Controlled Activation of NLR-Mediated Immunity in Roots Using a Copper-Inducible Expression System

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) function in both shoots and roots as key components of plant immunity, yet their biological roles in roots remain poorly understood. Here, we established a copper-inducible gene expression system for tomato (Solanum lycopersicum) hairy roots that enables precise, on-demand activation of immune signaling in the root tissue. The system integrates the copper-binding transcription factor CUP2, the CUP2-dependent chimeric CBS4-miniDFR promoter (CBS, copper-binding site; DFR, dihydroflavonol-4-reductase) driving the target gene, and a fluorescent marker for root identification, together within a single transferred DNA construct. This all-in-one vector system provided easy isolation of transgenic root cells and clear on-off control of immune activation with minimal basal expression. Using this platform, we induced autoactive NLR variants NRC4^DV-EGFP and Gpa2^NB-EGFP, which triggered reactive oxygen species accumulation and hypersensitive cell death in roots. Confocal imaging further revealed distinct subcellular dynamics of active and inactive NLRs in root cells upon induction, consistent with those in leaf cells. This system provides a robust and versatile tool for investigating root immune signaling and NLR dynamics under controlled conditions. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.