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PLANTS INTERACTING WITH OTHER ORGANISMS

The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells^1,[C]

UMR INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, F–21000 Dijon, France

The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) 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 minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. 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 15 min. In the 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 reactive oxygen species (ROS) production occurring within the first 15 min after elicitation. Moreover, in BY-2 cells expressing NtrbohD antisense cDNA, which are unable to produce 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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Nathalie Leborgne-Castel (nathalie.leborgne-castel{at}dijon.inra.fr).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.111716

^* Corresponding author; e-mail nathalie.leborgne-castel{at}dijon.inra.fr.

Received October 25, 2007; accepted December 21, 2007; published January 9, 2008.

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