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Figure S1 Chloroplast localization of tpΔP, P, and ΔP proteins. Transgenic tobacco protoplasts
expressing tpΔP, P, and ΔP were fixed, permeabilized, and subjected to immunofluorescence.
Fluorescence originated by the anti-phaseolin antiserum detected using FITC conjugated anti-rabbit
secondary antibody (green signal), red chlorophyll autofluorescence (chlorophyll), and the overlay
of the two fluorescences are shown. Bars: 4 µm.
Fig. S2. Analysis of the correct transgene insertion in supertransformed (SupT) tobacco plants.
Regeneration capacity of WT (a-b), SupT (c-d) and tpΔP explants (e-f) on regeneration medium
with or without antibiotic selection (kanamycin 50 mg/l, spectinomycin 500 mg/l). (g) PCR
amplification on DNA extracted from four SupT plants. In the upper panel primers 3M/3P were
used to verify the integration of the phaseolin gene into the plastome. The lower panel shows the
expected amplification fragment due to the integration of the TpΔP gene in the plant nucleus
(primers 35S/Zeo3).
C+nu: pGreenII.tpP-FLAG plasmid (positive control for nuclear
transformation); C+cl: a tobacco plant transformed with the pLD-CtV vector (positive control for
plastome transformation).
Fig. S3. Absence of phaseolin polypeptides in the immunoprecipitation supernatant fractions. In
Figure 6a, leaf protoplasts from a WT plant, or from transformed tobacco plants expressing tpΔP, P,
and SupT proteins, were pulse-labelled for 1 h in presence or absence of cycloeximide (CHX).
After protoplast immunoprecipitation with anti-phaseolin antibody, supernatants were recovered,
subjected to a second round of immunoprecipitation with the same antibody and analyzed by SDSPAGE and fluorography.
Fig. S4. Tridimensional conformation of tpΔP and SupT phaseolins in the chloroplast. Total leaf
proteins (0.12 ml of leaf homogenate) extracted from the nuclear-transformed plant tpΔP and from
the supertransformed plant SupT were fractionated by centrifugation on velocity sucrose gradient.
Different fractions were collected and each fraction was analyzed by SDS-PAGE and Western
blotting using anti-phaseolin antiserum. In SupT the large part of phaseolin was constituted by
trimeric tpP polypeptides, whereas P trimers and oligomers were only a minor fraction of the total
phaseolin polypeptides. Numbers on top indicate molecular mass, in kD, of sedimentation markers.
Table S1. Oligonucleotides used in this study
Name
5’NdeIP
3’NotIP
5’NdeIP
SphIP
EcoRIP-FLAG
Sp5’
Sp3’
3M
3P
Zeo3
35S
Sequence (5’-3’)
Tccatcggacatatgatgagagca
ccccctccggatcgcggccgctagtacacaaatgcaccctttcttccct
Tcactttctgcctcacatatgacttcactccgggag
Ccaagattttcgcatgctcacttcactccggg
gagcggccgcgaattcctagtacacaaatgc
atgatgagagcaagggttcc
ggcaaatgaggcagaaagtg
ccgcgttgtttcatcaagccttacg
aaaacccgtcctcagttcggattgc
ccaagacgagtatggcgctc
catggagtcaaagattcaaa
Application
ORF phaseolin in pCR 2.1-5’UTR
ORF phaseolin in pCR 2.1-5’UTR
ORF phaseolin in pCR 2.1-5’UTR
Phaseolin with transit peptide in pJIT117
Phaseolin with transit peptide in pJIT117
Phaseolin signal peptide as a probe
Phaseolin signal peptide as a probe
Verify transgene cassette insertion
Verify transgene cassette insertion
Verify transgene cassette insertion
Verify transgene cassette insertion