Single-molecule analysis reveals the phosphorylation of FLS2 governs its spatiotemporal dynamics and immunity
- State Key Laboratory of Tree Genetics and Breeding, College of Biological Sciences and Technology, Beijing Forestry University, China
- National Engineering Research Center of Tree Breeding and Ecological Restoration, College of Biological Sciences and Technology, Beijing Forestry University, China
- College of Life Sciences, Hebei Agricultural University, China
- Biotechnology Research Institute, China
Figures
Figure 1 with 6 supplements
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 were analyzed. The 5-day-old transgenic Arabidopsis plant cells were observed under VA-TIRFM. The red balls indicate the positions of the …
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Figure 1—source data 1
The original VA-TIRFM image and single-particle tracking of FLS2-expressing hypocotyl cells are shown in Figure 1A.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig1-data1-v1.xlsx
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Figure 1—source data 2
The list of the diffusion coefficients of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 1D.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig1-data2-v1.zip
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Figure 1—source data 3
The list of the motion range of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 1E.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig1-data3-v1.zip
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Figure 1—source data 4
The list of the lifetime of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 1I.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig1-data4-v1.zip
Figure 1—figure supplement 1
The subcellular localization of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP was determined by confocal imaging.
The left images represent GFP signal showing the subcellular localization of FLS2 molecules under different phosphorylation states. The middle images represent red fluorescence signal showing plasma …
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Figure 1—figure supplement 1—source data 1
The original confocal images of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
Uniform Manifold Approximation and Projection (UMAP) visualization of FLS2S938A (control, n = 27 spots; flg22, n = 30 spots) and FLS2S938D (control, n = 11 spots; flg22, n = 14 spots) samples in the different conditions.
Dots represent individual images and are colored according to the reaction conditions.
Figure 1—figure supplement 3
The representative fluorescence recovery after photobleaching (FRAP) time course of FLS2-GFP, FLS2S938A, and FLS2S938D under control treatments.
White squares indicate bleached regions. Bar = 5 μm.
Figure 1—figure supplement 4
Images of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP with flg22 treatment were collected by VA-TIRFM.
For short presentation, one frame is displayed for every 10 frames captured (at 100 ms intervals). Bar = 300 nm.
Figure 1—figure supplement 5
Representative kymographs showing individual FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP dwell times under the control and flg22 treatment.
Bar = 2 s.
Figure 1—figure supplement 6
The single-molecule trajectories of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP analyzed by Imaris could be faithfully tracked under control and flg22 treatments.
Bar = 1 μm.
Figure 2 with 3 supplements
Different Ser-938 phosphorylation states of FLS2 affect its partitioning into AtRem1.3-associated nanodomains.
(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 …
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Figure 2—source data 1
The list of the fluorescence mean lifetimes (t) of FLS2/FLS2S938A/FLS2S938D-GFP and BAK1-mCherry under different treatments is shown in Figure 2A.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-data1-v1.zip
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Figure 2—source data 2
The list of the Pearson correlation coefficient of FLS2/FLS2S938A/FLS2S938D-GFP and BAK1-mCherry under different treatments is shown in Figure 2B.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-data2-v1.zip
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Figure 2—source data 3
The list of the mean protein proximity indexes of FLS2/FLS2S938A/FLS2S938D-GFP and BAK1-mCherry under different treatments is shown in Figure 2C.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-data3-v1.zip
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Figure 2—source data 4
The original intensity and lifetime maps of FLS2/FLS2S938A/FLS2S938D-GFP and AtRem1.3-mCherry under different treatments are shown in Figure 2H.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-data4-v1.zip
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Figure 2—source data 5
The list of the fluorescence mean lifetimes (t) of FLS2/FLS2S938A/FLS2S938D-GFP and AtRem1.3-mCherry under different treatments is shown in Figure 2G.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-data5-v1.zip
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Figure 2—source data 6
The list of the Pearson correlation coefficient of FLS2/FLS2S938A/FLS2S938D-GFP and AtRem1.3-mCherry under different treatments is shown in Figure 2I.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-data6-v1.zip
Figure 2—figure supplement 1
SR-Tesseler analysis shows the distribution and co-localization of all spots on FLS2-GFP, FLS2S938A-GFP and FLS2S938D-GFP and BAK1 under control or flg22 treatment.
Bar = 50 μm.
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Figure 2—figure supplement 1—source data 1
The list of the coordinates of FLS2/FLS2S938A/FLS2S938D-GFP and BAK1-mCherry under different treatments.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
Images show co-localization of FLS2-GFP, FLS2S938A-GFP, or FLS2S938D-GFP with BAK1 in N. benthamiana leaves cells under control or flg22 treatment.
Figure 2—figure supplement 3
3D plot of FLS2-GFP, FLS2S938A-GFP, or FLS2S938D-GFP and BAK1 cross-correlation versus pixel shift under control or flg22 treatment.
Figure 3 with 5 supplements
Ser-938 phosphorylation site affects flg22-induced endocytosis.
