# Virus‐induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics

## TL;DR

A virus-based gene editing method in plants allows rapid study of genes controlling stomata, linking genetic changes to plant water use and resilience.

## Contribution

A TRV-based gene editing platform enables rapid, heritable, and somatic genome editing of stomatal regulators in Nicotiana benthamiana.

## Key findings

- Somatic and heritable genome editing was achieved in multiple stomatal regulator genes with up to 95% editing efficiency.
- Mutations in stomatal genes caused gene-specific changes in stomatal density and leaf temperature.
- A reporter system allowed visualization of virus-infected tissues for phenotyping edited plant sectors.

## Abstract

Virus‐induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)‐based single‐guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9‐expressing lines. sgRNAs fused to a mobile RNA element and co‐delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra‐allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene‐specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus‐infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV‐based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.

We present a rapid method for functional genomics using a tobacco rattle virus‐based gene editing platform. This platform enables both somatic and heritable editing of stomatal regulators in Nicotiana benthamiana, including STOMAGEN, EPF2, YODA, and SPEECHLESS, linking mutations to changes in stomatal density and leaf thermoregulation. This versatile approach accelerates trait discovery and provides a foundation for improving plant water use efficiency and resilience to environmental stress.