1.9.12
CHARACTERIZATION AND GENE CLONING OF THE NOVEL MANNOSE-BINDING RICE LECTIN

K HIRANO, T TERAOKA, H TAKAHASHI and D HOSOKAWA

Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

Background and objectives
Through our studies on the interaction between rice and its pathogens Xanthomonas campestris pv. oryzae and Magnaporthe grisea, we found a novel rice lectin in the seedlings which has the same sugar specificity to mannosyl and glucosyl residue as concanavalin A (ConA) [1]. The purified rice lectin (MRL) agglutinated the intact cells of X. campestris pv. oryzae and also the spores and protoplasts of M. grisea. Now we focus on the hypothesis that MRL may function as plant antibodies or receptors in host-parasite interaction. Some reports [2, 3] have been presented to support our hypothesis. One of the essential approaches to verify our hypothesis and clarify the role of MRL in situ is to characterize the properties of MRL and clone the gene.

Materials and methods
MRL in rice seedlings (cv. Aichiasahi) was extracted with 0.05 N HCl containing 1% (w/v) ascorbic acid, precipitated with 10-50% (NH4)2SO4, fractionated by Q-Sepharose ion exchange chromatography, and finally purified by Sephadex G-50 affinity chromatography. In analysis of MRL, two-dimensional PAGE (firstly isoelectric focusing with immobiline, secondly SDS-PAGE) was applied with the Multiphor II system. In the Western blot analysis, MRL was detected by a monoclonal antibody (No. 9C5) against MRL. The amino acid and DNA sequence of MRL were determined by ABI 477A, 377 sequencer, respectively.

Results and conclusions
MRL finally purified by Sephadex G-50 was not homogeneous in two-dimensional PAGE; some spots were detected at pI4.30, pI4.44, pI4.56, pI4.66, pI4.74 and pI4.85 in the first direction, whereas all were located at MW of about 45,000 in the second direction. In some cases, regardless of protein concentration, only the major spots at pI4.66, pI4.74 and pI4.85 were detected. In other cases, faint spots at pI4.44, pI4.56, pI4.66 and pI4.74 were observed just below major spots at MW of 38,000-40,000. In Western blot analysis, all detected spots reacted with the monoclonal antibody against MRL. Major spots at pI4.56, pI4.66, pI4.74 and pI4.85 showed hemagglutination activity when recovered from the gel. The N-terminal amino acid sequences of both spots at pI4.85 and pI4.74 were TLVKIGPWGGNGGSAQDISV, which shows high homology to rice root-specific protein induced by salt and drought stress [4]. The homology is also conserved at the DNA level. These results suggested that MRL may consist of isolectins or related proteins which retain a common epitope and may belong to a family of stress-induced proteins.

References
1. Teraoka T, Sakakibara T, Den E et al., 1990. Agricultural and Biological Chemistry 54, 3053-3056.
2. Kano H, Toyoki T, Haga M, Sekizawa T, 1988. Agricultural and Biological Chemistry 52, 3163-3164.
3. Valent B, Hamer JE, Howard RJ, Chumley G, 1987. Science 239, 288-240.
4. Claes B, Dekeyser R, Villarroel R et al., 1990. Plant Cell 2, 19-27.