Effects of Ser-938 phosphorylation on the spatiotemporal dynamics of FLS2 at the plasma membrane.

(A) VA-TIRFM images of a FLS2-expressing hypocotyl cell was analyzed. The 5-day-old transgenic Arabidopsis plant cells were observed under VA-TIRFM. The red balls indicate the positions of the identified points that appeared. Trajectories represent the track length of the identified points. Bar = 10μm. (B) Time-lapse images of FLS, FLS2S938A, and FLS2S938D. Bar = 2 μm. The fluorescence intensity changes among different 3D luminance plots. (C) The trajectories of representative individual FLS2, FLS2S938A and FLS2S938D under 30 min for 10 μM flg22 processing. (D) Diffusion coefficients of FLS (control, n = 42 spots; flg22, n = 24 spots), FLS2S938A (control, n = 44 spots; flg22, n = 25 spots) and FLS2S938D (control, n = 27 spots; flg22, n = 23 spots) under different environments. Statistical significance was assessed using the Student’s t-test (***p < 0.001). Error bars represent the SD. (E) Frequency of long- and short-range motions for FLS, FLS2S938A, and FLS2S938D under different environments. Statistical significance assessed with the Student’s t-test (***p < 0.001). Error bars represent the SD. (F) UMAP visualization of FLS2S938D samples under different conditions (control, n = 32 spots; flg22, n = 21 spots). Dots represent the individual images, and are colored according to the reaction conditions. (G) Fluorescence recovery curves of the photobleached areas with or without the flg22 treatment. Three biological replicates were performed. (H) The single-molecule trajectories of FLS2 analyzed by Imaris could be faithfully tracked for 4 s under control and flg22 treatments. (I) Dwell times were analyzed for FLS2 (control, n = 18 spots; flg22, n = 16 spots), FLS2S938A (control, n = 19 spots; flg22, n = 24 spots), and FLS2S938D (control, n = 11 spots; flg22, n = 14 spots) under the control and flg22 treatments. Statistical significance assessed with Student’s t-test (*p < 0.05; **p < 0.01). Error bars represent the SD.

Different Ser-938 phosphorylation states of FLS2 affect its partitioning into AtRem1.3-associated microdomains.

(A) FLIM-FRET was used to detect the co-expression of FLS2/FLS2S938A/FLS2S938D-GFP and BAK1-mCherry in the N. benthamiana epidermal cells stimulated by 1/2 MS or flg22 (10 μM) for 30 min. Average fluorescence lifetime (t) and the FRET efficiency were analyzed for FLS2, FLS2S938A or FLS2S938D and BAK1. Statistical significance was assessed with Student’s t-test (***p < 0.001). Error bars represent the SD. (B) Pearson correlation coefficient values of co-localization between FLS2 (control, n = 24 images; flg22 = 15 images), FLS2S938A (control, n = 18 images; flg22 = 22 images), or FLS2S938D (control, n = 15 images; flg22 = 20 images) and BAK1 upon stimulation with control or flg22. Statistical significance was assessed with the Student’s t-test (*p < 0.05). Error bars represent the SD. (C) Mean protein proximity indexes showing FLS2 (control, n = 8 images; flg22 = 5 images), FLS2S938A (control, n = 4 images; flg22=3 images) or FLS2S938D (control, n = 4 images; flg22 = 3 images) and BAK1 degree of proximity under different conditions. Statistical significance was assessed with the Student’s t-test (*p < 0.05; **p < 0.01). Error bars represent the SD. (D) TIRF-SIM images of the Arabidopsis leaf epidermal cells co-expressing FLS2/FLS2S938A/FLS2S938D-GFP and AtRem1.3-mCherry. The white line represents the fluorescence signals. Bar = 5 μm. (E) The histogram shows the co-localization ratio of FLS2-GFP (control, n = 52 images; flg22 = 61 images), FLS2S938A-GFP (control, n = 30 images; flg22 = 36 images) or FLS2S938D-GFP (control, n = 34 images; flg22 = 54 images) and AtRem1.3-mCherry. Statistical significance assessed with the Student’s t-test (*p < 0.05). Error bars represent the SD. (F) FLS2/FLS2S938A/FLS2S938D-GFP and AtRem1.3-mCherry fluorescence signals as shown in D. (G) The fluorescence mean lifetime (T) and the corrected fluorescence resonance efficiency (Rate) of FLS2, FLS2S938D or FLS2S938A with co-expressed AtRem1.3. Statistical significance was assessed using the Student’s t-test (***p < 0.001). Error bars represent the SD. (H) Intensity and lifetime maps of the Arabidopsis leaf epidermal cells co-expressing FLS2/FLS2S938A/FLS2S938D-GFP and AtRem1.3-mCherry as measured by FLIM-FRET. (I) Quantification of co-localization between FLS2, FLS2S938A or FLS2S938D and AtRem1.3 with and without stimulation with ligands. Pearson correlation coefficient values (r) were obtained between FLS2 (control, n = 66 images; flg22 = 53 images) /FLS2S938A (control, n = 42 images; flg22 = 54 images) /FLS2S938D-GFP (control, n = 29 images; flg22 = 38 images) and AtRem1.3-mCherry. Statistical significance assessed with the Student’s t-test (*p < 0.05). Error bars represent the SD.

