MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein–protein interactions

Abstract

Background The bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein–protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed. Results Here we describe the development of a flexible modular cloning-based toolkit for BiFC (MoBiFC) and proximity labelling in the chloroplast and other cellular compartments using synthetic biology principles. We used pairs of chloroplast proteins previously shown to interact (HSP21/HSP21 and HSP21/PTAC5) and a negative control (HSP21/ΔPTAC5) to develop standardised Goldengate-compatible modules for the assembly of protein fusions with fluorescent protein (FP) fragments for BiFC expressed from a single multigenic T-DNA. Using synthetic biology principles and transient expression in Nicotiana benthamiana, we iteratively improved the approach by testing different FP fragments, promoters, reference FPs for ratiometric quantification, and cell types. A generic negative control (mCHERRY) was also tested, and modules for the identification of proximal proteins by Turbo-ID labelling were developed and validated. Conclusions MoBiFC facilitates the cloning process for organelle-targeted proteins, allows robust ratiometric quantification, and makes available model positive and negative controls. Development of MoBiFC underlines how Goldengate cloning approaches accelerate the development and enrichment of new toolsets, and highlights several potential pitfalls in designing BiFC experiments including the choice of FP split, negative controls, cell type, and reference FP. We discuss how MoBiFC could be further improved and extended to other compartments of the plant cell and to high throughput cloning approaches.

Structural Maintenance of Chromosome 3 interacts with the Topoisomerase VI complex and contributes to the oxidative stress response inArabidopsis thaliana

“…We designed two recombinant CDSs, BIN4 fused with the C-terminus of YFP as well as the CDS of SMC3(193-807) fused with the N-terminus of YFP and supplemented with a Simian-Virus 40 Nuclear Localization Signal (NLS) to ensure the nuclear location of the truncated SMC3 peptide. We then added both CDS of interest to the same multigenic expression vector that also contains the silencing repressor P19 and the chloroplast outer membrane OEP7-mTRQ as a transformation marker (Yong Jik Lee et al ., 2001; Scholthof, 2006; Velay et al ., 2022). Expression of BIN4-cYFP and SMC3(193-807)-nYFP resulted in reconstitution of YFP fluorescence in the nuclei of epidermal cells of Nicotiana benthamiana (Fig.…”

“…Multigenic BiFC vectors containing a repressor of silencing (P19), a transformation marker (OEP7-mTRQ), and the two coding sequences of interest fused with the two different parts of the 174/175 YFP split were made using the cloning toolbox and the protocol described in (Engler et al ., 2014; Velay et al ., 2022). The chimeric CDS of AtSMC3 was cloned following an optimized protocol due to the high instability of the construct in bacterial strains.…”

“…Transient expression in Nicotiana benthamiana leaves was performed following the protocol described in Velay et al . 2022.…”

“…BiFC assays were carried out in Nicotiana benthamiana lower epidermal cells. Sample preparation, imaging and analysis were performed following the protocol described in (Velay et al ., 2022). Exposure time and post-acquisition analyses were similar between all samples.…”

“…Transient expression in Nicotiana benthamiana leaves was performed following the protocol described in Velay et al 2022. For Arabidopsis stable expression, Agrobacterium tumefaciens GV3101 transformed with the plasmid containing GFP-SMC3 were grown at 28°C in LB medium supplemented with rifampicin and kanamycin.…”