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

SUMMARY 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 M 0 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.