Endocytosis and endosomal regulation of the S-receptor kinase during the self-incompatibility response in Brassica oleracea

Abstract

Intracellular trafficking of plant receptor kinases (PRKs) is a key step in regulation of cellular signaling. Our current knowledge in this field is based on systems that address signaling pathways affecting the whole cell. There are, however, signaling phenomena that add a further layer of complexity. In the Brassica self-incompatibility response, a single cell can adequately respond to two opposite stimuli: accepting cross-pollen and rejecting self-pollen simultaneously. To understand how PRK signaling can influence the coexistence of two seemingly exclusive states of the cell, we investigated the subcellular localization and internalization of the S-receptor kinase (SRK) involved in the self-incompatibility response of Brassica oleracea. Here, we describe the unusual subcellular distribution of SRK3, which localizes predominantly to intracellular compartments and to a much lesser extent to the plasma membrane. Using an anti-SRK antibody that fully substitutes for the natural ligand, we demonstrate that the interaction with the receptor takes place at the plasma membrane and is followed by SRK internalization in endosomes that are enriched in the SRK negative regulator Thioredoxin-h-like1.

Figure 1.

SRK₃ Localizes to PM and Sorting Endosomes in B. oleracea Stigmas. (A) Signal recognition by anti-SRK₃-N-ter in S₃-papilla cells. Inset presents the bright-field image. Bar = 50 μm. (B) Signal recognition by anti-SRK₃-N-ter in S₂₉-papilla cells. No specific signal can be detected. Inset presents the bright-field image. Bar = 50 μm. (C) Higher magnification of a S₃-stigma papilla cell shows fluorescent signals in the periphery of the cell and intracellular compartments. The experiment was reproduced five times. Bar = 10 μm. (D) to (H) Immunolocalization on electron microscopy sections. Bars = 100 nm. (D) SRK₃ signal at the PM (arrows) and in the cell wall (arrowhead). Signals visible in the cell wall probably correspond to the soluble splice variant eSRK₃. (E) SRK₃ signal in the multivesicular bodies (arrow). (F) SRK₃ signal in the ER (arrows). (G) SRK₃ signal in the Golgi apparatus (arrow). (H) SRK₃ signal in different small vesicles in the vicinity of the Golgi apparatus (arrows). cw, cell wall; cyt, cytoplasm; er, endoplasmic reticulum; Ga, Golgi apparatus; pm, plasma membrane; mvb, multivesicular body.

Figure 2.

SRK₃ Colocalizes with Sorting Endosome Markers in Papilla Cells. (A) to (C) Colocalization between SRK₃ (A) and sorting endosome marker VPS29 (C). Merged image is presented in (B). Images shown are representative of five independent experiments. Bar = 10 μm. (D) to (F) Colocalization between SRK₃ (D) and sorting endosome marker SYP21 (F). Merged image is presented in (E). Images shown are representative of three independent experiments. Bar = 10 μm. (G) to (I) Colocalization between SRK₃ (G) and trans-Golgi network marker SYP61 (I). Merged image is presented in (H). Cases of colocalization are rare and are indicated with arrowheads. Inset represents a case where signals are closely associated but do not colocalize. Images shown are representative of three independent experiments. Bar = 10 μm.

Figure 3.

SRK₃ Undergoes Degradation upon Self-Pollination. (A) Protein gel blot showing stability of full-length SRK₃ between 0 and 120 min following self- or cross-pollination. (B) Quantification of (A). No significant change in signal intensity was observed. The bars represent the means (+sd) of three independent experiments. (C) Degradation of SRK₃ after self-pollination of stigmas treated with CHX (+) or a mock solution (−) between 0 and 120 min . The bars represent the means (+sd) of three independent experiments. The 62.8-kD splice variant eSRK₃ (*) is not sensitive to the CHX treatment, while the 59.4 kD variant (**) and the full-length receptor are sensitive. A typical protein gel blot is shown as an example below the graph. (D) CHX treatment affects SRK₃ specifically upon self-pollination. The bars represent the means (+ sd) of three independent experiments. The 62.8 kD eSRK₃ form (*) is not sensitive to the CHX treatment, while the 59.4-kD eSRK₃ (**) is sensitive irrespective of the pollination type. A typical protein gel blot is shown as an example below the graph. np, nonpollinated; sp, self-pollination; cp, cross-pollination.

