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LEGENDS OF SUPPORTING INFORMATION Supplemental figure legends: Figure S1: The large GOLD36 aggregates are not nuclei. Confocal images of GOLD36 cotyledon epidermal cells treated with propidium iodide (PI) showing nuclei (arrowheads) and cell wall (empty arrow) labeled by the dye. A large aggregate (arrow) is distinguishable from the nucleus (arrowhead) (merged image). Bar = 20 μm. Figure S2: The GOLD36 structures are not the result of ST-GFP expression. Confocal images of cotyledon epidermal cells of nonmutagenized ST-GFP (n.m. ST-GFP) or a segregating Ler x GOLD36 F2 individual that did not express ST-GFP. Treatment with the general endomembrane dye DiOC6 shows the presence of GOLD36 structures (arrow) only in the mutant. Bar = 20 μm. Figure S3: The presence of GOLD36 structures in the gold36-1 line is not due to transgene overexpression. A. PCR of genomic DNA of Col0 and gold36-1 plants showing the presence of T-DNA (771 bp band) in gold36-1 (line 2) but not in Col0 (line 1), and the presence of partial gold36 genomic sequence (1011 bp) in Col0 (line 3) but not in gold36-1 (line 4). These results indicate that the gold36-1 line isolated here is homozygous for the TDNA insertion. B. Confocal images of cotyledon epidermal cells of either gold36-1 or complemented gold36-1 treated with DiOC6 showing the presence of the aberrant structures only in the gold36-1 line (arrow). Bar = 10 μm. Figure S4: GOLD36 is not secreted to the apoplast. Images of cells GOLD36/35S::gold36mRFP plasmolyzed in mannitol (0.5 M for 20 min) show that the fluorescent signal is present within the cells rather than in the apoplast. For clarity, only the GOLD36-mRFP signal is shown. Arrows point to cell areas with evident plasmolysis. An arrowhead points to a nucleus visible in negative contrast with the vacuolar fluorescence signal. Bar = 50 μm. Figure S5: GOLD36-mRFP is partially redistributed to the apoplast in the presence of wortmannin. To further ensure that GOLD36-mRFP was directed to the vacuole, we used wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, which has been used to perturb traffic to the lytic vacuole in cells of yeast (Schu et al., 1993; Wurmser et al., 1999), mammals (Davidson, 1995; Arighi et al., 2004) and plants (Matsuoka et al., 1995; daSilva et al., 2005; Wang et al., 2009). Because in the presence of the drug, proteins destined to the vacuole are secreted (Kim et al., 2001; Pimpl et al., 2003; daSilva et al., 2005), we hypothesized that GOLD36-mRFP would be at rerouted to the apoplastic space if the vacuole was its final destination. Upon wortmannin treatment of GOLD36::GOLD36-mRFP cotyledons, we found that GOLD36-mRFP was partially secreted to the apoplastic space, confirming our hypothesis that GOLD36-mRFP is destined to post-ER compartments, specifically the vacuole. As shown in this figure, confocal images of GOLD36/GOLD36mRFP cotyledons plasmolyzed in mannitol (0.5 M) either untreated (control) or treated with wortmannin, and images of treated and plasmolyzed cotyledons stably expressing the ER lumenal marker [ssGFP-HDEL; (Brandizzi et al., 2003a)]. The mRFP signal is exclusively visible in the vacuoles (stars) in the control, while it is also visible in the apoplast (arrows) in the wortmannin-treated sample. Consistent with the known effect of the drug on rerouting vacuole-destined proteins to the apoplast, these data support post-ER traffic of GOLD36mRFP. As expected, wortmannin did not have an effect on the distribution of the ER lumenal marker ssGFP-HDEL, which was retained in the ER (empty arrows). In the GOLD36/35S::GOLD36-mRFP only the mRFP channel is shown for clarity. Note the presence of chloroplasts (arrowheads) that are visible in the GOLD36/35S::GOLD36-mRFP images but not in the ssGFP-HDEL image because the microscopy imaging settings for mRFP enable also the capture of the chlorophyll autofluorescence signal. Bar = 20 μm. Figure S6: GOLD36-mRFP can be found in the ER in transient expression. To establish whether GOLD36-mRFP could be transiently localized in the ER, we transformed Arabidopsis seedlings using a transient expression procedure used previously (Marion et al., 2008; Agee et al., 2010). We found that in transient expression, GOLD36-mRFP was localized in the ER. As shown in this figure, confocal images of cotyledons of Arabidopsis thaliana plants stably expressing the ER luminal marker ssGFP-HDEL (Brandizzi et al., 2003a). Plants were transformed transiently with GOLD36-mRFP as described earlier (Marion et al., 2008). The images show that in these conditions of expression GOLD36mRFP is localized in the ER (arrows). In addition to the ER localization, GOLD36-mRFP is also present in the vacuole (star). Bar = 5 μm. Figure S7: Additional evidence that GOLD36-mRFP may be localized in the ER in transient expression.Three-dimensional reconstruction of consecutive confocal optical sections of tobacco leaf epidermal cells transiently expressing GOLD36-mRFP upon Agrobacterium tumefaciens mediated transformation. This