Supplementary materials Oligonucleotides used in this work. Name Sequence (5 ’-3’) PGL 35-F gat ttc aaa atc ccc atg gcg gac atc gac gac g PGL 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 gc Strep-F gat ata ccc atg gct agc atg act gg Strep-R cg cgc cat ggg ctg aac ggc gtc gag c HCV-F ccg tgc cat atg agc acg aat cc HCV-R gga aga tct aga aag agc aac cag g HCV-BclI cct agg ttg atc atc tag aaa gag caa ccg g PGL35-BclI cca cga atg atc acc cat ggc gga cat cg P24-BclI cct agg ttg atc atc tag agg aat tct act cta gc P24-F cgc cct tca tat ggc tag cgg atc c P24-R cgc cct ttc tag agg aat tct act cta gc

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.

(a) WT 1.1 2.5 (b) 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 WT 1.1 2.5 (c) WT 1.1 2.5

PGL25 PGL29 Thylakoid PG/thylakoid Isoelectric point (pI) Strep-PGL35-HIVp24 PG core PGL45 PGL34-YFP PGL35 PGL34 PGL40 Hydrophobicity (GRAVY Index) 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 97 66 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).

TableS1Chlorophyll content and fluorescence in leaves of tobacco wild-type and Streptavidin-PGL35-fusion lines pVSB1 (1.1) and pVSB2 (2.5) Plant Total Chlorophyll (mg/g fresh weight±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 Fv/Fm – maximum photochemical quantum yield of photosystem II