The Plant Steroid Receptor Kinase BRI1 Signals from Endosomes

Endosomal signaling of BRI1 receptor kinase, Geldner, page 1

Supplementary table 1: BRI1-GFP at endogenous expression levels rescues bri1 null mutant

Segregation ratios of independent crosses between BRI1-GFP (endog.) and bri1-116 null mutant lines. The segregation ratio indicates that the BRI1-GFP complements the recessive bri1-116 allele in a dominant fashion (6.25% expected ratio for complementation, versus 25% for non-complementation). In addition, BRI1-GFP expression was strictly correlated with wt phenotypes in the segregating lines.

Supplementary figure 1: Comparison of BRI1-GFP signal intensities

A-D Intensity comparison and controls for BRI1-GFP signals in root meristem epidermal cells. A BRI1-GFP overexpressing line B BRI1-GFP endogenous expresser, same detection settings as A. C same as B, adapted detection settings. D wild-type control for C. E,F Individual channels of overlays in Figure 1. E BRI1-GFP (green, left) partially co-localises (overlay, right) with the endocytic tracer FM4-64 (red, middle) after 5-10 min uptake. F BRI1-GFP (green, left) also co-localises partially (overlay, right) with VHA-a1-RFP (middle, red). Scale bars = 10 m.

Supplementary figure 2: Quantification of BRI1-YFP levels.

Example of quantification using Leica SP/2 software package. Several equal sized regions-of-interest were defined along root meristems. Pixel intensities were controlled to avoid saturation at peak expression and background subtracted. Peak intensities were set to 100% and degradation rate was then determined as the average loss of relative fluorescence from the time point of peak intensity values. 4-5 ROIs were measured and averaged per root meristem and at least five root meristems were averaged per experiment.

Supplementary figure 3: Cartoon indicating endosomal trafficking of BRI1 and the sites of BFA action.

BFA leads to aggregation of individual endosomal compartments and interferes as much with recycling back to the plasma membrane as with translocation into the vacuole.

Supplementary figure 4: Model of the influence of sub-cellular localization on BRI1 singaling activity.

Our data supports a model whereby ligand binding and initial BRI1-activation takes place at the plasma membrane, but that BRI1 signal transduction to BES1 takes place more efficiently, perhaps exclusively, from endosomal compartments, perhaps due to preferential or exclusive presence as yet unidientified downstream signaling components.

Supplementary video 1: Movie of BRI-GFP localisation in root meristem cells.
Movie is 10 seconds per frame real-time, shown at 5 frames per second (fps). It visualizes the dynamics and pleiomorphic nature of the BRI1-positive intracellular compartments, which seem to continuously undergo fusion and fission. Time-stack was XY-drift and bleach corrected using ImageJ. Scale bar: 10 μm (QuickTime; 2.58 MB).

Supplementary video 2: Movie of Figure 2A.

Movie is 1 min per frame real-time, shown at 5 fps. It shows unaltered localization and levels of BRI1-GFP upon addition of BL after first frame. This time-stack was XY-drift corrected using ImageJ software bundle. Scale bar: 10 μm (QuickTime; 940 KB).

Supplementary video 3: Movie of Figure 2B.

Movie is 2 min per frame real-time, shown at 4 fps. It shows the unaltered localization and levels of BRI1-GFP in ligand-depleted roots (time-point 0) and upon addition of BL after first frame. Stack shows z-axis drift, XY-drift was corrected using ImageJ software bundle. Scale bar: 10 μm (QuickTime; 979 KB).

Supplementary video 4: Transient expression of BRI1-YFP after heat-shock.

This movie demonstrates that heat-shock does not interfere with continued growth of roots. Movie is 5 min per frame real-time, shown at 24 fps. Scale bar: 100 μm (QuickTime; 9.85 MB).

Supplementary video 5: Movie of Figure 2C.

Movie is 2 min per frame real-time, shown at 24 fps. It is a representative time-stack used to determine BRI1-YFP degradation rate in untreated root meristems (see Suppl. Fig. 1). Recording was started 4.5-5 hours after a 20-30 min heat-shock. Scale bar: 100 μm (QuickTime; 9.72 MB).

Supplementary video 6: Movie of Figure 3A.

Movie is 1 min per frame real-time, shown at 5 fps. Time of BFA addition is indicated in movie. BFA induces aggregation of BRI1-labeled endosomes and increases the endosomal pool of BRI1-GFP. Scale bar: 10 μm (QuickTime; 2.29 MB).