Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors
- Institute of Plant and Microbial Biology and Zurich-Basel Plant Science Center, University of Zurich, Switzerland
- The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, United Kingdom
- Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences Vienna, Austria
- FAFU-UCR Joint Center for Horticultural Biology and Metabolomics Center, Haixia, Institute of Science and Technology, Fujian Agriculture and Forestry University, China
Figures
Figure 1 with 5 supplements
FER regulates the nanoscale organization of FLS2-GFP and BAK1-mCherry.
(A, E) FLS2-GFP and BAK1-mCherry nanodomain organization. Pictures are maximum projection of 20 variable angle total internal reflection fluorescence microscopy (VA-TIRFM) images obtained at 5 …
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Figure 1—source data 1
Source data points for the graphs in Figure 1C and G.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Analysis of FLS2-GFP single-particle dynamics in fer-4.
(A) Representative images of FLS2-GFP single particles tracked in Col-0 and fer-4 cotyledon epidermal cells of 5-day-old seedlings. (B) Quantification of FLS2-GFP diffusion coefficient (D). Graph is …
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Figure 1—figure supplement 1—source data 1
Source data points for the graph in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Analysis of FLS2-GFP organization and dynamics in fer-2.
(A) Representative images of FLS2-GFP single particles tracked in Col-0 and fer-2 cotyledon epidermal cells of 5-day-old seedlings. (B) Quantification of FLS2-GFP diffusion coefficient (D). Graph is …
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Figure 1—figure supplement 2—source data 1
Source data points for the graphs in Figure 1—figure supplement 2B and D.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
FLS2-GFP accumulation at the PM is altered in fer mutants.
(A–D) Representative confocal microscopy pictures of FLS2-GFP driven by its native promoter in Col-0 and fer-2 (A) and fer-4 (C) cotyledon epidermal cells of 5-day-old seedlings. Scale bar indicates …
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Figure 1—figure supplement 3—source data 1
Source data points for the graphs in Figure 1—figure supplement 3B and D.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig1-figsupp3-data1-v2.xlsx
Figure 1—video 1
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of FLS2-GFP in Col-0 and fer-4.
Representative stream images acquisition of FLS2-GFP particles observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 5 frames per second.
Figure 1—video 2
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of BAK1-mCherry in Col-0 and fer-4 with or without RALF23 treatment.
Representative stream images acquisition of BAK1-mCherry observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 2.5 frames per second, in Col-0 (A, B) and fer-4 (C, D…
Figure 2 with 7 supplements
LRX3, LRX4, and LRX5 regulate pattern-triggered immunity (PTI) and BAK1-mCherry organization.
(A) flg22-induced FLS2-BAK1 complex formation. Immunoprecipitation of FLS2 in Arabidopsis Col-0 and lr3/4/5 seedlings either untreated or treated with 100 nM flg22 for 10 min. Blot stained with …
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Figure 2—source data 1
Source data points for the graphs in Figure 2B, C and F.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-data1-v2.xlsx
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Figure 2—source data 2
Source blots images for the co-immunoprecipitation (co-IP) in Figure 2A.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-data2-v2.pdf
Figure 2—figure supplement 1
Subcellular localization of BAK1-mCherry in fer-4 and lrx3/4/5.
(A) Representative confocal microscopy pictures of BAK1-mCherry driven by its native promoter in Col-0, fer-4, and lrx3/4/5 cotyledon epidermal cells of 5-day-old seedlings. Scale bar indicates 20 …
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Figure 2—figure supplement 1—source data 1
Source data points for the graph in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
LRX3, LRX4, and LRX5 are dispensable for FER plasma membrane localization and accumulation.
(A) Accumulation of endogenous FER detected by western blot. Total proteins were extracted from 2-week-old seedlings. Blot stained with Coomassie brilliant blue (CBB) is presented to show equal …
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Figure 2—figure supplement 2—source data 1
Source blots images for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-figsupp2-data1-v2.pdf
Figure 2—figure supplement 3
LRX3, LRX4, and LRX5 are dispensable for FER-GFP nanoscale organization.
(A) FER-GFP nanodomain organization. Pictures are maximum projection images (20 variable angle total internal reflection fluorescence microscopy [VA-TIRFM] images obtained at 5 frames per second) of …
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Figure 2—figure supplement 3—source data 1
Source data points for the graph in Figure 2—figure supplement 3B.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-figsupp3-data1-v2.xlsx
Figure 2—figure supplement 4
LRX3, LRX4, and LRX5 contribute to RALF23 responsiveness.
(A) Reactive oxygen species (ROS) production in Col-0 and lrx3/4/5 leaf discs treated with 100 nM elf18 and with or without 1 µM RALF23 co-treatment in 2 mM MES–KOH pH 5.8. Values are means of total …
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Figure 2—figure supplement 4—source data 1
Source data points for the graph in Figure 2—figure supplement 4A and B.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-figsupp4-data1-v2.xlsx
Figure 2—figure supplement 5
RALF23 does not modulate constitutive association between FER and LRX4.
