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. 2024 Jul 30;196(2):1196–1213. doi: 10.1093/plphys/kiae404

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© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — SlCOP1-1 interacts with SlOpaque2 in the nucleus. A) Yeast 2-hybrid assay confirming the interaction between SlCOP1-1 and SlOpaque2. SlCOP1-1 fused with the BD domain of GAL4 (BD-SlCOP1-1) was co-expressed with SlOpaque2 fused with the AD domain of GAL4 (AD-SlOpaque2) in yeast. The recombinant yeasts were selected on SD/-Leu/-Trp (-LW), SD/-Leu/-Trp/-Trp (-LWH), and SD/-Leu/-Trp/-His/-Ade (-LWHA) cultural media, with or without X-α-gal. Negative controls include parallel co-expression of BD-SlCOP1-1/AD, BD/AD-SlOpaque2, and AD/BD. B) Luciferase complementation imaging assay revealing the interaction between SlCOP1-1 and SlOpaque2. SlCOP1-1 fused with the N-terminus of luciferase (SlCOP1-1-nLUC) was transiently co-expressed SlOpaque2 fused with the C-terminus of luciferase (cLUC-SlOpaque2) in Nicotiana benthamiana leaves. Scale bar, 1 cm. C) A pull-down assay revealing the interactions between SlCOP1-1 and SlOpaque2. Recombinant GST-SlOpaque2, MBP-SlCOP1-1, and MBP tag protein (as a negative control) were mixed as indicated, and incubated with anti-MBP magnetic beads. Immunoblots were conducted to detect the eluted proteins using anti-MBP or anti-GST antibodies. IB, immunoblot. D) Co-immunoprecipitation assay revealing the interaction between SlCOP1-1 and SlOpaque2. SlCOP1-1-HA was transiently co-expressed with SlOpaque2-GFP in N. benthamiana leaves. The mCherry-HA served as a negative control. Total proteins extracted from transformed leaves were immunoprecipitated with anti-HA beads, followed by immunoblot analysis using anti-GFP or anti-HA antibodies. IP, immunoprecipitation. E) Subcellular colocalization of SlCOP1-1 and SlOpaque2. Fusion proteins of SlCOP1-1-eGFP and SlOpaque2-mCherry were co-expressed in N. benthamiana leaves. Non-fused eGFP and mCherry were used as a control. Scale bars, 10 μm.