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

(A) VA-TIRFM image of a hypocotyl cell expressing FLS2 was analyzed. The red balls indicate the positions of the identified points that appeared. Trajectories represent the track length of the identified points. (B) Time-lapse images of FLS, FLS2S938A, and FLS2S938D. Bar = 200 μm. The changes of fluorescence intensity among different 3D luminance plots. (C) The trajectories of representative individual FLS2, FLS2S938A and FLS2S938D under flg22 processing. (D) Diffusion coefficients of FLS, FLS2S938A and FLS2S938D under different environments. (E) Frequency of long- and short-range motions for FLS, FLS2S938A and FLS2S938D under different environments. (F) UMAP visualization of FLS2S938D samples in the same conditions. Dots represent individual images and are colored according to the reaction conditions. (G) Fluorescence recovery curves of the photobleached areas with or without flg22 treatment. (H) The single molecule trajectories of FLS2 analyzed by Imaris could be faithfully tracked for 10 s under control and flg22 treatments. (I) Dwell times were analyzed for FLS2, FLS2S938A and FLS2S938D in the presence of the control and flg22 treatment.

Different Ser-938 phosphorylation states of FLS2 affects its partitioning into membrane nanodomains.

(A) Average fluorescence lifetime (t) and the FRET efficiency were analyzed of FLS2, FLS2S938A or FLS2S938D and BAK1. (B) Pearson correlation coefficient values of co-localization between FLS2, FLS2S938A or FLS2S938D and BAK1 upon stimulation with CK or flg22. (C) Mean protein proximity indexes showing FLS2, FLS2S938A or FLS2S938D and BAK1 degree of proximity. (D) TIRM-SIM images of the leaf epidermal cells co-expressing FLS2 and AtRem1.3. The white line represents the fluorescence signals. Bar = 5 μm. (E) FLS2 and AtRem1.3 fluorescence signals as shown in A. (F) The histogram shows the co-localization ratio of FLS2, FLS2S938D or FLS2S938A and AtRem1.3-mCherry. (G) Intensity and lifetime maps of the cells co-expressing FLS2 and AtRem1.3 as measured by FLIM-FRET. (H) The fluorescence mean lifetime (T) and the corrected fluorescence resonance efficiency (Rate) of FLS2, FLS2S938D or FLS2S938A with co-expressed AtRem1.3. (I) Quantification of co-localization between FLS2, FLS2S938A or FLS2S938D and AtRem1.3 with and without stimulation with ligands.

Ser-938 phosphorylation site affects flg22-induced endocytosis.

(A) Images of FLS2, FLS2S938D and FLS2S938A in leaf epidermal cells of Arabidopsis thaliana. Transgenic seedlings were pretreated with CHX for 30 min, then treated with CHX + BFA for 60 min or CHX + BFA + FM4-64 for 30 min, followed by 10 μM flg22 treatment for a total of 30 minutes. White arrows indicate BFA bodies. Bar=3μm. (B) Images of FLS2, FLS2S938A and FLS2S938D in cells treated with flg22 for 15, 30, and 60 min. Bar=3μm. (C) Number analysis of FLS2, FLS2S938A and FLS2S938D endocytosis in cells treated with flg22 treatment over time. (D) The signal density of FLS2, FLS2S938A and FLS2S938D in cells after treatment with flg22 for different times as measured by FCS. (E) Immunoblot analysis of FLS2 protein in PM levels upon stimulation with or without flg22.

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

(A) The flg22-induced transient Ca2+ flux in leaf cells. The Ca2+ flux was continuously recorded in test medium for 12 min. (B) MAPKs phosphorylation in FLS2, FLS2S938A, and FLS2S938D seedlings incubated with flg22 for 0min, 5min, and 15min. (C) mRNA levels of the PTI marker genes FRK1 were significantly different between FLS2, FLS2S938A and FLS2S938D Arabidopsis after treatment with 10 μM flg22 for 30 min. (D) Phenotypes of 5-day old etiolated seedlings grown in the presence of 1/2 MS (CK) or 10 μM flg22 solid medium. Scale bar=0.5cm. (E) Hypocotyl length of FLS2, FLS2S938A and FLS2S938D plants. (F) The amount of callose stained with aniline blue per unit area on each image was quantitatively analyzed. (G) Working model for the spatiotemporal dynamic regulation of FLS2 phosphorylation at the plasma membrane upon stimulation with flg22. (H) Dynamic model of FLS2 with different Ser-938 phosphorylation states upon stimulation with flg22.