Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells

Immunodetection of transiently expressed nRFP/cRFP-fused proteins.

<p>Using protein extracts prepared from tobacco leaves of Agrobacterium-infiltrated plants analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160717#pone.0160717.g002" target="_blank">Fig 2</a>, the accumulation of nRFP/cRFP-fused proteins by Agrobacterium infiltration was confirmed by immunoblot analysis with anti-Myc or anti-HA antibodies. Lane 1, nRFP; Lanes 2 and 5, PMP38-nRFP with PMP38-cRFP; Lanes 3 and 6, nRFP-PEX7 with PTS2-cRFP; Lane 4, cRFP. Arrowheads indicate the positions of untagged or tagged polypeptides. Asterisks represent extra polypeptides, which are considered to be degradation products of the fusion proteins.</p

Detection of multiple protein—protein interactions using transient expression assays.

<p>(A, B) Two types of vectors were introduced as controls. Representative images of interactions of nRFP-PEX7 with PTS2-cRFP (A) and AtSEC31A-nYFP with AtSEC13A-cYFP (B). (C-E) Four types of vectors, nRFP-PEX7, PTS2-cRFP, AtSEC31A-nYFP, and AtSEC13A-cYFP, were introduced simultaneously into onion epidermal cells. Representative images of interactions of nRFP-PEX7 with PTS2-cRFP (C) and AtSEC31A-nYFP with AtSEC13A-cYFP (D) in the same cell. (E) Merged image of (C) with (D). Bar: 50 μm.</p

Schematic diagrams of the Gateway cloning technology-compatible vectors.

<p>A. ECFP, EGFP, EYFP and mRFP1 can be divided into two fragments. The letters ‘n’ and ‘c’ represent N- or C-terminal fragments of a split fluorescent protein, and the letters ‘C’, ‘G’, ‘Y’ and ‘R’ represent the type of fluorescent protein (ECFP, EGFP, EYFP or mRFP1). Since the nucleotide sequences of C-terminal CFP and C-terminal GFP are identical, we designated the fragment cCG. The letters ‘myc’ and ‘HA’ in the N- and C-terminal fragment from mRFP1 represent myc- and hemagglutinin-epitope tags, respectively. B. The structures of the region indicated as ‘Gateway’ in (C) and (D). GWnX and GWcX contain N- or C-terminal split fluorescent protein downstream of the <i>att</i>R2 site, respectively, whereas nXGW and cXGW contain N- or C-terminal split fluorescent protein upstream of the <i>att</i>R1 site, respectively. C. Outline of the pUGW-based vectors for BiFC. pGWnX, pGWcX, pnXGW, and pcXGW vectors, which bear the DNA fragment shown in (B) downstream of the 35S promoter from cauliflower mosaic virus. D. Outline of the binary vectors for BiFC. The pB4 and pB5 series contain Km^r and Hyg^r markers, respectively, which are placed in reverse orientation to the genes cloned via LR recombination. Details of plasmid construction and the vector backbone are given in Materials and Methods. <i>Cm</i>^<i>r</i>, chloramphenicol-resistance marker; <i>ccd</i>B, negative selection marker used in bacteria; <i>35Sp</i>, 35S promoter; <i>Tnos</i>, nopaline synthase terminator; myc, c-myc affinity tag; HA, hemagglutinin affinity tag.</p

Detection of protein—protein interactions between PMP38–PMP38 and PEX7–PTS2 in tobacco leaves by Agrobacterium infiltration.

<p>Various combinations of fusion genes were introduced by Agrobacterium infiltration. (A, C, E, G, I and K) Detection of red fluorescent signals from reconstituted mRFP1. (B, D, F, H, J and I) Merged images of reconstituted fluorescent signals with differential interference contrast settings. (A, B) PMP38-nRFP with PMP38-cRFP. (C, D) nRFP-PEX7 with PTS2-cRFP. (E, F) nRFP-PEX7 with PMP38-cRFP. (G, H) PMP38-nRFP with cRFP-PEX12. (I, J) PMP38-nRFP with untagged cRFP. (K, L) nRFP-PEX7 with untagged cRFP. Bar: 20 μm.</p