Download LEGENDS OF SUPPORTING INFORMATION Supplemental figure

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.
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