Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

被引:121
作者
Ito, Y [1 ]
Kaku, H [1 ]
Shibuya, N [1 ]
机构
[1] NATL INST AGROBIOL RESOURCES,DEPT BIOTECHNOL,TSUKUBA,IBARAKI 305,JAPAN
关键词
D O I
10.1046/j.1365-313X.1997.12020347.x
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identi fied by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [I-125]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([I-125]-GN(8)-AzPEA) and a [I-125]-labeled 2-(4-aminophenyl)ethylamino conjugate ([(125)]-GN(8)-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size dependent manner, and N-acetylchito-octaose (GlcNAc)(8) showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)(8), cellopentaose nor alpha-1,4 linked N-acetylgalactosamine octamer (GalNAc)(8) at concentrations as high as 25 mu M inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the 'high-affinity binding site' previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.
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页码:347 / 356
页数:10
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共 32 条
[11]  
Dixon R A, 1990, Adv Genet, V28, P165
[12]   SOLUBILIZATION OF PLANT MEMBRANE-PROTEINS FOR ANALYSIS BY TWO-DIMENSIONAL GEL-ELECTROPHORESIS [J].
HURKMAN, WJ ;
TANAKA, CK .
PLANT PHYSIOLOGY, 1986, 81 (03) :802-806
[13]  
KoomanGersmann M, 1996, PLANT CELL, V8, P929, DOI 10.1105/tpc.8.5.929
[14]   EPR EVIDENCE FOR GENERATION OF HYDROXYL RADICAL TRIGGERED BY N-ACETYLCHITOOLIGOSACCHARIDE ELICITOR AND A PROTEIN PHOSPHATASE INHIBITOR IN SUSPENSION-CULTURED RICE CELLS [J].
KUCHITSU, K ;
KOSAKA, H ;
SHIGA, T ;
SHIBUYA, N .
PROTOPLASMA, 1995, 188 (1-2) :138-142
[15]   N-ACETYLCHITOOLIGOSACCHARIDES, BIOTIC ELICITOR FOR PHYTOALEXIN PRODUCTION, INDUCE TRANSIENT MEMBRANE DEPOLARIZATION IN SUSPENSION-CULTURED RICE CELLS [J].
KUCHITSU, K ;
KIKUYAMA, M ;
SHIBUYA, N .
PROTOPLASMA, 1993, 174 (1-2) :79-81
[16]  
KUCHITSU K, 1997, IN PRESS PLANT CELL
[17]   CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].
LAEMMLI, UK .
NATURE, 1970, 227 (5259) :680-+
[18]   Two novel genes rapidly and transiently activated in suspension-cultured rice cells by treatment with N-acetylchitoheptaose, a biotic elicitor for phytoalexin production [J].
Minami, E ;
Kuchitsu, K ;
He, DY ;
Kouchi, H ;
Midoh, N ;
Ohtsuki, Y ;
Shibuya, N .
PLANT AND CELL PHYSIOLOGY, 1996, 37 (04) :563-567
[19]   One-step purification of the beta-glucan elicitor-binding protein from soybean (Glycine max L) roots and characterization of an anti-peptide antiserum [J].
Mithofer, A ;
Lottspeich, F ;
Ebel, J .
FEBS LETTERS, 1996, 381 (03) :203-207
[20]   Involvement of jasmonic acid in elicitor-induced phytoalexin production in suspension-cultured rice cells [J].
Nojiri, H ;
Sugimori, M ;
Yamane, H ;
Nishimura, Y ;
Yamada, A ;
Shibuya, N ;
Kodama, O ;
Murofushi, N ;
Omori, T .
PLANT PHYSIOLOGY, 1996, 110 (02) :387-392