Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis

Abstract

Pattern-recognition receptors (PRRs) trigger innate immune responses in animals and plants. One such PRR is the flagellin receptor FLS2 in Arabidopsis. Here, we demonstrate that a functional fusion of FLS2 to the green fluorescent protein (GFP) resides in cell membranes of most tissues. Stimulation with the flagellin epitope flg22 induces its transfer into intracellular mobile vesicles, followed by degradation. FLS2 internalization depends on cytoskeleton and proteasome functions, and receptor activation. A variant FLS2 mutated in Thr 867, a potential phosphorylation site, binds flg22 normally, but is impaired in flg22 responses and FLS2 endocytosis. We propose that plant cells regulate pathogen-associated molecular pattern (PAMP)-mediated PRR activities by subcellular compartmentalization.

Figure 1.

FLS2 expression analysis revealed by confocal fluorescence microscopy of Ws-0/FLS2-GFP lines. Micrographs show optical cross-sections (A–D,G) or merged cross-sections (E,F) of FLS2 cell membrane localization in different tissues. (A) Leaf epidermal cells (pavement and stomata cells). (B) Leaf mesophyll cells. (C) Stem. (D) Flower petal. (E) Root. (F) Root tip. Background autofluorescence of chloroplasts appears in yellow/red. (G) Retraction of FLS2-GFP-labeled cell membrane from root cell walls upon addition of 1 M sorbitol. Bars: A–D, 20 μM; E,F, 100 μM; G, 10 μM.

Figure 2.

Dynamics of FLS2 localization in response to flg22. (A) Detached leaves of Ws-0/FLS2-GFP seedlings were incubated in an aqueous solution supplied with 10 μM flg22. Fluorescence micrographs show GFP signals of optical cross-sections in epidermal cells (top) and mesophyll cells (bottom) at 30 min. Background autofluorescence of stomata inner sites stays green; background autofluorescence of chloroplasts appears in yellow/red. Leaves of Ws-0/FLS2-GFP were either incubated continuously in 10 μM flg22 (B) or pulse-incubated for 20 min, intensively rinsed, and further incubated in water or cyclohexamide (CHX) (C). Fluorescence micrographs show GFP signals at the indicated time points (depicted are cross-sections of epidermal cells). Bars, 10 μm.

Figure 3.

Specificity of FLS2 internalization. Fluorescence micrographs show optical cross-sections of GFP signals in epidermal cells of Ws-0/FLS2-GFP-derived detached leaves at 30 min of incubation with the elicitor active peptides flg22 and elf26, the inactive variants flg22^A.tum and flg22Δ2, and a cotreatment with flg22 and flg22Δ2 (all at 10 μM). Bar, 10 μm. Background autofluorescence of chloroplasts appears in yellow.

Figure 4.

Regulation of FLS2 internalization. Representative fluorescence micrographs of FLS2-GFP localization in epidermal cells (cross-sections) 30 min pretreated with the indicated inhibitors, or cotreated with K-252a shown 30 min after the addition of 10 μM flg22 in 1% DMSO. Bar, 10 μm. Arrows indicate movement of fluorescent vesicles in a time lapse of 3.5 sec. Bar, 5 μm. Background autofluorescence of chloroplasts appears in yellow/red.

Figure 5.

(A) Photographs presenting seedling growth of Ws-0 plants and lines expressing FLS2 and FLS2^T867V in the absence or presence of 0.1 μM flg22. Each two independent lines are represented. (B) Dose dependence of flg22-induced seedling growth inhibition in Ws-0 plants and lines expressing FLS2 and FLS2^T867V; n = 6 seedlings of each representative line. (C) FLS2-mediated disease resistance. Ws-0 plants and lines expressing FLS2 and FLS2^T867V were spray-inoculated with 5 × 10⁸ CFU/mL bacteria of Pseudomonas syringae pv. tomato DC3000. Photographs were taken 4 d post-inoculation and each shows a representative line. Disease occurs visible by leaf yellowing. (D) Subcellular localization of FLS2^T867V-GFP expressed in Ws-0. Confocal microscopy revealed a fluorescence signal at the cell membrane that stayed mostly unchanged in the presence of 10 μM flg22 (cross-section of epidermal cells). Bars, 10 μM.