 (as-1), can bind to the nuclear factor ASF-1 as well as to the transcription factor TGA1a. Here, we show that microinjection of 104 molecules of TGA1a per cotyledon cell activated transgenes containing as-1-linked promoters. Transgenes with promoters linked to the octopine synthase (ocs) element, which also binds TGA1a, responded similarly. The acidic, N-terminal segment of TGA1a is important for transcription activation in vivo because a deletion mutant without the first 80 amino acids was inactive. Finally, we show that the -90 35S-[beta]-glucuronidase (GUS) fusion gene conferred GUS expression in cotyledon cells when injected at 50,000 copies per cell. Collectively, these results provide support for the hypothesis that the undetectable expression of the as-1-linked transgene in cotyledon cells is most likely a result of its inability to compete for a limiting amount of its cognate transcription factor(s), presumably TGA1a or related proteins

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with beta-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca(2+) concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and beta-glucan elicitors caused a rapid increase in cytosolic Ca concentration, which peaked within 2-2.5 min of treatment. The Ca(2+) concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca(2+) concentration with a maximum occurring after about 7-8 min was observed in the case of beta-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca(2+) concentration from resting concentrations as low as 0.1 mu M to highest levels of about 2 mu M. Dose-response experiments showed that the concentration of elicitors giving a Ca(2+) response at the 50% level was 0.4 nM for the chitin fragment and 28 mu M and 72 mu M, respectively, for a synthetic hepta-beta-glucoside and a fungal beta-glucan fraction. The beta-glucan- or N,N'.N ",N"'-tetraacetyl chitotetratose (CH4)-induced Ca(2+) signals were inhibited by both the Ca(2+) chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) and by the Ca(2+)-channel inhibitor La(3+). Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca(2+) curve following beta-glucan treatment. Bacterial cyclic beta-glucans known to elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses

Induction of H₂O₂ synthesis by β-glucan elicitors in soybean is independent of cytosolic calcium transients

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca2+ concentration in β-glucan elicitors-induced defence responses, such as H2O2 and phytoalexin production. The intracellular Ca2+ concentration was monitored in transgenic cells expressing the Ca2+-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca2+ concentration is not necessarily involved in the induction of H2O2 generation: (i) a Bradyrhizobium japonicum cyclic β-glucan induced the H2O2 burst without increasing the cytosolic Ca2+ concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja β-glucan elicitor-induced H2O2 synthesis but did prevent a cytosolic Ca2+ concentration increase. Moreover, A9C and NPPB inhibited P. sojae β-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae β-glucan-induced transient cytosolic Ca2+ increase is not necessary for the elicitation of H2O2 production but is very likely required for phytoalexin synthesi