Present address: Regierung von Oberbayern, Technischer Umweltschutz – Gentechnik, Maximilianstraße 39, 80538 München, Germany.
Functional anatomy of the Arabidopsis cytokinesis-specific syntaxin KNOLLE
Article first published online: 19 SEP 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 68, Issue 5, pages 755–764, December 2011
How to Cite
Touihri, S., Knöll, C., Stierhof, Y.-D., Müller, I., Mayer, U. and Jürgens, G. (2011), Functional anatomy of the Arabidopsis cytokinesis-specific syntaxin KNOLLE. The Plant Journal, 68: 755–764. doi: 10.1111/j.1365-313X.2011.04736.x
- Issue published online: 25 NOV 2011
- Article first published online: 19 SEP 2011
- Accepted manuscript online: 12 AUG 2011 02:07PM EST
- Received 10 May 2011; revised 19 July 2011; accepted 3 August 2011; published online 19 September 2011.
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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