An integrated framework to elucidate mechanisms underlying host-branched broomrape infection.

Branched broomrape (Phelipanche ramosa) is an obligate root parasitic weed that threatens tomato production in many regions. Progress in understanding host resistance mechanisms has been hindered by the parasite's subterranean life cycle and the technical limitations of traditional soil-based assays. Here, we introduce an integrated experimental framework that enables molecular, genetic, and cellular analysis of broomrape parasitism in tomato under controlled conditions. We implemented a transparent, soil-less co-cultivation system for non-destructive, real-time monitoring of broomrape development on tomato roots, and a dual-compartment in vitro co-culture system supporting parasite infection of transgenic hairy roots. This methodology enabled rapid functional testing of candidate host resistance genes, exemplified by CRISPR-edited mutants of the tomato transcription factor SCHIZORIZA (SlSCZ), which displayed localized lignin accumulation at the parasite entry site in the root. The observed lignification suggests a role for this gene in regulating inducible cell wall lignification against broomrape. Together, these tomato-focused integrated methods enable reproducible imaging, genetic perturbation, and high-resolution analysis of host-parasite interfaces. These provide a scalable platform for dissecting broomrape resistance and accelerating resistance gene discovery in tomato and a critical tool for combating the devastating consequences of this parasite on agriculture.