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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.


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 co-localisation analysis for FLS2-GFP or BRI1-GFP, respectively, with FLS2-mCherry after transient co-expression in epidermal leaf cells of N. benthamiana and BRZ-treatment. 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 (original or ori.) were significant, co-localisation analysis was also carried out after image randomisation. For this purpose, one of the two image channels was rotated by 90 degrees prior to co-localisation analysis (rotated or rot.). In addition, the co-localisation values of a reciprocal experiment are shown. Here, co-localisation of FLS2-GFP or BRI1-GFP was determined with regard to BRI1-mRFP. As emphasised by the represented p-values, we observed a specific co-localisation for FLS2-GFP and FLS2-mCherry. In contrast, the co-localisation of BRI1-GFP with FLS2-mCherry was not different from randomised images, and thus non-specific. Similar observations were made after BRZ-treatment for 2 days to deplete endogenous BRs as well as for reciprocal experiments using BRI1-mRFP as reference. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction.DOI:http://dx.doi.org/10.7554/eLife.25114.013
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fig5s1: 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 co-localisation analysis for FLS2-GFP or BRI1-GFP, respectively, with FLS2-mCherry after transient co-expression in epidermal leaf cells of N. benthamiana and BRZ-treatment. 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 (original or ori.) were significant, co-localisation analysis was also carried out after image randomisation. For this purpose, one of the two image channels was rotated by 90 degrees prior to co-localisation analysis (rotated or rot.). In addition, the co-localisation values of a reciprocal experiment are shown. Here, co-localisation of FLS2-GFP or BRI1-GFP was determined with regard to BRI1-mRFP. As emphasised by the represented p-values, we observed a specific co-localisation for FLS2-GFP and FLS2-mCherry. In contrast, the co-localisation of BRI1-GFP with FLS2-mCherry was not different from randomised images, and thus non-specific. Similar observations were made after BRZ-treatment for 2 days to deplete endogenous BRs as well as for reciprocal experiments using BRI1-mRFP as reference. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction.DOI:http://dx.doi.org/10.7554/eLife.25114.013

Mentions: To address whether FLS2 and BRI1 clusters coincide or are spatially separated within the PM, we performed co-localisation studies. As positive control, we first determined the overlap of two differently tagged FLS2 receptor populations. We co-expressed FLS2-GFP and FLS2-mCherry (Mbengue et al., 2016) in leaf epidermal cells of N. benthamiana and, as shown in Figure 5A–D, both fluorescently tagged FLS2 populations showed similar PM localisation patterns and also co-localised (Figure 5C and D). Based on quantitative co-localisation analysis, we determined moderate to high Pearson correlation coefficients for FLS2-GFP and FLS2-mCherry fluorescence signals (Figure 5I and Figure 5—figure supplement 1).10.7554/eLife.25114.012Figure 5.FLS2 and BRI1 show distinct plasma membrane localisation patterns.


Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains
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 co-localisation analysis for FLS2-GFP or BRI1-GFP, respectively, with FLS2-mCherry after transient co-expression in epidermal leaf cells of N. benthamiana and BRZ-treatment. 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 (original or ori.) were significant, co-localisation analysis was also carried out after image randomisation. For this purpose, one of the two image channels was rotated by 90 degrees prior to co-localisation analysis (rotated or rot.). In addition, the co-localisation values of a reciprocal experiment are shown. Here, co-localisation of FLS2-GFP or BRI1-GFP was determined with regard to BRI1-mRFP. As emphasised by the represented p-values, we observed a specific co-localisation for FLS2-GFP and FLS2-mCherry. In contrast, the co-localisation of BRI1-GFP with FLS2-mCherry was not different from randomised images, and thus non-specific. Similar observations were made after BRZ-treatment for 2 days to deplete endogenous BRs as well as for reciprocal experiments using BRI1-mRFP as reference. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction.DOI:http://dx.doi.org/10.7554/eLife.25114.013
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383397&req=5

fig5s1: 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 co-localisation analysis for FLS2-GFP or BRI1-GFP, respectively, with FLS2-mCherry after transient co-expression in epidermal leaf cells of N. benthamiana and BRZ-treatment. 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 (original or ori.) were significant, co-localisation analysis was also carried out after image randomisation. For this purpose, one of the two image channels was rotated by 90 degrees prior to co-localisation analysis (rotated or rot.). In addition, the co-localisation values of a reciprocal experiment are shown. Here, co-localisation of FLS2-GFP or BRI1-GFP was determined with regard to BRI1-mRFP. As emphasised by the represented p-values, we observed a specific co-localisation for FLS2-GFP and FLS2-mCherry. In contrast, the co-localisation of BRI1-GFP with FLS2-mCherry was not different from randomised images, and thus non-specific. Similar observations were made after BRZ-treatment for 2 days to deplete endogenous BRs as well as for reciprocal experiments using BRI1-mRFP as reference. The indicated p-values were obtained using a two-tailed heteroscedastic t-test and a Bonferroni multiple hypothesis correction.DOI:http://dx.doi.org/10.7554/eLife.25114.013
Mentions: To address whether FLS2 and BRI1 clusters coincide or are spatially separated within the PM, we performed co-localisation studies. As positive control, we first determined the overlap of two differently tagged FLS2 receptor populations. We co-expressed FLS2-GFP and FLS2-mCherry (Mbengue et al., 2016) in leaf epidermal cells of N. benthamiana and, as shown in Figure 5A–D, both fluorescently tagged FLS2 populations showed similar PM localisation patterns and also co-localised (Figure 5C and D). Based on quantitative co-localisation analysis, we determined moderate to high Pearson correlation coefficients for FLS2-GFP and FLS2-mCherry fluorescence signals (Figure 5I and Figure 5—figure supplement 1).10.7554/eLife.25114.012Figure 5.FLS2 and BRI1 show distinct plasma membrane localisation patterns.

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.