Name Sequence (5 ’-3’)PGL 35-F GAT TTC AAA ATC CCC ATG GCG GAC ATC GAC GAC GPGL 35-R CTT GTT CTA GAT CAT ATG GCC TTG CAG ATA GAC CAG TTC GGG CCC CTG CCC GTA GAA

CAG CTC CCC CTG CTT GTA CAT CAT TTC AGG GTT TAA GAG AGA GCStrep-F GAT ATA CCC ATG GCT AGC ATG ACT GG

Strep-R CG CGC CAT GGG CTG AAC GGC GTC GAG CHCV-F CCG TGC CAT ATG AGC ACG AAT CC

HCV-R GGA AGA TCT AGA AAG AGC AAC CAG GHCV-BclI CCT AGG TTG ATC ATC TAG AAA GAG CAA CCG GPGL35-BclI CCA CGA ATG ATC ACC CAT GGC GGA CAT CGP24-BclI CCT AGG TTG ATC ATC TAG AGG AAT TCT ACT CTA GCP24-F CGC CCT TCA TAT GGC TAG CGG ATC CP24-R CGC CCT TTC TAG AGG AAT TCT ACT CTA GC

Oligonucleotides used in this work.

Supplementary materials

Figure. S1. a Amino acid sequence of coding regions of the Streptavidin-PGL35-HCV core protein

Figure. S1. b Amino acid sequence of coding regions of the Streptavidin-PGL35-HIVp24

Figure. S2 Alignment of amino acid sequences for PGL35 from Arabidopsis, tobacco, rice, tomato, Medicago and potato. The N-terminal transit peptide amino acids were removed and sequence alignment was performed by CLUSTALW.

WT1.1 WT2.5

1.1WT

(a)

(c)

WT 1.1 2.5

(b)2.5

Figure. S3 Phenotype of wild- type and transplastomic lines(a) The wild-type and transplastomic seedlings pVSB1 (1.1), pVSB2 (2.5) 7 days after sowing on soil.(b) Wild-type, pVSB1 (1.1) and pVSB2 (2.5) transplastomic plants were grown on soil for 4 weeks.(c) 12-week-old plants (T1 generation) from transplastomic lines pVSB1 (1.1) and pVSB2 (2.5) grown in a greenhouse are phenotypically indistinguishable from wild-type tobacco

-0.38 -0.28 -0.18 -0.083.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

10

PGL35

PGL40

PGL45

PGL25PGL29

Thylakoid

PG coreStrep-PGL35-HIVp24

PG/thylakoid

Hydrophobicity (GRAVY Index)

PGL34

PGL34-YFP

Isoe

lect

ric

poin

t (pI

)

Figure. S4 Plastoglobulin and Streptavidin-PGL35-HIVP24 fusion protein localization is determined by isoelectric

point and hydrophobicity. The graph adopted from (Lundquist et al. 2012).

TC TC TM S

pVSB2 (2.5)

Amidoblack

31

9766

44

kDa

116

Anti-PGL40

Anti-RLSU

Anti-Lhcb2

WT

Figure. S5 Total chloroplast (TC) were isolated from wild type (WT) and pVSB2 (2.5). The transplastomic pVSB2 (2.5) chloroplasts were lysed and separated into total membranes (TM) and stroma (S). 10µg of protein were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antisera to the proteins as indicated. PGL40 (plastoglobule marker), RLSU (stroma marker) and Lhcb2 (thylakoid marker).

Plant

Total Chlorophyll(mg/g freshweight±SD)

  Fv/Fm

 Wild type

 1.18±0.02

 0.786±0.018

 1.1

 1.19±0.04

 0.776±0.011

 2.5

 1.17±0.02

 0.786±0.019

TableS1Chlorophyll content and fluorescence in leaves of tobacco wild-type and Streptavidin-PGL35-fusion lines pVSB1 (1.1) and pVSB2 (2.5)

Fv/Fm – maximum photochemical quantum yield of photosystem II