is a well-established method for following the distribution of fluorescent protein fusions in dose-response experiments (Batoko et al., 2000; Zheng et al., 2005; Stefano et al., 2006; Samalova et al., 2008; Osterrieder et al., 2010). The subcellular localization of GOLD36-mRFP was followed in two conditions of optical density (OD600 = 0.02 and 0.2) of agrobacteria bearing the GOLD36mRFP plasmid and using identical imaging conditions (i.e. laser configuration and intensity settings, detector gain amplitude, and pinhole settings). As a marker of the ER we used ssGFP-HDEL (OD600 = 0.02). Imaging of tissue expressing ssGFP-HDEL alone represents the control for autofluorescence in the mRFP channel. The bacterial optical density used for GOLD36-mRFP transformation is indicated at the left side of the images. We hypothesized that at low levels of bacterial optical density (OD600 = 0.02), GOLD36mRFP would be mainly visible in the vacuole; however at a higher bacterial optical density (OD600 = 0.2), the signal would be clearly accumulated also in the ER due to higher levels of expression. Differences in expression levels would be indicated by the intensity of the fluorescence signal in the confocal imaging. Consistent with our hypothesis, in conditions of transformation at a lower bacterial optical density, GOLD36-mRFP was distributed in the vacuole (arrowheads) and faintly in the ER (arrows). Compared to these experimental conditions, cells transformed with a higher optical density showed a marked ER localization of GOLD36-mRFP (arrows). In both conditions of optical density, GOLD36-mRFP also appeared in the apoplast (stars), most likely because of saturation of the vacuolar traffic route (Tormakangas et al., 2001; Pimpl et al., 2003). Empty arrows indicate chloroplasts, which are visible with the settings used for mRFP acquisition. Individual frames of each 3D reconstruction are presented as a sequence in Supplemental Video S3. Bar = 20 μm. Supplemental video legends: Video S1: ST-GFP signal is restricted to Golgi stacks in non-mutagenized plants, whereas it is also distributed to other structures in GOLD36. Sequential confocal optical 1-μm thick sections of cotyledon epidermal cells of non-mutagenized ST-GFP (Video S1A) and GOLD36 (Video S1B). Arrow points either to Golgi stacks (Video S1A) or to the large globular structure (Video S1B). Video S2: The aberrant GOLD36 structures are motile. Time-lapse confocal images (time of acquisition is indicated at the top left corner) show motility of the structures. Video S3: In transient expression, GOLD36-mRFP is partially localized in the ER. Sequential confocal optical 1-μm thick sections of tobacco leaf epidermal cells either expressing ssGFP-HDEL alone (Video S3A) or co-expressing GOLD36-mRFP (Video S3BC) upon transformation using two different bacterial optical density: OD600 = 0.02 (Video S3B) and OD600 = 0.2 (Video S3C). The depth of each confocal section is indicated at the top left corner of the videos. Supplemental tables legends Table S1: SNPs identified through Solexa sequencing for which ≥ 80% of the overlapping reads supported the SNP call. Table S2: List of primers used in this work. References Arighi, C.N., Hartnell, L.M., Aguilar, R.C., Haft, C.R. and Bonifacino, J.S. (2004) Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J. Cell Biol. 165, 123–133. Batoko, H., Zheng, H.Q., Hawes, C. and Moore, I. 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(2010) Golgi membrane dynamics after induction of a dominant-negative mutant Sar1 GTPase in tobacco. J. Exp. Bot. 61, 405–422. Pimpl, P., Hanton, S.L., Taylor, J.P., Pinto-daSilva, L.L. and Denecke, J. (2003) The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole. Plant Cell, 15, 1242–1256. Samalova, M., Fricker, M. and Moore, I. (2008) Quantitative and qualitative analysis of plant membrane traffic using fluorescent proteins. Methods Cell Biol. 85, 353–380. Schu, P.V., Takegawa, K., Fry, M.J., Stack, J.H., Waterfield, M.D. and Emr, S.D. (1993) Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science, 260, 88–91. daSilva, L.L., Taylor, J.P., Hadlington, J.L., Hanton, S.L., Snowden, C.J., Fox, S.J., Foresti, O., Brandizzi, F. and Denecke, J. (2005) Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. Plant Cell, 17, 132–148. Tormakangas, K., Hadlington, J.L., Pimpl, P., Hillmer, S., Brandizzi, F., Teeri, T.H. and Denecke, J. (2001) A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum. Plant Cell, 13, 2021–2032. Wang, J., Cai, Y., Miao, Y., Lam, S.K. and Jiang, L. (2009) Wortmannin induces homotypic fusion of plant prevacuolar compartments. J. Exp. Bot. 60, 3075–3083. Wurmser, A.E., Gary, J.D. and Emr, S.D. (1999) Phosphoinositide 3-kinases and their FYVE domain-containing effectors as regulators of vacuolar/ lysosomal membrane trafficking pathways. J. Biol. Chem. 274, 9129– 9132. Zheng, H., Camacho, L., Wee, E., Batoko, H., Legen, J., Leaver, C.J., Malho, R., Hussey, P.J. and Moore, I. (2005) A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis. Plant Cell, 17, 2020– 2036.