Additional file 1: of Proximity-dependent biotinylation screening identifies NbHYPK as a novel interacting partner of ATG8 in plants

Figure S1. Full immunoblot images for Fig. 1a using ATG8 antibody. (a) Mock. (b) TMV. (c) TMV 24A + UPD. Figure S2. Expression of BirA* and BirA_ATG8 constructs. (a) Graphical representation of control BirAand experimental plasmids BirA-ATG8. Full gel image for Fig. 2a carried out using anti-mCherry antiseri, for both (b) & (c) and anti-ATG8 antiserum (* indicates a weak band). (d) to ascertain fusion protein expression. (e) Detection of RFP signals from agroinfiltrated N.benthamiana leaves to confirm expression of RFP-fused BirA* and BirA*-ATG8. Figure S3. Full gel Immunoblot analysis of total biotinylated proteins for Fig. 2b and c. (a) Streptavidin-HRP blot of crude protein lysate. (b)Strep-HRP blot after DynabeadsTM purification. Abbreviations: b, beads; s, supernatant. Lane 1. WT- Biotin only. Lanes 2–3,-BirA*, lanes 4–5, BirA*-ATG8, lane 6-BirA*, lane 7, BirA*-ATG8, lane 8, BirA*, lane 9, BirA*-ATG8. Different treatments as follows: Samples 2–5, biotin was infiltrated 3 days after agroinfiltration. Samples 6–9, biotin-infiltrated concurrently with agroinfiltration buffer. Samples 6 and 7, proteins extracted 3 dpi and samples 8 and 9 were collected 4 dpi. Treatments 6 and 7 were used for the final experiment. Figure S4. ATG8 directly interacts with NbHYPK but not NbHYPKΔUBA and UBA-NbHYPK (Fig. 4). (a) GFP-NbHYPK, GFP-NbHYPKΔUBA, GFP-UBA-HYPK and RFP-ATG8 fusion proteins were detected in Western blot using GFP and RFP antibodies, respectively. (b) RFP-ATG8 aggregates with GFP-NbHYPK but not GFP -NbHYPKΔUBA or GFP-UBA-NbHYPK. (c) Bimolecular fluorescence complementation (BiFC) analysis showed that ATG8 was able to associate with NbHYPK but ATG8-Yn did not associate with x-Yc (plasmid without insert). NbHYPK-Yc also did not associate with x-Yn (plasmid without insert). Figure S5. Analysis of gene down-regulation in ATG5/7/8-VIGS plants (Fig. 5). (a) Semi-quantitative RT-PCR expression analysis of ATG8 isoforms in ATG8-silenced and non-silenced Nicotiana benthamiana plants. (b) qRT-PCR analysis of ATG5 and ATG7 genes in control and VIGS-plants. Figure S6. TMV accumulation and NbHYPK relative expression in VIGS-NbHYPK plants (Fig. 6). (a) TMV coat protein expression as detected by anti-TMV antibody. TMV coat protein is ~ 17.5 kDa. Statistical significance was determined by Student’s t test (p = .39). (b) Semi-quantitative qRT-PCR analysis of NbHYPK expression in VIGS silenced and non-silenced Nicotiana benthamiana plants. (ZIP 3798 kb)