GST Pull-Down, Co-Immunoprecipitation, Yeast Two-Hybrid, Bimolecular Fluorescence Complementation and Subcellular Localization Protocols for Studying SsCP1-AtPR1 Interaction

There are three protocols mentioned in the text.

Protocol 1: GST Pull-Down and Co-Immunoprecipitation

To study the interaction between SsCP1 and AtPR1 using GST pull-down, the plasmids pGEX-6p-1::Sscp1 and pET28a::AtPR1 were expressed in Escherichia coli. Equal amounts of GST-SsCP1 and His-AtPR1 lysates were incubated with high-affinity GST resin overnight. Bound proteins were eluted with glutathione elution buffer and analyzed by SDS-PAGE followed by immunoblotting with anti-GST or anti-His antibodies.

For co-immunoprecipitation, pCNG-Sscp1-GFP with or without pCNF3-AtPR1 constructs were transiently coexpressed in N. benthamiana. Total proteins were isolated using RIPA buffer and subjected to a-FLAG IP. Eluted proteins were analyzed by immunoblotting with anti-GFP antibody.

Protocol 2: Yeast Two-Hybrid System

The Matchmaker Gold Yeast Two-Hybrid System was used to verify SsCP1-interacting proteins from IP-LC-MS/MS. Sscp1 (without signal peptide) was introduced into pGBKT7 as bait, and AtPR1 (without signal peptide) was introduced into pGADT7 as prey. The bait and prey plasmids were co-transformed into yeast strain Y2H Gold. Truncated Sscp1 mutants were generated and subcloned into pGBKT7. Transformed yeast cells were assayed for growth on selective media and for the expression of reporter genes.

Protocol 3: Bimolecular Fluorescence Complementation and Subcellular Localization

Split-mCherry system was used for bimolecular fluorescence complementation (BiFC) analysis. N-terminal and C-terminal fragments of mCherry were subcloned into pCNF3 and pCNM3, respectively. Coding sequences of Sscp1 and AtPR1 were introduced into these vectors to generate pCNRN-Sscp1 and pCNRC-AtPR1. BiFC was conducted by agroinfiltration in leaves of N. benthamiana, and protein expression was confirmed by Western blot.

For subcellular localization, coding sequences of Sscp1 and AtPR1 were introduced into pCNG and pCNR, respectively. Transient expression was carried out by agroinfiltration. Plasmolysis of N. benthamiana cells was performed using 1 M NaCl, and then confocal microscopic analysis was performed. Fluorescence in leaves of N. benthamiana was monitored at 3 d post-agroinfiltration and imaged using a confocal laser scanning microscope (CLSM).