Implementation of Ribo-BiFC method to plant systems using a split mVenus approach

Abstract

Translation is a fundamental process for every living organism. In plants, the rate of translation is tightly modulated during development and in response to environmental cues. However, it is difficult to measure the actual translation state of the tissues in vivo. Here, we report the implementation of an in vivo translation marker based on bimolecular fluorescence complementation, the Ribo-BiFC. We combined method originally developed for fruit-fly with an improved low background split-mVenus BiFC system previously described in plants. We labelled Arabidopsis thaliana small subunit ribosomal protein (RPS) and large subunit ribosomal protein (RPL) with fragments of the mVenus fluorescent protein. Upon the assembly of the 80S ribosome, the mVenus fragments complemented and were detected by fluorescent microscopy. We show that these recombinant proteins are in close proximity in the tobacco epidermal cells, although the signal is reduced when compared to BiFC signal from known interactors. This Ribo-BiFC method system can be used in stable transgenic lines to enable visualisation of translational rate in plant tissues and could be used to study translation dynamics and its changes during plant development, under abiotic stress or in different genetic backgrounds.