In vivo imaging of tagged mRNA in plant tissues using the bacterial transcriptional antiterminator BglG.

Eduardo J Peña,Manfred Heinlein,Gabriel Robles Luna

doi:10.1111/tpj.15035

Abstract

RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.

Highlights

MRNA localization is an evolutionary conserved mechanism observed in a wide range of organisms and cell types, and by which mRNA species carrying RNA localization signals (“zipcodes”) are localized to discrete locations in the cytoplasm (Ryder and Lerit, 2018)
Imaging of movement protein (MP) mRNA with MS2, λN and BglG systems Using the MS2 method to study Tobacco mosaic virus (TMV) RNA transport and localization, it was shown that MP colocalizes with MP mRNA in mobile particles and at PD, suggesting that the mobile particles are ribonucleoprotein complexes (RNPs) destined to PD (Sambade et al, 2008)
We started by testing the λN system, which is based on the specific binding of a 22 amino acids long peptide derived from the phage lambda protein N to an RNA hairpin formed by 15 nucleotides derived from the nut N binding site in the N mRNA

Summary

Introduction

MRNA localization is an evolutionary conserved mechanism observed in a wide range of organisms and cell types, and by which mRNA species carrying RNA localization signals (“zipcodes”) are localized to discrete locations in the cytoplasm (Ryder and Lerit, 2018). A likely candidate for such a common feature is synaptotagmin 1 (SYT1) This protein occurs at ER:PM contact sites as well as at PD (Ishikawa et al, 2020; Levy et al, 2015) and is a target of MP required for virus movement (Levy et al, 2015; Yuan et al, 2018; Lewis and Lazarowitz, 2010) While proteins can be localized by translational fusion to fluorescent reporters, the development of comparable techniques to localize RNA molecules by transcriptional fusion to a fluorescent RNA molecule remain to be developed. Using the MS2 system in plants, an SL array-tagged mRNA encoding the MP of TMV in fusion to red fluorescent protein (RFP) was shown to localize to the mobile MP particles in the cortical cytoplasm (Sambade et al, 2008). Only a small number of RNA-containing MP particles could be detected

Methods

Results

Discussion

Conclusion