Contamination of Chitin Oligosaccharides in a Laminarioligosaccharide Preparation Can Cause a Confused Interpretation of Its Elicitor Activity

Abstract

Chitinase treatment of a commercial laminarioligosaccharide preparation from a mushroom resulted in a loss of previously reported elicitor activity in rice cells, indicating that the activity was attributable not to the laminarioligosaccharide but rather to the contaminating chitin fragments. This suggests that the elicitor activity of laminarioligosacchraides from such sources containing chitinaceous substances should be carefully interpreted.Key words: microbe-associated molecular pattern (MAMP); -glucan; chitin; elicitor; rice Branched -glucans are a typical component of the cell walls of various fungi and oomycetes, and hence are recognized as a representative MAMP (microbeassociated molecular pattern) for these microbes.1,2) In fact, their fragments, -glucan oligosaccharides, have been found to act as potent elicitors in various plant species. A hepta--glucoside from Phytophthora megasperma f. sp. glycinea and a tetraglucosylglucitol from the rice blast fungus Pyricularia oryzae are two well characterized examples of elicitor-active -glucan oligosaccharides, acting specifically on certain legumes and rice, respectively.3,4) Both of these oligosaccharides are branched at the O-3 or the O-6 positions of glucosyl residues in their 1,6-linked or 1,3-linked backbone chains and the positions of branching are critical to their biological activities. On the other hand, several papers have reported that linear laminarioligosaccharides also serve as an elicitor in some plant cells, including rice. 5,6)As we did not find any elicitor activity for linear laminarioligosaccharides in rice, 4) we tried to clarify the reason for the discrepancy, and found that contamination by minute amounts of chitin oligosaccharides in some commercial laminarioligosaccharides prepared from a mushroom caused the difference, at least in one case. Figure 1 shows the elicitor activity of two commercial products of laminaripentaose prepared from pachyman, a 1,3-linked -glucan from the cell walls of a mushroom, or curdlan, also a 1,3-linked -glucan from a bacterium, on suspension-cultured rice cells. In both the generation of reactive oxygen species (ROS; Fig. 1, top) and the induction of a defense gene (Fig. 1, bottom), OsKSL4, encoding an enzyme involved in the biosynthesis of phytoalexin, 7,8) only the laminaripentaose preparation from pachyman showed extensive elicitor activity comparable to that of N-acetylchitooctaose (GN8). 9)Considering the reason for this difference in elicitor activity between these two products, we noticed that the one prepared from pachyman might contain a small amount of chitin oligosaccharides, because chitin is a typical cell wall component of fungal cell walls including mushrooms, whereas bacterial cell walls do not contain chitin. To examine this possibility, laminaripentaose from pachyman was treated with rice chitinase expressed in yeast 10) and purified by affinity chromatography, and analyzed for elicitor activity. As shown in Fig. 2, the elicitor activity shown by the limanaripentaose preparation disappea...