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Published on January 9, 2008; 10.1104/pp.107.111716

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Received October 25, 2007
Accepted December 21, 2007

The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with ROS production in BY-2 tobacco cells

Nathalie Leborgne-Castel *, Jeannine Lherminier , Christophe Der , Jerôme Fromentin , Valerie Houot , and Francoise Simon-Plas

UMR Plante-Microbe-Environnement Universite de Bourgogne / 1088 INRA / 5184 CNRS, INRA, BP 86510, 21065 DIJON Cedex, France

* Corresponding author; email: nathalie.leborgne.castel{at}dijon.inra.fr.

The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few min after addition of cryptogein to tobacco BY-2 cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein since a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis.

In order to define the nature of the stimulated endocytosis, we quantified clathrin-coated pits (CCPs) forming on the plasma membrane of BY-2 cells. A transitory stimulation of this morphological event by cryptogein occurs within the first fifteen min. In presence of cryptogein, increases in both FM4-64 internalization and clathrin-mediated endocytosis are specifically blocked upon treatment with 5 µM tyrphostin A23, a "Receptor-Mediated Endocytosis" inhibitor. The kinetics of the transient increase in CCPs at the plasma membrane coincides with that of transitory ROS production occurring within the first fifteen min after elicitation. Moreover, in BY-2 cells expressing NtrbohD antisense cDNA, which are unable to produce reactive oxygen species (ROS) when treated with cryptogein, the CCP stimulation is inhibited. These results indicate that the very early endocytic process induced by cryptogein in tobacco is due, at least partly, to clathrin-mediated endocytosis and is dependent on ROS production by the NADPH oxidase NtrbohD.




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