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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 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 well as the merged image (D). (E–H) Confocal micrographs of BSK1-CFP (E), BSK1-nYFP/FLS2-cYFP (BiFC) (F), and FLS2-mCherry (G) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (H). (I) Quantitative co-localisation analysis for the reconstituted YFP fluorescence intensities (BiFC) with BSK1-CFP (BSK1) as well as BRI1-mRFP (BRI1) or FLS2-mCherry (FLS2) and the quantified co-localisation between BSK1-CFP with BRI1-mRFP or FLS2-mCherry. The coloured data points represent the technical replicates of 3 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. BiFC stands for bimolecular fluorescence complementation and the labelled image panels show YFP fluorescence signals for the respective protein complexes. Yc and Yn indicate the C- and N-terminal fragments of the split YFP fluorophore, respectively. Scale bars represent 5 µm. Black dots represent outliers.DOI:http://dx.doi.org/10.7554/eLife.25114.019
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fig8: 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 well as the merged image (D). (E–H) Confocal micrographs of BSK1-CFP (E), BSK1-nYFP/FLS2-cYFP (BiFC) (F), and FLS2-mCherry (G) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (H). (I) Quantitative co-localisation analysis for the reconstituted YFP fluorescence intensities (BiFC) with BSK1-CFP (BSK1) as well as BRI1-mRFP (BRI1) or FLS2-mCherry (FLS2) and the quantified co-localisation between BSK1-CFP with BRI1-mRFP or FLS2-mCherry. The coloured data points represent the technical replicates of 3 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. BiFC stands for bimolecular fluorescence complementation and the labelled image panels show YFP fluorescence signals for the respective protein complexes. Yc and Yn indicate the C- and N-terminal fragments of the split YFP fluorophore, respectively. Scale bars represent 5 µm. Black dots represent outliers.DOI:http://dx.doi.org/10.7554/eLife.25114.019

Mentions: To investigate the spatial organisation of FLS2 and BRI1 signalling units, we made use of bimolecular fluorescence complementation (BiFC). Since epitope-tagging of BAK1/SERK3 (and potentially other SERKs) compromises its function in immune signalling (Ntoukakis et al., 2011) we decided to omit these co-receptors for our study. Instead, we visualised FLS2 and BRI1 in complex with BSK1 or BIK1 using CLSM. As shown in Figure 8 and Figure 9, in addition to the ligand binding receptors, the two RLCKs also appeared heterogeneously distributed, and BSK1 and BIK1 clusters became evident.10.7554/eLife.25114.019Figure 8.FLS2 and BRI1 signaling complexes also undergo cluster formation within the plasma membrane.


Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains
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 well as the merged image (D). (E–H) Confocal micrographs of BSK1-CFP (E), BSK1-nYFP/FLS2-cYFP (BiFC) (F), and FLS2-mCherry (G) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (H). (I) Quantitative co-localisation analysis for the reconstituted YFP fluorescence intensities (BiFC) with BSK1-CFP (BSK1) as well as BRI1-mRFP (BRI1) or FLS2-mCherry (FLS2) and the quantified co-localisation between BSK1-CFP with BRI1-mRFP or FLS2-mCherry. The coloured data points represent the technical replicates of 3 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. BiFC stands for bimolecular fluorescence complementation and the labelled image panels show YFP fluorescence signals for the respective protein complexes. Yc and Yn indicate the C- and N-terminal fragments of the split YFP fluorophore, respectively. Scale bars represent 5 µm. Black dots represent outliers.DOI:http://dx.doi.org/10.7554/eLife.25114.019
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fig8: 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 well as the merged image (D). (E–H) Confocal micrographs of BSK1-CFP (E), BSK1-nYFP/FLS2-cYFP (BiFC) (F), and FLS2-mCherry (G) plasma membrane localisation after transient co-expression in epidermal leaf cells of N. benthamiana as well as the merged image (H). (I) Quantitative co-localisation analysis for the reconstituted YFP fluorescence intensities (BiFC) with BSK1-CFP (BSK1) as well as BRI1-mRFP (BRI1) or FLS2-mCherry (FLS2) and the quantified co-localisation between BSK1-CFP with BRI1-mRFP or FLS2-mCherry. The coloured data points represent the technical replicates of 3 independent experiments. No statistical differences were observed based on two-tailed heteroscedastic t-tests and a Bonferroni multiple hypothesis correction. BiFC stands for bimolecular fluorescence complementation and the labelled image panels show YFP fluorescence signals for the respective protein complexes. Yc and Yn indicate the C- and N-terminal fragments of the split YFP fluorophore, respectively. Scale bars represent 5 µm. Black dots represent outliers.DOI:http://dx.doi.org/10.7554/eLife.25114.019
Mentions: To investigate the spatial organisation of FLS2 and BRI1 signalling units, we made use of bimolecular fluorescence complementation (BiFC). Since epitope-tagging of BAK1/SERK3 (and potentially other SERKs) compromises its function in immune signalling (Ntoukakis et al., 2011) we decided to omit these co-receptors for our study. Instead, we visualised FLS2 and BRI1 in complex with BSK1 or BIK1 using CLSM. As shown in Figure 8 and Figure 9, in addition to the ligand binding receptors, the two RLCKs also appeared heterogeneously distributed, and BSK1 and BIK1 clusters became evident.10.7554/eLife.25114.019Figure 8.FLS2 and BRI1 signaling complexes also undergo cluster formation 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.