New GATEWAY vectors for High Throughput Analyses of Protein–Protein Interactions by Bimolecular Fluorescence Complementation

Abstract

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

Pseudomonas syringae type III effector HopAF1 suppresses plant immunity by targeting methionine recycling to block ethylene induction

“…HopAF1 and MTN1 coding sequences were cloned into BiFC constructs that contain a UBQ promoter (86). HopAF1-nYFP, HopAF1 H186A -nYFP, or HopAF1 G2AC4S -nYFP was transiently coexpressed with MTN1-cYFP, MTN2-cYFP, or PLC2-cYFP in N. benthamiana leaves using Agrobacterium-mediated transient transformation.…”

Arabidopsis Kelch Repeat F-Box Proteins Regulate Phenylpropanoid Biosynthesis via Controlling the Turnover of Phenylalanine Ammonia-Lyase

“…The bright cytosolic chimeric BiFC signals of the pairs of the truncated KFB-and PAL-half fluorescence proteins in the living cells and the co-IP of nYFP-KFB fusion protein by PAL(s)-CFPc coincide with our observations in the Y2H assay. The observed complementary fluorescent signal in BiFC and the immunodetection of KFB in the co-IP assay are not due to the artificial interaction of the tagged fluorophores, since the nonfunctional fluorophore fragments in general do not reassemble spontaneously and the refolding and reconstitution of a fluorescing protein only occurs at the fused partner proteins tightly interact (Kerppola, 2006(Kerppola, , 2008Gehl et al, 2009). The impossibility of an artificial interaction resulted from spontaneous reassembly of fluorescent fragments was validated further by our inclusion of a similar F-box protein, CPR30, as the negative control in both the BiFC and co-IP assays (Figures 2 and 3; see Supplemental Figure 3 online), where the CPR30 was tagged with fragment of fluorescence protein in a manner similar to the KFBs and coexpressed with the PAL fusion proteins.…”

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus

“…1) prompted us to test whether IQD proteins recruit CaM to diverse subcellular sites. We selected eight IQD proteins representing the phylogenetic clades and different localization patterns of the Arabidopsis IQD family and performed bimolecular fluorescence complementation (BiFC) assays, which detect protein-protein interactions in planta with subcellular resolution (Gehl et al, 2009;Kudla and Bock, 2016). Coexpression in N. benthamiana of nYFP-IQD1 and cYFP-CaM2 reconstitutes YFP fluorescence at MTs and in the nucle(ol)us (Fig.…”

“…Surface images of epidermal cells were acquired with a 633 water-immersion objective. BiFC assays were carried out with a Zeiss LSM 710 inverted microscope according to the protocol described by Gehl et al (2009). Imaging was performed with identical laser settings for all samples, and emission spectra of reconstituted fluorescence were recorded by lambda scan.…”