Affinity cross-linking of the plasma membrane fraction to an 125 I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization ϭ 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nm. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 m. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).

Binding experiments as well as affinity labeling with an (125)I-labeled 2-(4-aminophenyl)ethylamino derivative of N-acetylchitooctaose revealed the presence of high-affinity binding sites/proteins for N-acetylchitooligosaccharide elicitor in the plasma membrane preparation from suspension-cultured carrot cells, barley cells and wheat leaves. Their binding specificity corresponded with the elicitor activity of N-acetylchitooligosaccharides and related sugars in these plant cells/tissues, and was similar to that reported for the binding site/protein previously reported for suspension-cultured rice cells. The molecular size of the binding proteins identified in carrot, barley and wheat was slightly smaller than that of rice. These plant cells were shown to respond to N-acetylchitooligosaccharides and generate reactive oxygen species, induced medium alkalinization, or previously shown to initiate lignification (wheat leaves, Barber et al. (1989) Physiol. Mol. Plant Pathol. 34: 3). No elicitor-binding protein nor the elicitor-induced cellular responses was detected for a cell line of tobacco BY-2 (BY-2T). On the other hand, another cell line of tobacco BY-2 (BY-2N) showed the presence of elicitor-binding protein and also elicitor-induced medium alkalinization. Thus, there was a good correlation between the existence of high-affinity binding proteins for the elicitor and elicitor-induced cellular responses among tested plant cells. These results indicated the wide distribution of N-acetylchitooligosaccharide elicitor-binding protein among various plants and added further support for the function of these plasma membrane proteins in the perception of the elicitor signal.

The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.

O 2 signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative 1 O 2 at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the 1 O 2 signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change. Conclusions:The results of this study suggested that the monitoring of 1 O 2 enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.

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