Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots

Abstract

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in "transformed hairy roots". The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.