(A) Confocal images of FLS2/FLS2S938A/FLS2S938D-GFP in Arabidopsis thaliana leaf epidermal cells. Firstly, experiments were performed after pretreatment with CHX (50 μM) for 30 min. Subsequently, to …
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Figure 3—source data 1
The original confocal images of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments are shown in Figure 3A.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-data1-v1.zip
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Figure 3—source data 2
The original confocal images of FLS2/FLS2S938A/FLS2S938D-GFP in Arabidopsis thaliana leaf epidermal cells treated with 10 μM flg22 for 15, 30, and 60 min are shown in Figure 3B.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-data2-v1.zip
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Figure 3—source data 3
The list of the endocytic vesicle numbers of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 3C.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-data3-v1.xlsx
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Figure 3—source data 4
The list of the signal density of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 3D.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-data4-v1.zip
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Figure 3—source data 5
The original file for the western blot analysis in Figure 3E.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-data5-v1.zip
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Figure 3—source data 6
The original file for the western blot analysis with highlighted bands and sample labels is shown in Figure 3E.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-data6-v1.zip
Figure 3—figure supplement 1
Distribution and co-localization with FM4-64 of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP in leaves epidermal cells treated with or without 10 μM flg22 for 30 min after 30 min pretreatment with CHX.
Bar = 5 μm.
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Figure 3—figure supplement 1—source data 1
The list of the number, diameter, and fluorescence intensity of FLS2/FLS2S938A/FLS2S938D-GFP BFA bodies under different treatments.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
Confocal images of FLS-GFP, FLS2S938A-GFP, and FLS2S938D-GFP in leaves epidermal cells treated with or without 10 μM flg22 after 30 min pretreatment with BFA.
Arrows indicate BFA bodies of FLS2, inset images show details of BFA bodies. White arrows indicate BFA bodies. Bar = 5 μm.
Figure 3—figure supplement 3
Effects of Ser-938 phosphorylation on the endocytosis of FLS2.
(A) Number analysis of FLS2-GFP (control, n = 19 images; flg22, n = 9 images), FLS2S938A-GFP (control, n = 28 images; flg22, n = 9 images) and FLS2S938D-GFP (control, n = 21 images; flg22, n = 7 …
Figure 3—figure supplement 4
The fluorescence intensity of FLS2-GFP (0 min, n = 9 images; 15 min, n = 5 images; 30 min, n = 7 images; 60 min, n = 9 images), FLS2S938A-GFP (0 min, n = 5 images; 15 min, n = 5 images; 30 min, n = 5 images; 60 min, n = 5 images), and FLS2S938D-GFP (0 min, n = 10 images; 15 min, n = 6 images; 30 min, n = 7 images; 60 min, n = 8 images) in the cytoplasm relative to the sum of the cytoplasm and PM in leaves epidermal cells treated with flg22 treatment over time; Three biological replicates were performed.
Statistical significance was assessed using Student’s t-test (*p<0.05, ***p<0.001). Error bars represent the SD.
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Figure 3—figure supplement 4—source data 1
The list of the fluorescence intensity of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP in the cytoplasm relative to the sum of the cytoplasm and PM in leaves epidermal cells under different treatments.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig3-figsupp4-data1-v1.zip
Figure 3—figure supplement 5
The confocal images of FLS2-GFP signal density in Arabidopsis leaf epidermal cells upon ligand stimulation were analyzed using fluorescence correlation spectroscopy.
Control (CK) plants received no treatment. All the samples except CK were pretreated with flg22 for different times. Bar = 10 μm.
Figure 4 with 3 supplements
Ser-938 phosphorylation is essential for various flg22-induced pattern-triggered immunity (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 …
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Figure 4—source data 1
The list of the transient Ca2+ flux of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 4A.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-data1-v1.zip
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Figure 4—source data 2
The list of the hypocotyl length of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments is shown in Figure 4C.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-data2-v1.zip
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Figure 4—source data 3
The list of the mRNA levels of the PTI marker genes FRK1/WRKY33/CYP81 of the FLS2, FLS2S938A, and FLS2S938D 10-day-old transgenic Arabidopsis plants under different treatments is shown in Figure 4D–F.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-data3-v1.zip
Figure 4—figure supplement 1
MAPKs phosphorylation in FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP 10-day-old seedlings incubated with 10 μM flg22 for 0 min, 5 min, and 15 min.
CBB, a loading control dyed with Coomassie brilliant blue.
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Figure 4—figure supplement 1—source data 1
The original file for the western blot analysis.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
The original file for the western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
Ser-938 phosphorylation affects flg22-induced callose deposition.
(A) Detection of callose deposition in leaves of FLS2-GFP, FLS2S938A-GFP, and FLS2S938D-GFP Arabidopsis seedlings after treated with CK (1/2 MS liquid medium) or 10 μM flg22 for 12 hr. Callose was …
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Figure 4—figure supplement 2—source data 1
The list of the callose deposition of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-figsupp2-data1-v1.zip
Figure 4—figure supplement 3
Phenotypes of 5-day-old etiolated seedlings grown in the 1/2 MS (CK) or solid medium with or without 10 μM flg22.
Scale bar = 0.5 cm.
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Figure 4—figure supplement 3—source data 1
The original images of FLS2/FLS2S938A/FLS2S938D-GFP under different treatments.
- https://cdn.elifesciences.org/articles/91072/elife-91072-fig4-figsupp3-data1-v1.zip
