Abstract
We describe a method which allows the visualization of elicitor-binding loci at the surface of plant protoplasts. Prerequisites for this method are the preparation of protoplasts under conditions of minimal proteolysis, and the availability of antibodies against either the elicitor itself or against the fluorescein portion of elicitor-fluorescein conjugates. Silver enhancement is used to amplify the visibility of 5-nm gold particles which are attached to an appropriate secondary antibody. Bound elicitor can then be visualized by epipolarization microscopy. This method, designated SEIG-EPOM (for silver enhanced immunogold as viewed by epipolarization microscopy), has been applied to protoplasts of parsley (Petroselineum cirspum L.), using the Phytophthora megasperma elicitor, and soybean (Glycine max Merr.) using polygalacturonic acid elicitor isolated from citrus pectin. We have been able to estimate the number of specific binding loci as being less than 100 per protoplast. Such loci possibly represent clusters of individual elicitor-receptor complexes. Structurally related elicitors have been shown to compete effectively for binding sites. The latter are sensitive to proteolysis, as is the elicitation response of protoplasts.
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Abbreviations
- BSA:
-
bovine serum albumin
- IgG:
-
immunoglobulin G;
- PEP-13(15):
-
13(15) amino-acid-containing oligopeptides of Pmg-elicitor
- PGA:
-
polygalacturonic acid
- PM:
-
plasma membrane
- Pmg:
-
Phytophthora megasperma f.sp. glycinea
- SEIG-EPOM:
-
silver enhanced immunogold as viewed by epipolarization microscopy
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Dedicated to Professor Peter Sitte on the occasion of his 65th birthday
We thank Professor Elmar Weiler (Lehrstuhl Pflanzenphysiologie, Universität Bochum, Germany) for sending us the monoclonals against bean leaf plasma membrane. We also are indebted to Dr. A. Hille-Rehfeld (Institut für Biochemie und Molekulare Zellbiologie, Universität Göttingen, Germany) for the Confocal laser scan micrographs. We also thank Renate Hagemann and Heike Freundt for typing the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to D.G.R. and D.S.) and the National Science Foundation (to P.S.L.). W. Diekmann was a recipient of a stipend from the Hanns-Seidel-Stiftung (München, Germany).
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Diekmann, W., Herkt, B., Low, P.S. et al. Visualization of elicitor-binding loci at the plant cell surface. Planta 195, 126–137 (1994). https://doi.org/10.1007/BF00206300
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DOI: https://doi.org/10.1007/BF00206300