Immunoprecipitation of LRX4ΔE-FLAG in Arabidopsis seedlings untreated or treated with 1 µM RALF23 for 10 min. Western blots were probed with α-FLAG or α-FER antibodies. Blot stained with Coomassie …
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Figure 2—figure supplement 5—source data 1
Source blots images for the co-immunoprecipitation (co-IP) in Figure 2—figure supplement 5.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig2-figsupp5-data1-v2.pdf
Figure 2—video 1
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of BAK1-mCherry in Col-0 and lrx3/4/5.
Representative stream images acquisition of BAK1-mCherry observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 2.5 frames per second, in Col-0 (A) and lrx3/4/5 (B). …
Figure 2—video 2
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of FER-GFP in fer-4 and fer-4;lrx3/4/5.
Representative stream images acquisition of FER-GFP observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 5 frames per second in fer-4 (A) and fer-4;lrx3/4/5 (B). …
Figure 3 with 1 supplement
FER malectin A domain regulates cell morphogenesis not pattern-triggered immunity (PTI).
(A) Graphical representation of RALF23 perception by FER-LLG1 complex. (B) Morphology of 4-week-old Arabidopsis plants; scale bar indicates 5 cm. (C, D) Confocal microscopy pictures of 5-day-old …
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Figure 3—source data 1
Source data points for the graphs in Figure 3F–H.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig3-data1-v2.xlsx
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Figure 3—source data 2
Source blots images for the co-immunoprecipitation (co-IP) in Figure 3E.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig3-data2-v2.pdf
Figure 3—figure supplement 1
Overexpression of LRX4ΔE does not affect pattern-triggered immunity (PTI).
(A) Flg22-induced FLS2-BAK1 complex formation. Immunoprecipitation of FLS2 in Arabidopsis seedlings either untreated or treated with 100 nM flg22 for 10 min. Blot stained with Coomassie brilliant …
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Figure 3—figure supplement 1—source data 1
Source data points for the graphs in Figure 3—figure supplement 1B–E.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Source blots images for the co-immunoprecipitation (co-IP) in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig3-figsupp1-data2-v2.pdf
Figure 4 with 11 supplements
RALF23 perception regulates BAK1-mCherry organization.
(A) BAK1-mCherry nanodomain organization (pBAK1::BAK1-mCherry). Pictures are maximum projection images (10 variable angle total internal reflection fluorescence microscopy [VA-TIRFM] images obtained …
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Figure 4—source data 1
Source data points for the graph in Figure 4C.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
FER kinase activity is dispensable to support pattern-triggered immunity (PTI) signaling.
(A) Flg22-induced FLS2-BAK1 complex formation. Immunoprecipitation of FLS2 in Arabidopsis Col-0, fer-4, and fer-4/pFER::FERKD-GFP seedlings that were either untreated or treated with 100 nM flg22 …
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Figure 4—figure supplement 1—source data 1
Source data points for the graphs in Figure 4—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Inhibition of pattern-triggered immunity (PTI) signaling by RALF23 requires FER kinase activity.
(A) Accumulation of endogenous FER and FERK565R-GFP detected by western blot. Total proteins were extracted from 2-week-old seedlings. Blot stained with Coomassie brilliant blue (CBB) is presented …
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Figure 4—figure supplement 2—source data 1
Source data points for the graph in Figure 4—figure supplement 2C.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Analysis of FLS2-GFP single-particle dynamics upon RALF23 treatment.
(A) Representative images of FLS2-GFP single particles tracked in Col-0 and fer-2 cotyledon epidermal cells of 5-day-old seedlings with or without 1 µM RALF23 treatment. (B) Quantification of …
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Figure 4—figure supplement 3—source data 1
Source data points for the graph in Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp3-data1-v2.xlsx
Figure 4—figure supplement 4
Analysis of FLS2-GFP organization upon RALF23 treatment.
(A) Pictures are maximum projection images (20 variable angle total internal reflection fluorescence microscopy [VA-TIRFM] images obtained at 20 frames per second) of FLS2-GFP in Col-0 and fer-2 …
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Figure 4—figure supplement 4—source data 1
Source data points for the graph in Figure 4—figure supplement 4C.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp4-data1-v2.xlsx
Figure 4—figure supplement 5
Time-resolved analysis of the spatial clustering index.
Data points correspond to the average of the spatial clustering index measured over 5 min windows for FLS2-GFP (A) and BAK1-mCherry (B) with or without 1 µM RALF23 treatment. The first data point (5 …
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Figure 4—figure supplement 5—source data 1
Source data points for the graphs in Figure 4—figure supplement 5A and B.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp5-data1-v2.xlsx
Figure 4—figure supplement 6
Working model for the regulation of FLS2 and BAK1 nanoscale organization by RALFs receptor complexes.
Figure 4—figure supplement 7
Linear regression analysis of the relationship between the spatial clustering index and fluorescence intensity.
Spatial clustering index is calculated as the ratio of the mean of the 5% highest values to the mean of 5% lowest values of fluorescence intensity obtained on a plot line. Here, spatial clustering …
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Figure 4—figure supplement 7—source data 1
Source data points for the graphs in Figure 4—figure supplement 6A–C.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp7-data1-v2.xlsx
Figure 4—figure supplement 8
Replicates of co-immunoprecipitation experiments.
