1.2.9
OLIGOGLUCAN-ELICITOR SIGNALLING IN RICE: CHARACTERIZATION OF THE ELICITOR MOLECULE AND CELLULAR RESPONSES

N SHIBUYA, T YAMAGUCHI and E MINAMI

National Institute for Agrobiological Resources, Tsukuba, Japan

Background and objectives
Several oligosaccharides derived from the cell surface polymers of pathogenic fungi have been known to act as potent elicitor signals for plants. We have shown that fragments of chitin, N-acetylchito-oligosaccharides, induce various defence responses in suspension-cultured rice cells, and have been analysing the molecular machinery involved in the perception and transduction of this elicitor signal [1, 2]. Little is known about the elicitor-active gluco-oligosaccharides (oligoglucans) which act on monocot plants. We show here that a specific fragment of b-glucan obtained from rice blast disease fungus, Pyricularia oryzae, acts as a potent elicitor signal in rice cells. Characterization of this elicitor-active glucan fragment, cellular responses induced by the oligosaccharide, and the differences in the recognition of oligoglucan elicitors between rice and soybean are the subjects of this paper.

Materials and methods
A b-glucan preparation from P. oryzae was digested with a highly purified endo-b-1,3-glucanase. The fragments obtained were subjected to gel filtration and reversed-phase HPLC. The structure of the purified oligosaccharides was analysed by methylation, acetolysis and mass spectrometry. Phytoalexin was analysed by GC and GC/MS. Induction of phytoalexin biosynthesis was examined by adding the elicitor to suspension-cultured rice cells (var. BL-1). Reactive oxygen generation was analysed by the luminol method. Expression of elicitor-responsive genes was analysed by Northern blotting.

Results and conclusions
A highly elicitor-active glucopentaose was isolated from the enzymatic digest of a b-glucan from P. oryzae. The oligosaccharide could induce phytoalexin (momilactone A) biosynthesis in rice cells at 10 nM. Structural analysis of the glucopentaose revealed the following structure; Glcb1, 3Glcb1, 3[Glcb1,6]Glcb1, 3Glc. The structure contrasts with that of the elicitor-active glucan fragment for soybean, hepta-b-glucoside from the cell walls of Phytophthora megasperma, in the linkage of backbone as well as the position of the branching. In fact, the hepta-b-glucoside was not an active elicitor for rice cells, indicating a significant difference between the recognition of oligoglucan elicitors in rice and soybean. We reported that the oligochitin elicitor induces, in addition to phytoalexin biosynthesis, various cellular responses including reactive oxygen generation in the rice cells. However, the ability of oligoglucan elicitor to induce reactive oxygen generation is negligibly low even at a concentration syfficient to induce phytoalexin biosynthesis, suggesting differences in the signal transduction cascade downstream of the putative receptors for these two oligosaccharide signals. The expression patterns of various elicitor-responsive genes by these two elicitors are now being examined.

References
1. Stacey G et al., eds, 1996. Biology of Plant-Microbe Interactions, pp. 83-88.
2. 1997. Plant Journal 12, 347-356.