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Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains

View Article: PubMed Central - PubMed

ABSTRACT

Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

Doi:: http://dx.doi.org/10.7554/eLife.25114.001

No MeSH data available.


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 analysis of VAEM micrograph shown in (C). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. (E–G) Plasma membrane localisation of BRI1-GFP (E) and FLS2-mCherry (F) after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (G). (H) Kymograph analysis of VAEM micrograph shown in (G). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. The presented images were acquired using variable angle epi-fluorescence microscopy (VAEM). In the merged images FLS2-GFP or BRI1-GFP signals are shown in green and FLS2-mCherry signals are shown in magenta. The scale bar represents 5 µm. The colour bar represents the colour code for fluorescence intensities. Two independent experiments with similar results were performed.DOI:http://dx.doi.org/10.7554/eLife.25114.016
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fig6: 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 analysis of VAEM micrograph shown in (C). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. (E–G) Plasma membrane localisation of BRI1-GFP (E) and FLS2-mCherry (F) after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (G). (H) Kymograph analysis of VAEM micrograph shown in (G). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. The presented images were acquired using variable angle epi-fluorescence microscopy (VAEM). In the merged images FLS2-GFP or BRI1-GFP signals are shown in green and FLS2-mCherry signals are shown in magenta. The scale bar represents 5 µm. The colour bar represents the colour code for fluorescence intensities. Two independent experiments with similar results were performed.DOI:http://dx.doi.org/10.7554/eLife.25114.016

Mentions: To obtain a more dynamic view on the co-localisation or spatial separation between the FLS2 and BRI1 receptor populations, we additionally applied dual-colour VAEM on leaf epidermal cells that co-expressed BRI1-GFP and FLS2-mCherry (Video 3 and 4, Figure 6). We hardly observed overlap between the two LRR-RKs as indicated by the kymograph representation in Figure 6H.Video 3.Visualization of FLS2 receptor cluster dynamics within the plasma membrane.


Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains
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 analysis of VAEM micrograph shown in (C). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. (E–G) Plasma membrane localisation of BRI1-GFP (E) and FLS2-mCherry (F) after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (G). (H) Kymograph analysis of VAEM micrograph shown in (G). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. The presented images were acquired using variable angle epi-fluorescence microscopy (VAEM). In the merged images FLS2-GFP or BRI1-GFP signals are shown in green and FLS2-mCherry signals are shown in magenta. The scale bar represents 5 µm. The colour bar represents the colour code for fluorescence intensities. Two independent experiments with similar results were performed.DOI:http://dx.doi.org/10.7554/eLife.25114.016
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Related In: Results  -  Collection

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fig6: 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 analysis of VAEM micrograph shown in (C). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. (E–G) Plasma membrane localisation of BRI1-GFP (E) and FLS2-mCherry (F) after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (G). (H) Kymograph analysis of VAEM micrograph shown in (G). The spatial dimension of the kymograph is indicated by the white arrow in (C). The acquisition time of a single channel was 0.25 s. For each channel 200 frames were collected. The presented images were acquired using variable angle epi-fluorescence microscopy (VAEM). In the merged images FLS2-GFP or BRI1-GFP signals are shown in green and FLS2-mCherry signals are shown in magenta. The scale bar represents 5 µm. The colour bar represents the colour code for fluorescence intensities. Two independent experiments with similar results were performed.DOI:http://dx.doi.org/10.7554/eLife.25114.016
Mentions: To obtain a more dynamic view on the co-localisation or spatial separation between the FLS2 and BRI1 receptor populations, we additionally applied dual-colour VAEM on leaf epidermal cells that co-expressed BRI1-GFP and FLS2-mCherry (Video 3 and 4, Figure 6). We hardly observed overlap between the two LRR-RKs as indicated by the kymograph representation in Figure 6H.Video 3.Visualization of FLS2 receptor cluster dynamics within the plasma membrane.

View Article: PubMed Central - PubMed

ABSTRACT

Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

Doi:: http://dx.doi.org/10.7554/eLife.25114.001

No MeSH data available.