The Ser-938 phosphorylation site affects flg22-induced endocytosis.

(A) Confocal images of FLS2/FLS2S938A/FLS2S938D-GFP in Arabidopsis thaliana leaf epidermal cells. The 5-day-old transgenic seedlings were pre-treated with 50 μM CHX for 30 min, then treated with 50 μM BFA for 60 min or CHX + BFA + 5 μM FM4-64 for 30 min, followed by 10 μM flg22 treatment for a total of 30 min. White arrows indicate BFA bodies. Bar = 3μm.

(B) Images of FLS2/FLS2S938A/FLS2S938D-GFP in Arabidopsis thaliana leaf epidermal cells treated with 10 μM flg22 for 15, 30, and 60 min. Bar = 3μm. (C) Analysis of FLS2, FLS2S938A, and FLS2S938D endocytic vesicle numbers in cells treated with 10 μM flg22 treatment over time. Statistical significance assessed using the Student’s t-test (***p < 0.001). Error bars represent the SD. (D) The signal density of FLS2, FLS2S938A and FLS2S938D in cells after control or 10 μM flg22 treatments for 30 min as measured by FCS. Statistical significance was assessed using Student’s t-test (**p < 0.01). Error bars represent the SD. (E) Immunoblot analysis of FLS2 protein in 10-day-old transgenic Arabidopsis plant upon stimulation with or without 10 μM flg22. CBB, a loading control dyed with Coomassie Brilliant Blue.

Ser-938 phosphorylation is essential for various flg22-induced PTI responses.

(A) The flg22-induced transient Ca2+ flux in 20-day-old transgenic leaf cells. The Ca2+ flux was continuously recorded for 12 min in the test medium. Each point represents the average value for about 12 individual plants ± SEM (B) Phenotypes of 5-day old etiolated seedlings grown in the presence of 1/2 MS (control) or 10 μM flg22 solid medium. Bar=0.5cm. (C) Hypocotyl length of FLS2 (control, n = 13 seedlings; flg22=13 seedlings), FLS2S938A (control, n = 13 seedlings; flg22=13 seedlings) and FLS2S938D (control, n = 13 seedlings; flg22=13 seedlings) transgenic plants. Statistical significance was assessed with the Student’s t-test (***p < 0.001). Error bars represent the SD. (D-F) mRNA levels of the PTI marker genes FRK1/WRKY33/CYP81 were significantly different between the FLS2, FLS2S938A, and FLS2S938D 10-day-old transgenic Arabidopsis plant after treatment with 10 μM flg22 for 30 min. Total RNA was extracted from 10-day-old seedlings and analyzed using qRT-PCR, with transcript levels being normalized to UBQ5. Error bars represent SD. (G) The working model for the spatiotemporal dynamic regulation of FLS2 phosphorylation at the plasma membrane upon flg22 stimulation. (H) Dynamic model of FLS2 with different Ser-938 phosphorylation states upon stimulation with flg22.