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. 2018 Apr 4;9:613. doi: 10.3389/fmicb.2018.00613

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Copyright © 2018 Zhang, Zhao, Li, Xiang, Dong, Zhang, Wang, Li, Yu and Han.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Self-interaction identification of P3a proteins in vivo. (A) BiFC assays. P3a-YFPN and P3a-YFPC were co-expressed by agro-infiltration in leaf epidermal cells of N. benthamiana plants. The upper panel shows a YFP fluorescence image showing the generation of the intracellular fluorescence. The lower panel shows an overlay of a bright-field image and the upper panel. Bars = 10 μm. (B) Co-immunoprecipitation immunoblot analysis. Dual combinations of P3a tagged with 3× Flag epitope and GFP epitope were co-infiltrated in N. benthamiana leaves and extracts were analyzed at 3 dpi. Co-immunoprecipitation analysis were performed, and the input and immunoprecipitated (IP) proteins were analyzed using anti-Flag and anti-GFP antibodies. The blue arrow indicates monomer and the red arrow indicates dimer. (C) The self-interaction of P3a in the split ubiquitin yeast two-hybrid assay. P3a was used as the fused bait protein (P3a-Cub) and the fused prey protein (NubG- P3a). Yeast strain NMY51 co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions, and grown on a SD/-Leu/-Trp/-His/-Ade or SD/-Leu/-Trp medium. The interaction of P3a-Cub/NubI and P3a^P18L-Cub/NubI are shown as positive control. The interaction of P3a-Cub/NubG and P3a^P18L-Cub/NubG are shown as negative control. (D) Prokaryotic expression of P3a and P3a^P18L. The prokaryotic expression of His.MBP-P3a and His.MBP-P3a^P18L fusion proteins were induced. U, uninduced protein. I, 0.1 mM IPTG induced protein. M, protein marker. The expression of the two fusion proteins were analyzed and identified by 12.5% SDS-PAGE (lower panel) and western blotting (upper panel), respectively. Red and blue arrow marks show that the fusion proteins migrated in their dimeric and monomeric forms, respectively. The MBP monoclonal antibody in mouse was used to detect the expression of fusion protein.