Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. Identification of the punctate structures labeled by secGFP in the presence of RAB-E1^d[NI]. (A) The large faint punctate structures (arrows) labeled by secGFP (green) in the presence of RAB-E1^d [NI] colocalize with Golgi stacks labeled by ST-YFP (red), but the small bright punctate structures (arrowheads) do not. (B) The small bright punctate structures (arrows) labeled by secGFP (green) in the presence of RAB-E1^d [NI] colocalize with a subpopulation of FM4-64-labeled compartments (red); the diffuse red signal is from the cell wall. (C) Z-projection of a series of confocal sections showing that the small bright punctate structures (arrows) labeled by secGFP (green) in the presence of RAB-E1^d [NI] colocalize with a subpopulation of YFP-RAB-F2^b-labeled compartments (red).
• Supplemental Figure 2 - Supplemental Figure 2. secGFP but not GFP-HDEL accumulates in the vacuole in the presence of E64-d. Confocal images of tobacco leaf epidermal cells expressing secGFP (A) or GFP-HDEL (B) following treatment with E64-d in darkness for 14 hours.
• Supplemental Figure 3 - Supplemental Figure 3. Dominant-negative mutants in the Rab-E, Rab-D2, and Rab-F2 subclasses have differing effects on aleu-GFP traffic. (A)-(F) Single confocal sections, taken with identical imaging parameters, of aleu-GFP fluorescence in E64-d-treated leaf tissues in which expression of aleu-GFP is sufficiently high that apoplastic accumulation is clearly seen in most control cells (A and B). (A) aleu-GFP only (B) aleu-GFP co-expressed with RAB-E1^d. (C and D) aleu-GFP co-expressed with two independent RAB-E1^d [NI] clones; fluorescence from the vacuole is increased relative to (A) and (B), while apoplastic fluorescence is reduced. (E) aleu-GFP co-expressed with RAB-F2^b [SN]; apoplastic accumulation of aleu-GFP is dramatically increased. (F) aleu-GFP co-expressed with RAB-D2^a [NI]; aleu-GFP accumulates to high levels in the ER. Bar in (A) = 100μM for all images.
• Supplemental Movie 1 - Supplemental Figure 3. Dominant-negative mutants in the Rab-E, Rab-D2, and Rab-F2 subclasses have differing effects on aleu-GFP traffic. (A)-(F) Single confocal sections, taken with identical imaging parameters, of aleu-GFP fluorescence in E64-d-treated leaf tissues in which expression of aleu-GFP is sufficiently high that apoplastic accumulation is clearly seen in most control cells (A and B). (A) aleu-GFP only (B) aleu-GFP co-expressed with RAB-E1^d. (C and D) aleu-GFP co-expressed with two independent RAB-E1^d [NI] clones; fluorescence from the vacuole is increased relative to (A) and (B), while apoplastic fluorescence is reduced. (E) aleu-GFP co-expressed with RAB-F2^b [SN]; apoplastic accumulation of aleu-GFP is dramatically increased. (F) aleu-GFP co-expressed with RAB-D2^a [NI]; aleu-GFP accumulates to high levels in the ER. Bar in (A) = 100μM for all images.
• Supplemental Movie 2 - Supplementary video 2. In the presence of RAB-E1^d [NI], secGFP (green) accumulates in two morphologically distinct mobile punctate compartments that are not labeled by the ER marker, YFP-HDEL (red). This cell is different from that shown in Figure 3A-F. Time-series of 34 confocal sections collected at 1.6 second intervals; played at 3 frames per second; signals in the red and green channels have been merged.
• Supplemental Movie 3 - Supplementary video 3. The small bright punctate structures (arrows) labeled by secGFP (green) in the presence of RAB-E1^d [NI] colocalize with a subpopulation of mobile FM4-64-labeled compartments (red); the diffuse red signal is from the cell wall. Time-series of 11 confocal sections collected at 1.6-second intervals; played at 3 frames per second.
• Supplemental Movie 4 - Supplementary video 4. The small bright punctate structures (arrows) labeled by secGFP (green) in the presence of RAB-E1^d[NI] colocalize with a subpopulation of mobile YFP-RAB-F2^b-labeled compartments (red). Time-series of 25 confocal sections collected at 0.9-second intervals.
• Supplemental Movie 5 - Supplementary video 5. YFP-RAB-E1^d (red) colocalizes with the Golgi-stacks (red arrows) that are weakly labeled by secGFP (green), but not with the PVC compartments that accumulate secGFP. Time-series of 10 confocal sections collected at 4-second intervals.
• Supplemental Movie 6 - Supplementary video 6. secYFP exhibits weaker fluorescence than secGFP in the PVC and cell wall. The movie shows a 3-dimensional reconstruction of the upper portion of epidermal cells co-expressing secGFP and its spectral variant secYFP. The image was collected approximately 50h after infiltration, when intracellular accumulation and transport of the fluorescent markers are at their maximum (Figure 1A). Both markers are clearly evident in the ER network where signals of similar intensity were detected under these conditions, but the YFP signal is reduced in the cell wall and PVC which appear green.