Figure 4.

Ligand-Induced Internalization of SRK₃. (A) to (D) Anti-SRK₃-N-ter can functionally substitute for the natural SRK₃ ligand in vivo. B. oleracea S₃- and S₂₉- stigmas were treated with anti-SRK₃-N-ter or mock solution and pollinated with S₁₅-pollen, which is normally compatible for both haplotypes. Pollen germination was visualized under UV light after aniline blue staining. Five independent experiments were performed, yielding similar results. (A) Germination of S₁₅-pollen on S₃-stigma treated with mock solution. Bar = 500 μm. (B) Rejection of S₁₅-pollen on S₃-stigma treated with anti-SRK₃-N-ter solution. Bar = 500 μm. (C) Germination of S₁₅-pollen on S₂₉-stigma treated with mock solution. Bar = 500 μm. (D) Germination of S₁₅-pollen on S₂₉-stigma treated with anti-SRK₃-N-ter solution. No rejection can be observed. Bar = 500 μm. (E) and (F) B. oleracea S₃- and S₂₉-stigmas were treated with anti-SRK₃-N-ter solution and pollinated with S₁₅-pollen (normally compatible for both haplotypes). Antibody internalization was followed by immunofluorescence on fixed sections. The results could be reproduced in three independent experiments. (E) Internalization in S₃-papilla cells. Anti-SRK₃-N-ter is found in intracellular compartments after 2.5 h, detected directly by the secondary anti-mouse antibody. Bar = 10 μm. (F) No internalization is observed in S₂₉-papilla cells. Bar = 10 μm. (G) B. oleracea S₃-stigmas were treated with secondary donkey anti-mouse Alexa555 solution and pollinated with S₁₅-pollen. No internalization is observed in S₂₉-papilla cells after 2.5 h. Bar = 10 μm. (H) to (J) Detection of SRK₃ on S₃ stigma papilla sections. SRK₃ is detected in intracellular compartments and unspecifically on the cell wall (arrow) by anti-SRK₃C-dom antibody. Bright-field image is presented in (J) and the merge image in (I). Similar results were observed in three independent experiments. Bar = 10 μm. (K) to (M) Absence of SRK₃ from S₂₉-stigma papilla sections (control experiment). The unspecific cell wall staining probably corresponds to the bands detected on protein gel blot with the same antibody. Bright-field image is presented in (M) and the merge image in (L). Similar results were observed in three independent experiments Bar = 10 μm. (N) to (P) Simultaneous detection of SRK₃ in S₃-papilla cells using anti-SRK₃-N-ter (recognizing the N terminus of SRK₃) and anti-SRK₃C-dom antibody (recognizing the C terminus of SRK₃). Anti-SRK₃C-dom does not recognize the PM-localized SRK₃, possibly due to an interaction blocking the antibody epitope and shows a weak unspecific cell wall signal. Similar results were observed in three independent experiments. Bar = 10 μm. (Q) to (S) Colocalization of internalized anti-SRK₃-N-ter (detected directly by the secondary anti-mouse antibody) and SRK₃ (detected by anti-SRK₃C-dom and secondary anti rabbit antibody) in S₃-papilla cells. Similar results were observed in three independent experiments. Bar = 10 μm.

Figure 5.

Internalized SRK₃ Localizes in Sorting Endosomes with THL1. (A) to (C) Colocalization of internalized anti-SRK₃-N-ter (A) and VPS29 (C). Merged picture is shown in (B). Similar results were observed in three independent experiments. Bar = 10 μm. (D) to (F) THL1 (F) colocalizes partially with SRK₃ (D) in endosomes, but not at the PM. Merged picture is shown in (E). Arrows in the inserts indicate the PM-localized SRK₃. Similar results were observed in six independent experiments. Bar = 10 μm in the panels and 5 μm in the insets. (G) to (I) Colocalization of internalized anti-SRK₃-N-ter (G) and THL1 (I). Merged picture is presented in (H). Similar results were observed in three independent experiments Bar = 10 μm.