Protein Interaction Studies of fmkj1 and xydb2: GST Pull-Down, Co-Immunoprecipitation, Yeast Two-Hybrid, and BiFC Analysis

Protocol 1: GST Pull-Down and Co-Immunoprecipitation

For GST pull-down, plasmids pGEX-6p-1, pGEX-6p-1::fmkj1, pET28a, and pET28a::xydb2 were introduced (separately) into the Escherichia coli strain BL21 (DE3), and the expression of proteins was induced with 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at 20°C for 8 h. Equal amounts of GST-fmkj1 and His-xydb2 sonicated lysates were mixed with high-affinity GST resin (GenScript, Piscataway, NJ, USA) and incubated at 4°C overnight with rotation. The bound proteins were then eluted with fresh 10 mM glutathione elution buffer. Next, the proteins were separated by 12% SDS-PAGE and immunoblotted with anti-GST or anti-His antibody (Proteintech, Rosemont, IL, USA).

To validate the protein interactions of fmkj1 and xydb2, co-immunoprecipitation (Co-IP) assays were performed as follows. pCNG-fmkj1-GFP with or without pCNF3-xydb2 constructs was transiently coexpressed in N. benthamiana by agroinfiltration. Total proteins were isolated by homogenizing tissues with RIPA buffer 3 d after agroinfiltration. Proteins were extracted from leaves using RIPA buffer, and subjected to a-FLAG IP. Eluted proteins (25 ul) were subjected to immunoblot analysis with anti-GFP antibody. Approximately 25 ul of RIPA buffer containing the total proteins was loaded as an input control.

Protocol 2: Yeast Two-Hybrid System

The Matchmaker Gold Yeast Two-Hybrid (Y2H) System (Clontech, Palo Alto, CA, USA) was used to verify fmkj1-interacting proteins from IP-LC-MS/MS. Fmkj1 (without signal peptide, SP) was introduced into pGBKT7 as bait, while xydb2 (without SP) was introduced into pGADT7 as prey. The bait and prey plasmids were co-transformed into yeast strain Y2H Gold according to the manufacturer’s instructions. Truncated fmkj1 mutants with different deletions in the N-terminal region were generated by PCR amplification and were subcloned into pGBKT7 (Table S1). Transformed yeast cells were assayed for growth on synthetic dropout (SD)/-Trp-Leu plates and SD/-Trp-Leu-His-Ade plates containing X-a-galactosidase (X-a-gal) and aureoba-sidin A (AbA).

Protocol 3: Bimolecular Fluorescence Complementation and Subcellular Localization

A split-mCherry system was used for bimolecular fluorescence complementation (BiFC) analysis as described previously (Fan et al., 2008; Li et al., 2012). N-terminal and C-terminal fragments of monomer RFP (mCherry variant) were amplified with primers RNSmF/RNSR and RCSmF/RCSR, and then subcloned into SmaI-digested pCNF3 and pCNM3 (3 x Myc instead of 3 x FLAG in pCNF3) to generate pCNRNF3 and pCNRCM3. Coding sequences of fmkj1 and xydb2 were introduced into pCNRNF3 and pCNRCM3 to generate pCNRN-fmkj1 and pCNRC-xydb2, respectively (Fig. S2). BiFC was conducted with agroinfiltration in leaves of N. benthamiana, and protein expression was confirmed by Western blot using the appropriate antibody.

For subcellular localization, coding sequences of fmkj1 and xydb2 were introduced into pCNG and pCNR, respectively. Transient expression was carried out by agroinfiltration. Plasmolysis of N. benthamiana cells was performed by immersing pieces of leaves in 1 M NaCl for 10 min, followed by confocal microscopic analysis. Fluorescence in leaves of N. benthamiana was monitored at 3 d post-agroinfiltration, and then imaged directly using a confocal laser scanning microscope (CLSM) (Olympus FluoView FV1000). The excitation wavelengths used were 488 nm for eGFP and 587 nm for mCherry.