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Updated: May 25, 2026

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

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A Visual Reporter System for Analyzing Small RNA-Triggered Local and Systemic Silencing of an Endogenous Plant Gene.

Adriana E Cisneros¹, Alberto Carbonell²

¹Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València), Valencia, Spain.

Methods in Molecular Biology (Clifton, N.J.)

|May 16, 2025

Summary

Researchers developed a visual system to study plant small RNAs (sRNAs). This system tracks how sRNAs trigger gene silencing locally and systemically, aiding the discovery of factors influencing this process.

Keywords:

Local silencing Nicotiana benthamiana RNA mobility Small RNAs Sulfur Systemic silencing amiRNA syn-tasiRNA

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RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

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Area of Science:

Plant molecular biology
Gene silencing mechanisms
RNA interference (RNAi) pathways

Background:

Plant small RNAs (sRNAs) regulate gene expression by silencing target messenger RNAs (mRNAs).
sRNAs can act locally within cells or move to distant tissues to induce systemic gene silencing.
Identifying factors controlling sRNA-induced silencing and spread has been challenging due to limited experimental systems.

Purpose of the Study:

To establish a visual reporter system for studying local and systemic gene silencing induced by plant sRNAs.
To facilitate the identification of genetic, molecular, and environmental factors governing sRNA silencing activities.

Main Methods:

Agroinfiltration of an artificial small RNA (sRNA) construct into Nicotiana benthamiana plants.
Utilizing the Nicotiana benthamiana SULFUR (NbSu) gene as a visual reporter for gene silencing.
Observing and characterizing visual phenotypes (bleaching and chlorosis) indicative of local and systemic silencing.

Main Results:

Agroinfiltrated areas exhibited strong, uniform bleaching, indicating effective local gene silencing.
Apical, non-agroinfiltrated leaves displayed near-vein chlorosis, demonstrating systemic gene silencing.
The visual phenotypes provide a clear readout for sRNA-induced silencing and its spread.

Conclusions:

The developed visual reporter system effectively visualizes both local and systemic gene silencing triggered by plant sRNAs.
This system provides a powerful tool for dissecting the regulatory mechanisms underlying sRNA-mediated gene silencing.
It will accelerate the discovery of factors controlling the induction and systemic spread of sRNA activity in plants.