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Figure  S1. The coiled-coil region of MYA1 consists of two major coiled-coil sections. (a) Two major sections of coiled-coil structures are predicted in MYA1 by COILS and Paircoil2 algorithms. (b) Schematic representation of a parallel dimeric coiled-coil section 1 (CC1). (c) Schematic representation of a parallel dimeric coiled-coil section 2 (CC2). Only the residues at core (a and d) positions or e and g positions are indicated. The underlined residues stabilize the coiled-coil dimerization while asterisks indicate residues predicted to destabilize the coiled-coil dimerization.

Figure  S2. The coiled-coil regions of MYA2, XI-I and XI-K affect the targeting of their globular tails in Arabidopsis leaf epidermis. All constructs were observed in Arabidopsis leaf epidermal cells following transient expression after particle bombardment. (a, b, c) YFP-2GT, YFP-iGT and YFP-kGT showed diffuse cytoplasmic distribution. (d, e, f) YFP-2CCGT, YFP-iCCGT and YFP-kCCGT labeled punctate structures in the cytosol. Scale bar = 20 μm.

Figure  S3. BiFC assays indicate that the coiled-coil region mediates MYA1 dimerization in tobacco leaf epidermis. (a) Restored YFP fluorescence was detected in BiFC combination of YN-GCN4CC + YC-GCN4CC. (b, c, e) No restored YFP fluorescence was detected in MYA1 BiFC combinations YN-CC + YC-CC, IQCC-YN + IQCC-YC and YN-CCGT + YC-KCA1CCGT. The inset shows peroxisome-labeling CFP fluorescence in the same cell as transformation marker at half-size. (d) Restored YFP fluorescence was detected in MYA1 BiFC combination YN-CCGT + YC-CCGT. All constructs were observed following transient expression after agrobacteria co-infiltration. Scale bar = 20 μm.

Figure  S4. Dimerization of MYA1 coiled-coil constructs in tobacco leaf epidermis measured by FRET. (a–d) The combinations YFP-CC + Cerulean-CC, IQCC-YFP + IQCC-Cerulean, YFP-CCGT + Cerulean-CCGT and YFP-KCA1CCGT + Cerulean-CCGT demonstrated FRET signal with variable intensities. Nfret represents a corrected FRET value and is reflected by the pseudocolor coded for a range from 0.0 (blue) to 1.0 (red). Arrows point to the representative punctate structures labeled by fluorescently tagged CCGT constructs. All constructs were observed following transient expression after agrobacteria co-infiltration. Scale bar = 20 μm.

Appendix  S1.Experimental procedures (bioinformatics analysis, plasmid construction, transient expression in tobacco leaves), results and discussion (additional considerations of MYA1 coiled-coil prediction, prediction of coiled-coil dimerization strength based on primary sequence).

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TPJ_3522_sm_fig2.tif2017KSupporting info item
TPJ_3522_sm_fig3.tif1084KSupporting info item
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