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An Immunocytochemical Procedure for Protein Localization in Various Nematode Life Stages Combined with Plant Tissues Using Methylacrylate-Embedded Specimens

October 2012 , Volume 102 , Number  10
Pages  990 - 996

Paulo Vieira, Mohamed Youssef Banora, Philippe Castagnone-Sereno, Marie-Noëlle Rosso, Gilbert Engler, and Janice de Almeida Engler

Institut National de la Recherche Agronomique, UMR 1355 ISA; Centre National de la Recherche Scientifique, UMR 7254 ISA; Université de Nice-Sophia Antipolis, UMR ISA; 400 Route des Chappes, Sophia-Antipolis, France.

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Accepted for publication 7 June 2012.

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.

© 2012 The American Phytopathological Society