Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

Abstract

SummaryThe specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such proteinprotein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calciummediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.

Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

“…For BiFC assays the full length cDNA of SOS1 was transferred to the pSPYCE(M) and pSPYNE173 plasmids 41 using the XbaI and KpnI sites to create C-terminal translational fusions of SOS1 to the N-terminal and C-terminal moieties of YFP, respectively. The cDNA encoding the Arabidopsis thaliana HKT1 transporter was cloned into the pSPYNE 173 using the XhoI-KpnI sites.…”

“…The cDNA of SOS2 cloned into pSPYNE(R)173 41 was used to assess the interaction with SOS1. All the YFP fusions were expressed under the control of the 35S promoter.…”

Pumilio-Based RNA In Vivo Imaging

“…BiFC can be performed with various derivatives of both GFP and red FPs (31,(34)(35)(36)(37)(38)(39). This allows selection of an FP that will facilitate co-localising the RNA of interest with other fluorescent markers that may already be available.…”

Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

“…BiFC analyses were conducted as described previously (Waadt et al, 2008). To generate the BiFC constructs, cDNAs encoding AvrBsT and CaHSP70a without termination codons were PCR amplified and subcloned into the binary vectors pVYNE(R) (XbaI/XhoI) and pSCYCE (XbaI/XhoI) under the control of the cauliflower mosaic virus 35S promoter (Supplemental Table S1).…”

“…CaHSP70a and AvrBsT were fused to the C-terminal 84-amino acid Super Cyan Fluorescent Protein 3A (SCFP3A) region and the N-terminal 173-amino acid Venus region, respectively (Waadt et al, 2008). A. tumefaciens cells harboring the corresponding constructs were mixed and coinfiltrated into N. benthamiana leaves.…”