Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains
- Norwich Research Park, United Kingdom
- Institute of Genetics, Germany
Figures
Figure 1 with 2 supplements
FLS2 and BRI1 form receptor clusters within the plasma membrane.
(A, B) Plasma membrane localisation of FLS2-GFP (A) and BRI1-GFP (B) in epidermal cells of Arabidopsis seedling cotyledons. (C, D) Plasma membrane localisation of FLS2-GFP (C) and BRI1-GFP (D) after …
Figure 1—figure supplement 1
Illustration of the image processing steps for emphasising receptor cluster formation.
(A) Raw data of a confocal micrograph showing the fluorescence intensity of FLS2-GFP in grey scale. (B) Confocal micrograph of FLS2-GFP fluorescence intensity as shown in (A) but using the ‘fire’ …
Figure 1—figure supplement 2
Plasma membrane localisation of FLS2 and BRI1 with respect to the cytoskeleton.
(A I–A IV) Confocal micrographs of actin filaments visualised using LifeAct-tRFP (A I) and FLS2-GFP (A II) in epidermal leaf cells of Arabidopsis seedling cotyledons as well as the merged image (A …
Figure 2 with 1 supplement
FLS2 receptor clusters are more stable than BRI1 clusters.
(A, B) Plasma membrane localisation of FLS2-GFP (A) and BRI-GFP (B) in epidermal cells of Arabidopsis seedling cotyledons. (C, D) Plasma membrane localisation of FLS2-GFP (C) and BRI1-GFP (D) after …
Figure 2—figure supplement 1
The stability of FLS2 clusters depends on the PM lipid composition.
(A, B) VAEM micrograph of FLS2-GFP (A) and a kymograph of the corresponding VAEM time series (B) after mock treatment. (C, D) VAEM micrograph of BRI1-GFP (C) and a kymograph of the corresponding …
Figure 3
BRs reduce BRI1 cluster displacement within the plasma membrane.
(A) Quantification of FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after 2 days in liquid medium containing 5 µM BRZ. (B) Quantification …
Figure 4
Activation of FLS2 results in reduced lateral receptor cluster displacement.
(A) Time-dependent quantification of short-range FLS2-GFP and BRI1-GFP receptor cluster displacements in epidermal cells of Arabidopsis seedling cotyledons after application of 100 nM flg22. (B) …
Figure 5 with 1 supplement
FLS2 and BRI1 show distinct plasma membrane localisation patterns.
(A–D) Confocal micrographs of FLS2-GFP (A) and FLS2-mCherry (B) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (C) …
Figure 5—figure supplement 1
Control experiments for verifying the specific localisation patterns of FLS2 and BRI1.
(A) Quantitative co-localisation analysis for FLS2-GFP or BRI1-GFP, respectively, with FLS2-mCherry after transient co-expression in epidermal leaf cells of N. benthamiana. (B) Quantitative …
Figure 6
FLS2 and BRI1 clusters are spatiotemporally separated.
(A–C) Plasma membrane localisation of FLS2-GFP (A) and FLS2-mCherry (B) after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (C). (D) Kymograph …
Figure 7 with 1 supplement
FLS2 and BRI1 co-localize differentially with remorin markers.
(A I–A III) Confocal micrographs of mRFP-REM1.2 (A I) and FLS2-GFP (A II) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged …
Figure 7—figure supplement 1
Control experiments for verifying the specific co-localisation of FLS2 and BRI1 with remorin markers.
The coloured data points indicate the values of technical replicates; black dots indicate the position of outliers. To assess whether the determined co-localisation values (orig.) were significant, …
Figure 8
FLS2 and BRI1 signaling complexes also undergo cluster formation within the plasma membrane.
(A–D) Confocal micrographs of BSK1-CFP (A), BSK1-nYFP/BRI1-cYFP (BiFC) (B), and BRI1-mRFP (C) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as …
Figure 9 with 1 supplement
BRI1-BIK1, but not FLS2-BIK1, complexes associate with cortical microtubules.
(A–D) Confocal micrographs of BIK1-CFP (A), BIK1-nYFP/BRI1-cYFP (BiFC) (B), and BRI1-mRFP (C) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as …
Figure 9—figure supplement 1
BIK1 and BRI1-BIK1 complexes associate with cortical microtubules.
(A–D) Confocal micrographs of BIK1-CFP (A), BIK1-nYFP/BRI1-cYFP (B), and BRI1-mRFP (C) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as …
Author response image 1
Subpopulations of FLS2 and BRI1 co-localise with BAK1 in plasma membrane nanodomains.
The images show the plasma membrane localisation of FLS2-GFP or BRI1-GFP with BAK1- mCherry. The white arrowheads indicate plasma membrane nanodomains that contain fluorescently labelled receptor …
Author response image 2
FLS2-GFP forms receptor clusters in fls2 mutant background.
The images show the plasma membrane localisation of pFLS2::FLS2-GFP stably transformed in the fls2 mutant or Columbia wild type background, respectively. The images were acquired 5 days post …
Author response image 3
Actin depolymerisation does not abolish FLS2 cluster formation.
https://doi.org/10.7554/eLife.25114.025
Author response image 4
FLS2 still undergoes cluster formation in serk mutant backgrounds.
https://doi.org/10.7554/eLife.25114.026Videos
Video 1
Dynamics of FLS2 receptor clusters within the plasma membrane.
The presented time series was acquired from an epidermal cell of an Arabidopsis seedling cotyledon expressing FLS2-GFP under its native promoter using variable angle epi-fluorescence microscopy …
Video 2
Dynamics of BRI1 receptor clusters within the plasma membrane.
The presented time series was acquired from an epidermal cell of an Arabidopsis seedling cotyledon expressing BRI1-GFP under its native promoter using variable angle epi-fluorescence microscopy …
Video 3
Visualization of FLS2 receptor cluster dynamics within the plasma membrane.
The presented time series was acquired from an epidermal leaf cell after transient co-expression of FLS2-GFP and FLS2-mCherry in N. benthamiana using variable angle epi-fluorescence microscopy …
Video 4
Simultaneous visualization of FLS2 and BRI1 receptor cluster dynamics within the plasma membrane.
The presented time series was acquired from an epidermal leaf cell after transient co-expression of BRI1-GFP and FLS2-mCherry in N. benthamiana using variable angle epi-fluorescence microscopy …
Additional files
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Supplementary file 1
Summary of quantitative image analysis.
In this table a summary of the quantitative image analysis is given providing the number of independent biological experiments, the number of technical replicates as well as the results of t-tests and the corresponding final p-values. The final p-values were obtained by multiplying the t-test results with a Bonferroni factor of 2. For the comparison of FLS2 and BRI1 two-tailed heteroscedastic t-tests were applied. For the comparison of original and rotated images two-tailed homoscedastic t-tests were applied. For time series experiments one-tailed heteroscedastic t-tests were applied. The results of t-tests and final p-values were based on the analysis of the corresponding mean values for respective independent biological experiments. The presented mean values are in the units shown in the respective figures. ‘SD’ stands for standard deviation based on the technical replicates. ‘At’ stands for Arabidopsis thaliana and ‘Nb’ stands for Nicotiana benthamiana. ‘FLS2/BSK1’, ‘BRI1/BSK1’, ‘FLS2/BIK1’, and ‘BRI1/BIK1’ represent the respective BiFC complexes.
- https://doi.org/10.7554/eLife.25114.022