Replicate of immunoprecipitation experiments corresponding to Figure 2 (A, B), Figure 3 (C, D), Figure 3—figure supplement 1 (E, F), Figure 4—figure supplement 1 (G), and Figure 4—figure supplement 2…
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Figure 4—figure supplement 8—source data 1
Source blots images for the replicate of co-immunoprecipitation (co-IP) experiments.
- https://cdn.elifesciences.org/articles/74162/elife-74162-fig4-figsupp8-data1-v2.pdf
Figure 4—video 1
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of FLS2-GFP in Col-0 with or without RALF23 treatment.
Representative stream images acquisition of FLS2-GFP particles observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 5 frames per second. Scale bar indicates 2 µm.
Figure 4—video 2
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of FLS2-GFP in fer-4 with or without RALF23 treatment.
Representative stream images acquisition of FLS2-GFP particles observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 5 frames per second. Scale bar indicates 2 µm.
Figure 4—video 3
Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) imaging of FLS2-GFP in Col-0 and fer-2 with or without RALF23 treatment.
Representative stream images acquisition of FLS2-GFP particles observed at the surface of 5-day-old cotyledon epidermal cells by VA-TIRF microscopy at 20 frames per second. Scale bar indicates 2 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Arabidopsis thaliana) | Col-0/pFLS2::FLS2-GFP#1 | Göhre et al., 2008 | See Materials and methods | |
| Genetic reagent (A. thaliana) | Col-0/pFLS2::FLS2-GFP#2 | This paper | See Materials and methods | |
| Genetic reagent (A. thaliana) | fer-2/pFLS2::FLS2-GFP#1 | Stegmann et al., 2017 | See Materials and methods | |
| Genetic reagent (A. thaliana) | fer-4/pFLS2::FLS2-GFP#2 | This paper | See Materials and methods | |
| Genetic reagent (A. thaliana) | fer-4 | Duan, 2010 | See Materials and methods | |
| Genetic reagent (A. thaliana) | fer-4/pFER::FER-GFP | Duan, 2010 | See Materials and methods | |
| Genetic reagent (A. thaliana) | fer-4/pFER::FERKD-GFP | Chakravorty et al., 2018 | See Materials and methods | |
| Genetic reagent (A. thaliana) | lrx3/4/5 | Dünser, 2019 | See Materials and methods | |
| Genetic reagent (A. thaliana) | p35S::LRX4ΔE-Citrine | Dünser, 2019 | See Materials and methods | |
| Genetic reagent (A. thaliana) | p35S::LRX4ΔE-FLAG | Dünser, 2019 | See Materials and methods | |
| Genetic reagent (A. thaliana) | Col-0/pBAK1::BAK1-mCherry | Bücherl et al., 2013 | See Materials and methods | |
| Genetic reagent (A. thaliana) | fer-4/ pBAK1::BAK1-mCherry | This paper | See Materials and methods | |
| Genetic reagent (A. thaliana) | lrx3/4/5/ pBAK1::BAK1-mCherry | This paper | See Materials and methods | |
| Genetic reagent (A. thaliana) | lrx3/4/5/ pFER::FER-GFP | This paper | See Materials and methods | |
| Antibody | anti-FLAG-HRP | Sigma-Aldrich | A8592 | WB (1:4000 dilution) |
| Antibody | Monoclonal rabbit anti-FLS2 | Chinchilla et al., 2007 | WB (1:1000 dilution) | |
| Antibody | Polyclonal rabbit anti-BAK1 | Roux, 2011 | WB (1:5000 dilution) | |
| Antibody | Polyclonal rabbit anti-BAK1 pS612 | Perraki, 2018 | WB (1:3000 dilution) | |
| Antibody | Polyclonal rabbit anti-FER | Xiao et al., 2019 | WB (1:2000 dilution) | |
| Antibody | Anti-rabbit IgG-HRP Trueblot | Rockland | 18-8816-31 | WB (1:10,000 dilution) |
| Peptide, recombinant protein | Flg22 | Synthesized by EZBiolab(purity >95%) | See Materials and methods | |
| Peptide, recombinant protein | Elf18 | Synthesized by EZBiolab(purity >95%) | See Materials and methods | |
| Peptide, recombinant protein | RALF23 | Synthesized by EZBiolab(purity >95%) | See Materials and methods | |
| Chemical compound, drug | GFP-Trap agarose beads | ChromoTek | See Materials and methods | |
| Chemical compound, drug | M2 anti-Flag affinity gel | Sigma-Aldrich | A2220-5ML | See Materials and methods |
| Chemical compound, drug | Anti-rabbit Trueblot agarose beads | eBioscience | SML1656 | See Materials and methods |
| Software, algorithm | Fiji | https://imagej.net/Fiji | See Materials and methods |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/74162/elife-74162-transrepform1-v2.pdf
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Source data 1
Raw images of the western blots.
- https://cdn.elifesciences.org/articles/74162/elife-74162-supp1-v2.zip
