The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in bright yellow-2 tobacco cells

Abstract

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 microm 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.

Figure 1.

Cryptogein modifies spectral properties of FM4-64 on plasma membrane. A, Confocal sections of 7-d-old BY-2 tobacco cells labeled with FM4-64 (4.25 μm) for 5 min (top; 0 min), then incubated in the presence or absence of cryptogein for 5 min (middle; 5 min) or 15 min (bottom; 15 min) under constant agitation at 23°C in the dark. Left, Control cells; right, cells plus 50 nm cryptogein. All image acquisition parameters were kept constant for imaging control and cryptogein-treated cells. Bars = 20 μm. B, Measurements of FM4-64 fluorescence at the plasma membrane level using the Image J software. Data are mean values and sd from eight independent experiments.

Figure 2.

Increasing FM4-64 internalization within BY-2 cells in the presence of cryptogein. Seven-day-old tobacco BY-2 cells were labeled with FM4-64 (4.25 μm) for 5 min (time 0 min) at 23°C or treated in the presence or absence of cryptogein (50 nm). Cells were kept under constant agitation in the dark. A, Measurements of FM4-64 fluorescence of vesicles in control cells (continuous line), or subsequent to cryptogein treatment (dashed line). Data are mean values and sd from four independent experiments. B, Quantification of control and cryptogein-treated cells showing endocytosis during time. Data are presented as percentage of cells (n approximately 30–50) corresponding to class 0, no endocytosis (white columns); class 1 to 10, 1 to 10 vesicles (dashed columns); class >10, more than 10 vesicles (black columns). Data are mean values and sd from eight independent experiments. Inset, Images of BY-2 untreated (control) or cryptogein-treated cells after 5 min (corresponding to 10 min FM4-64 loading) are presented. Bars = 20 μm. [See online article for color version of this figure.]

Figure 3.

LTP1 from wheat does not modulate endocytosis. Effect of 500 nm LTP from wheat (LTP1; dashed columns) on the percentage of cells containing more than 10 vesicles. The kinetics of FM4-64 internalization in cells exposed to LTP is comparable to control cells (white columns), whereas cryptogein-treated cells show stimulated endocytosis (black columns). Mean and sd values are from three independent experiments.

Figure 4.

Cryptogein stimulates CCP formation at the plasma membrane. Plasma membrane of BY-2 cells was imaged by TEM. CCPs (arrows) were observed and quantified on 20 to 30 cell sections per 100-μm plasma membrane perimeter. A to E, Micrographs of different stages of CCP invagination before scission. Coated pits (arrows, A, B, C, E), coated vesicles (arrowheads, D). G, Golgi apparatus. Bars = 100 nm. F, Percentage of cell sections in the three classes of CCPs corresponding to no CCP (class 0; white portion), one CCP (class 1; dashed portion), or two or more CCPs (class 2; black portion) in control cells and at 10 and 20 min of cryptogein treatment. G, Relative increase of CCPs during the first 20 min in cells without or with 50 nm cryptogein treatment compared to control cells (ctl, 0 min).

Figure 5.

Tyrphostin A23, but not tyrphostin A51, prevents endocytosis in cryptogein-elicited BY-2 cells (A and B). Quantification of CCPs per 100-μm plasma membrane of 7-d-old BY-2 cells imaged by TEM. A, Relative increase of CCPs in elicited cells was compared in the presence and absence of tyrphostin A23. B, Relative increase of CCPs in elicited cells was compared in the presence and absence of tyrphostin A51. C, Seven-day-old BY-2 cells were labeled with FM4-64 (4.25 μm) for 5 min (0 min), then incubated with 0.1% DMSO (white columns), 0.01% DMSO plus 50 nm cryptogein (black columns), 5 μm tyrphostin A23 plus 50 nm cryptogein (spotted columns), or 5 μm tyrphostin A51 plus 50 nm cryptogein (dashed columns) under constant agitation at 23°C in the dark. BY-2 cells (n approximately 30–50) presenting full endocytosis (>10 vesicles) were quantified as described in Figure 2B. D, Extracellular ΔpH was measured in cells challenged with 50 nm cryptogein for 30 min in presence of 0.1% DMSO (control, black columns), 5 μm tyrphostin A23 (spotted columns), or 5 μm tyrphostin A51 (dashed columns). All treated cells exhibited extracellular alkalinization and no significant difference was observed. E, Effect of 5 μm tyrphostins on cryptogein-induced extracellular ROS production during the first 30 min after elicitor treatment. Total ROS production measured during 30 min of treatment by cryptogein was summed and expressed in percentage of ROS production in cells treated with cryptogein plus DMSO. Cells treated with cryptogein plus DMSO (black columns), cryptogein plus 5 μm tyrphostin A23 (spotted columns), or cryptogein plus 5 μm tyrphostin A51 (dashed columns).

Figure 6.

Cryptogein-stimulated endocytosis does not occur in cells inhibiting ROS production. The morphology of gp3 cells (antisense line of NADPH oxidase NtrbohD), which no longer produce ROS, was imaged by TEM. CCPs (arrows) were observed and quantified. A, Micrographs of three representative cell sections showing flat pits delimited at the intracellular face by a clathrin electron-dense coating (arrows). Bars = 100 nm. B, Relative increase of CCPs in gp3 cells in the absence or presence of 50 nm cryptogein; cell sections presenting no CCP (class 0), one CCP (class 1), or two or more CCP (class 2) within a 100-μm membrane perimeter (n = 20–30). C, Distribution of CCPs according to their shape in elicited (cry) or nonelicited (ctl) wild-type and gp3 cells after 15 min. Three classes of CCP were defined as a function of their diameter: flat (90–120 nm), curved (70–90 nm), and invaginated (40–70 nm). n = 20 to 30 cell sections.