Stomatal XVE: an inducible system for cell-stage-specific gene expression and editing in the stomatal lineage.

Stomatal development has emerged as a valuable model for studying developmental processes. Examining gene function along the stomatal lineage often requires gene perturbation in a controlled and cell-stage-specific manner, but this remains tedious without a dedicated genetic tool. Here, we describe Stomatal XVE, a modular, two-component XVE-based inducible system that enables user-controlled gene overexpression and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based knockout at defined stomatal cell stages in Arabidopsis thaliana. The system consists of a collection of estrogen-responsive XVE driver lines under cell-stage-specific promoters and effector vectors responsive to activated XVE. This design simplifies cloning and allows users to scale their investigation. We validated the cell-stage specificity and inducibility of the XVE driver lines and characterized key induction parameters. To test the system functionally, we employed it to study MAPKKK YODA and a pathogen effector AvrPtoB. While YODA overexpression reproduced known early- and late-stage phenotypes, stage-specific knockouts argued against its late-stage role in guard cell (GC) differentiation. Furthermore, AvrPtoB expression during later stages triggered striking disruptions in GC morphology and viability, revealing cell-type-specific effects of the pathogen protein. Overall, our Stomatal XVE system enables precise functional analysis of genes across defined stages of stomatal development and is particularly well suited for investigating genes with pleiotropic effects.