A Split Enzyme System for Plant RNA Imaging and Genetic Engineering

Greg Howard
10th February, 2025

A Split Enzyme System for Plant RNA Imaging and Genetic Engineering

The developed split ribozyme biosensor system enables the in vivo visualization of diverse RNA targets, successfully detecting cell-specific endogenous gene expression in stably transformed Arabidopsis thaliana (left) and the presence of both foreign transgenes and viral RNA in Nicotiana benthamiana (right).

Composite: Natural Science News / CC BY-SA. [Sources]
Adapted from photos by:

Key Findings

  • Researchers at Oak Ridge National Laboratory developed a new system to visualize RNA activity in living plants in real time, tested on tobacco and Arabidopsis plants
  • The system uses a synthetic split ribozyme to convert RNA signals into protein outputs, enabling RNA imaging at cellular and tissue levels
  • This approach overcomes traditional RNA analysis limitations, offering a scalable, non-destructive method to study plant responses and gene expression
RNA is a fundamental molecule in plants, regulating cellular and physiological processes essential for growth, development, and responses to environmental changes. Understanding the dynamic abundance of RNA in real time provides critical insights into how plants adapt to internal and external stimuli. However, traditional methods for analyzing RNA abundance in plants are often destructive, labor-intensive, and time-consuming. A recent study conducted by researchers at Oak Ridge National Laboratory[1] introduces a novel approach to overcome these challenges, enabling real-time, in vivo RNA imaging in plants. The study presents a synthetic split ribozyme system that transforms RNA signals into orthogonal protein outputs, allowing for the visualization of RNA dynamics directly in living plants. This innovative system was tested in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana, demonstrating its capability to detect RNA derived from both transgenes and viral sources, such as the tobacco rattle virus. The researchers also successfully engineered a biosensor in Arabidopsis for visualizing endogenous gene expression at the cellular level. By enabling RNA imaging at multiple scales—ranging from individual cells to entire tissues—this approach provides a versatile platform for studying RNA dynamics in plants. This study builds on earlier advancements in RNA visualization. For instance, the development of fluorescent RNA aptamer-based systems, such as 3WJ-4× Bro, has previously demonstrated the feasibility of non-protein-based RNA reporters for tracking mRNA expression patterns in plants[2]. These systems offered a protein-independent alternative for dynamic mRNA analysis, but their application was limited in terms of flexibility and scalability. The split ribozyme system introduced by Oak Ridge researchers expands on this by incorporating customizable protein outputs, allowing greater adaptability in detecting diverse RNA targets. Additionally, the study addresses the limitations of traditional RNA imaging methods, such as whole-mount in situ hybridization (WISH) protocols, which require extensive tissue preparation and are not suitable for dynamic, real-time analysis[3]. While WISH and fluorescent adaptations like F-WISH have provided subcellular resolution of mRNA localization, they are inherently static and cannot capture the temporal dynamics of RNA abundance. In contrast, the split ribozyme system enables continuous monitoring of RNA activity in living plants without the need for destructive sampling. The findings also complement insights from studies on transcriptional dynamics in plants. For example, research using PP7 and MS2 RNA-labeling technologies revealed that transcriptional responses in plants, such as during heat shock, exhibit cell-to-cell variability and stochastic behavior[4]. These studies highlighted the importance of single-cell resolution in understanding RNA dynamics. The split ribozyme system aligns with this need by enabling cellular-level visualization of RNA signals, providing a tool to further investigate how transcriptional variability contributes to plant responses under different conditions. The flexibility of the split ribozyme system lies in its modular design, which allows researchers to integrate various protein outputs for optimal RNA detection. This adaptability is particularly valuable for studying complex RNA interactions in plants, where different types of RNA—such as messenger RNA (mRNA), viral RNA, and regulatory RNA—play distinct roles in cellular processes. The ability to monitor these interactions in vivo opens new avenues for exploring the molecular mechanisms underlying plant resilience, development, and productivity. In practical terms, the study's approach has significant implications for plant biotechnology. By enabling real-time RNA imaging, researchers can better understand how plants respond to environmental stressors, such as drought or pathogen infection. This knowledge can inform the development of crops with improved stress tolerance and productivity. Moreover, the system's compatibility with different protein reporters makes it a versatile tool for functional genomics and synthetic biology applications, where precise control and monitoring of gene expression are essential. In conclusion, the split ribozyme system developed by Oak Ridge National Laboratory represents a transformative advancement in plant molecular biology. By enabling in vivo visualization of RNA dynamics at cellular and tissue levels, it addresses longstanding limitations of traditional RNA analysis methods. Building on prior innovations in RNA imaging[2][3][4], this system provides a flexible and scalable platform for studying RNA-mediated processes in plants, paving the way for advances in crop improvement and fundamental plant research.

BiotechGeneticsPlant Science

References

Main Study

1) A split ribozyme system for in vivo plant RNA imaging and genetic engineering.

Published 7th February, 2025

https://doi.org/10.1111/pbi.14612

Related Studies

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

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

3) Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule.

https://doi.org/10.1016/j.ymeth.2015.10.019

4) Quantitative imaging of RNA polymerase II activity in plants reveals the single-cell basis of tissue-wide transcriptional dynamics.

https://doi.org/10.1038/s41477-021-00976-0


Related Articles

An unhandled error has occurred. Reload 🗙