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Additional Supporting Information may be found in the online version ofthis article:

Figure S1: Overexpression of SYP31 has both an immediate and a long-term inhibitory effect on secretion. A–C) Co-electroporations of tobacco mesophyll protoplasts with plasmid DNAs encoding for α-amylase (5 μg) and HA-tagged SYP31 (5 μg) as well as control electroporations with α-amylase alone were performed over a 24-h expression period with samples being removed at the times indicated. In addition to the calculation of the secretory index (A), the total activity of α-amylase (B), the results of protein gel blotting for secreted α-amylase and the expression of the SNARE are given (C).

Figure S2: Control organelle marker signals in tobacco protoplasts. A–E) Electroporations of tobacco mesophyll protoplasts were performed as in Figure 2 but with standard marker proteins for the Golgi apparatus (A), the ER (C and E) and the plasma membrane (D). In addition, a protoplast from leaves stably expressing the Golgi marker ST–GFP is presented (B).

Figure S3: Movie showing Golgi localization of YFP–SYP31 and ST–CFP in tobacco leaf epidermis.

Figure S4: Movie showing the mobility of BFA-resistant YFP–SYP31 structures in tobacco leaf epidermis.

Figure S5: BFA resistance of SYP31 structures is independent of duration of SYP31 expression. A–E) Tobacco leaf protoplasts were electroporated with 5 μg plasmid DNA encoding for Man1–GFP (A, D and G), GFP–SYP31 (B, C, E and F) or SYP31–GFP (H and I) and observed in the CLSM after 7-h (A–D) or 24-h (E–I) expression. BFA (end concentration10 μg/mL) was added to the medium after 6-h (A–D) and 23-h (E–I) expression. Whereas the Golgi marker protein Man1–GFP is redistributed into the ER (D), the punctate fluorescent signals for GFP–SYP31 remain intact and become larger with increased expression (compare D with B and E). In the case of SYP31–GFP, BFA causes only part of the signal to move into the ER.

Figure S6: Secretion index measurements for HA-tagged SNAREs (BS14a, SEC22 and MEMB11). In agreement with the secretory index measurements for WT and YFP-tagged SNAREs, only overexpressed MEMB11 is effective in inhibiting secretion.

Figure S7: YFP–SYP81 localizes to the ER in tobacco mesophyll protoplasts. YFP–SYP81 was co-electroporated with ER markers (GFP–HDEL and calnexin–GFP). Although the YFP–SYP81 fluorescent signal was in the main punctate, the punctae were always distributed on the ER.

Table S1: SNARE sequence similarities (Tomato, Arabidopsis, Yeast and Human)

Table S2: SNARE sequence similarities (Arabidopsis, Yeast and Human)

Table S3: List of constructs used in this study together with sense and antisense oligonucleotides

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.