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Figure S1. Sequence alignment of Arabidopsis KNOLLE with putative orthologues from other flowering plant species (yellow background) and other Arabidopsis syntaxins representing the five syntaxin groups (green background). Identical amino-acid residues are colour-coded, positions are indicated on the right. Consensus sequence, degree of conservation, sequence logo and predicted domain organisation (Ha, Hb, Hc, N-terminal helices; SNARE, SNARE domain; TA, tail anchor) are shown below the alignment. *AtSYP121 and Oryza sativa Indica (EAY91785) sequences are 346aa and 521aa long but only amino-acid residues up to 336 and 322, respectively, are shown, although full-length sequences were used for the alignment (Clustal Plug-in, CLC Main-Workbench 6.0, CLC bio A/S, Denmark). Domains were added manually in Adobe Indesign (Adobe Systems Incorporated). All sequences were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/).

Figure S2. Expression analysis of chimeric proteins. Protein extracts from flower buds of transgenic plants (two independent lines for each chimera except #21) were subjected to western-blot analysis using anti-Myc monoclonal antibody. Alpha-tubulin was used as loading control. Numbers in brackets at the top identify transgenes (see Figures 1, 2, 4–6); Col, non-transformed plant; (KN), Myc-KN. Numbers on the left indicate molecular weights (kD, kilodaltons).

Figure S3. Subcellular localisation of chimeric proteins in BFA-treated seedling roots. Immunofluorescence images of the subcellular localisation of chimeric proteins (anti-Myc, red) in mitotic and non-mitotic cells of BFA-treated seedling roots. Numbers on the left indicate transgenes (depicted in Fig. 1a). Only chimeras encoded by transgenes 1, 3 and 5 localised to BFA compartments. Endogenous KNOLLE (KN, green) and DNA (blue, overlay) are also shown. Scale bars, 5 μm.

Figure S4. Subcellular localisation of chimeras with swapped N-terminal region. (a) Higher magnification of cell-plate areas in seedlings roots expressing chimeras from transgenes 12 and 13 (anti-Myc, red; compare with Fig. 4). Neither chimera co-localised with endogenous KNOLLE (green). (b) Subcellular localisation of chimeric proteins (anti-Myc, red) encoded by transgenes 10-13 (see Fig. 4a) in response to BFA treatment. All chimeras except #13 accumulated in BFA compartments. Endogenous KNOLLE (KN, green) and DNA (blue, overlay) are also shown. Scale bars, 5 μm.

Figure S5. Localisation of chimeric proteins relative to different subcellular markers in BFA-treated seedling roots. (a) Golgi marker γCOP (anti-SEC21, green) partially co-localised with chimera (anti-Myc, red) encoded by transgene #12 but not #13 (see Fig. 4). (b) TGN marker ARF1 (anti-ARF1, green) co-localised with chimeras (anti-Myc, red) encoded by transgenes #10 and #12 but not #13. DNA was DAPI-stained (blue, overlay). Scale bars, 5 μm.

Figure S6. Localisation of N-terminally truncated syntaxins relative to subcellular markers. (a) Higher magnification of cell-plate areas showing co-localisation of endogenous KNOLLE (anti-KN, green) with truncated KNOLLE (anti-Myc, red) encoded by transgene #17 but not with truncated PEP12 (anti-Myc, red) encoded by transgene #18 (see Fig. 5). In this case, the distribution of truncated PEP12 resembles PEP12 localisation (Müller et al., 2003). (b) Localisation of truncated KNOLLE (#17) and PEP12 (#18) relative to subcellular markers (green) in BFA-treated seedling roots. Truncated KNOLLE (anti-Myc, red, #17) co-localised with TGN marker ARF1 (anti-ARF1, green). In contrast, truncated PEP12 (anti-Myc, red, #18) did not co-localise with Golgi marker γCOP (anti-Sec21, green) and TGN marker ARF1 (anti-ARF1, green). DNA was DAPI-stained (blue, overlay). Scale bars, 5 μm.

Table S1. List of oligonucleotide primers used. *The first PCR with primers forward-1 and reverse-1 or forward-2 and reverse-2 from a specific template was followed by another PCR of the combined PCR products with primers forward-1 and reverse-2. #PCR 1 with primers forward-1 /reverse-1 and forward-2/reverse-2 using Pep12 template. PCR 2 with forward-3/reverse-3 and forward-4/reverse-4 from KNOLLE. Products from PCR1 were used for PCR3 using forward-1/reverse-2. PCR 2-products and product from PCR3 were used for final PCR using primers forward-3/reverse-4.

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