1.3.9
PURIFICATION OF A PUTATIVE RECEPTOR MOLECULE FOR OLIGOCHITIN ELICITOR FROM PLASMA MEMBRANE OF SUSPENSION-CULTURED RICE CELLS

H KAKU and N SHIBUYA

National Institute for Agrobiological Resources, Tsukuba, Japan

Background and objectives
N-acetylchito-oligosaccharides (DP 6-8, GN6-8), fragments of a backbone glycan of fungal cell walls, can induce the production of the antimicrobial agent phytoalexin in suspension cultured rice cells. A high-affinity binding site for this elicitor was detected in the plasma membrane of rice cells [1] and a corresponding binding protein was identified by affinity labelling [2]. In the present study, we report the purification of this elicitor-binding protein (EBP), an active form from the plasma membrane, by the use of affinity chromatography on elicitor-active sugar as ligand, and preparation/partial purification of an antibody against this protein.

Materials and methods
The conjugate of tyramine with N-acetylchito-octaose, (GlcNAc)8-Tyr, was synthesized and radio-iodinated. The plasma membrane of suspension-cultured rice (Oryza sativa L. cv. Nipponbare) cells were prepared by aqueous two-phase partitioning with the use of dextran and polyethylene glycol. The plasma membrane fraction was incubated with 0.5% Triton X-100 for 1 h at 4C. After ultracentrifugation (200,000 g, 1 h), the solubilized protein preparation was applied to an oligochitin-Sepharose column. The column was washed with buffer and successively with several elicitor-inactive sugar solutions, then the bound fraction was eluted with elicitor-active (GN8) solution or with glycine-HCl buffer (pH 2.3). As the EBP was found to bind to Con-A Sepharose (Y. Ito et al., unpublished), the Con A-bound PM proteins were used to raise an antiserum against this binding protein. The antibody against EBP was enriched with several affinity columns.

Results and conclusions
The detergent-solubilized fraction of plasma membrane was applied to a (GlcNAc)7-Lys-Sepharose column. The bound fraction eluted with elicitor-active (GN8) solution showed the specific binding activity to 125I-labelled GN8 derivative. SDS-PAGE showed the presence of a single protein band whose size corresponded to the 75-kDa plasma membrane protein detected by affinity labelling [2]. However, the binding efficiency of EBP for this column was quite low. A survey of more efficient ligands for affinity chromatography to increase the recovery of EBP is in progress.

Two-dimensional PAGE of the Con A-bound fraction, followed by Western blotting with partially purified antibody, detected four major and two minor spots. Among these, three major spots were identical to those detected by the affinity labelling. This antibody would be useful for the cloning of cDNA encoding the EBP.

References
1. Shibuya N, Ebisu N, Kamada Y et al., 1996. Plant Cell Physiology 37, 894-898.
2. Ito Y, Kaku H, Shibuya N, 1997. Plant Journal 12, 347-356.