A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering

Jiuyuan Bai,Rui Wang,Xin Wang,Yuanli Zuo,Mingdi Zhang,Junyu Yao,Meng Yin,Shi Li,Hua Yang,Yun Zhao,Wei Wei,Yao Luo,Maolin Wang,Guolin Li,Chunhai Fan,Mei Luo

doi:10.1038/s41467-020-17497-7

Jiuyuan Bai, Rui Wang + Show 14 more

Open Access

https://doi.org/10.1038/s41467-020-17497-7

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Abstract

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.

Highlights

Reporter systems are routinely used in plant genetic engineering and functional genomics research
We investigate the performance of the 3WJ-nBro series and the previously developed F30-Broccoli sequence (Bro) in RNA imaging both in vitro and in vivo. 3WJ-4 × Broccoli (3WJ-4 × Bro) shows bright fluorescence in N. benthamiana cells, whereas neither F30Bro nor the other aptamers we created produce a detectable signal, revealing 3WJ-4 × Bro to be a satisfactory aptamer for mRNA imaging in plant cells
In N. benthamiana cells, only cells expressing AtCLE-3WJ-4 × Bro mRNA exhibited bright fluorescence under the confocal microscope, whereas cells expressing any of the other aptamer-tagged mRNAs showed no detectable fluorescence (Fig. 2e). These results indicated that 3WJ-4 × Bro was the only fully competent aptamer that could be successfully used for mRNA imaging in both live E. coli cells and plant cells

Summary

Introduction

Reporter systems are routinely used in plant genetic engineering and functional genomics research. The Luc gene has been frequently used to monitor real-time gene expression in plant[10,11,12]; when measured based on bioluminescence, the Luc activity can be affected by substrate availability or inherent differences in local cell environments, making the Luc gene unsatisfactory for accurately monitoring tissue- or cell-specific expression patterns[13] These reporters can be used for selection of multiple generations of transgenic plants, these systems do not directly detect the presence of the target gene in progeny generations, in which the partial integration of T-DNA may occur[14]; the expression levels of target genes are not directly reflected by those of the reporter genes. There is a need for a reporter system to directly monitor the expression of different categories of target genes without the accumulation of foreign proteins

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