Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Separate intracellular and extracellular activities from figures 2B and 5A,B. Protoplasts were transfected with constant amount of plasmid (10μg) encoding either the secreted control cargo Amy or the vacuolar test cargo Amy-spo together with increasing concentrations of plasmids encoding effector molecules (amounts in μg are indicated below each lane). After 24 hours of incubation cells and medium were harvested and α-amylase activity was measured in each fraction. Intracellular activities are represented by grey bars whilst extracellular activities are shown in white bars. The mock-electroporated sample was used as blank for the α-Amylase assays. These individual activities were used to calculate the secretion indices in figures 2B and 5A,B. A) Effect of either SYP21δTM or full-length SYP21 on Amy secretion. Data are from five independent protoplast transfections ±SE. B) Effect of YFP-SYP21 on Amy-spo transport. Data are from three independent protoplast transfections ±SE. C) Effect of YFP-SYP21 on Amy transport. Data are from three independent protoplast transfections ±SE. Note that the secretion index reflects an increased secretion of Amy-spo caused by the effector YFP-SYP21 and that none of the effectors exhibit a noticeable shift in the distribution of the constitutive secretion marker Amy. Note also that total activities increase for Amy-spo when vacuolar sorting is inhibited, possibly by preventing proteolysis in the vacuoles. In contrast, total Amy activities are slightly reduced as a result of co-expression. The latter is well known in the field and thought to be caused by non-specific competition for transcription-translation machinery.
• Supplemental Figure 2 - YFP-SYP21 overproduction reduces vacuolar transport of GFP-spo. Transient expression experiment (24 hours incubation) with tobacco leaf protoplasts electroporated with plasmids encoding the vacuolar cargo molecule GFP-spo alone (left panel) or with YFP-SYP21 (right panel). The figure shows a cross-section of the two protoplasts to illustrate distribution of GFP-spo between the cell-cortex (containing cytosol, ER and organelles) and the central vacuole. Note the diffuse GFP fluorescence in the central vacuole, which is significantly reduced upon co-expression with YFP-SYP21 (right panel). Note also bright punctate fluorescent structures highlighted by both GFP-spo and YFP-SYP21 in the right panel (white arrows). These structures are easily appreciated in the cortex view (Figure 6B). The fluorescence intensity was measured across the middle section of the protoplasts. The graphs below represent the intensity of fluorescence expressed in arbitrary units (intensity) along the y axis, and the length of the transect along which the fluorescence measurements were gathered is indicated along the x axis in microns (Distance). Red line, YFP fluorescence intensity; green line, GFP fluorescence intensity. Scale bar 5 μm. The black arrow in the right graph indicates one of the bright punctate structures highlighted by GFP-spo and YFP-SYP21. Note that both red and green signals increase simultaneously.
• Supplemental Figure 3 - GFP-SYP21 overproduction does not influence plasma membrane targeting of YFP-SYP121. A) Cross-section of a protoplast transfected with YFP-SYP121. Note the typical plasma membrane localization. Scale bar 5 μm. B) Cortex of a protoplast transfected with YFP-SYP121 and GFP-SYP21. Note that both markers highlight distinct punctuate structures without overlap, showing that YFP-SYP121 is not trapped in the SYP21 compartment, in contrast to GFP-spo, YFP-SYP22 and GFP-BP80 (Figure 6). Scale bar 5 μm.
• Supplemental Movie 1 - YFP-SYP21 labels small highly mobile PVCs as well as large PVC-clusters. Cortex of a protoplast expressing YFP-SYP21. Note that large PVC-clusters undergo continuous morphological changes and are still mobile in the cell.
• Supplemental Movie 2 - YFP-SYP21 labels small highly mobile PVCs as well as large PVC-clusters. Epidermis cell expressing YFP-SYP21. Note that large PVC-clusters undergo continuous morphological changes and are still mobile in the cell.