Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model

Abstract

ORCID ID: 0000-0002-8568-7125 (J.B-S.).Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type-and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomatooptimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

“…Moneymaker. Here, we report the use of the A. rhizogenes strain ARqua1, but it should be noted that strain ATCC 15834 has also been reported to work in tomato 9 .…”

“…So-called hairy roots are a convenient, alternative system since a large number of independent, stably transformed materials can be generated within weeks, as opposed to months for stable plants. CRISPR vectors are very effective at inducing DNA mutations in hairy roots 9,10 . DNA assembly methods efficiently ligate DNA fragments containing overlapping ends 11 .…”

“…Hairy roots have been used to study a variety of plant functions including root development 9 , symbiotic associations 24,25 , and nematode resistance 26 and the use of hairy roots for screening immunity-related genes is an area of active research in our lab. In addition to overexpression, gene silencing, and other transgenic technologies, targeted gene deletion via CRISPR/Cas9 is a powerful tool for functional genomic studies.…”

Focus on Roots

“…In addition, Arabidopsis remains unrivaled in its ability to be imaged by confocal microscopy, a consequence of its very thin and low-fluorescent root system. In this Focus Issue, Ron et al (2014) present a set of inspiring tools for the tomato (Solanum spp.) hairy root system, demonstrating how cell type-specific transcriptome profiling, confocal imaging of fluorescent reporters, and the latest cluster regularly interspaced short palindromic repeats technology can be combined into a powerful system for investigating root biology outside Arabidopsis.…”

Targeted mutagenesis in the tetraploid switchgrass cultivar 'Alamo' using CRISPR-Cas9

“…Gao et al (2014) used Agrobacterium mediated transformation of leaf discs from six week old common tobacco plants. In tomato (Solanum lycopersicum) CRISPR Cas9 mediated mutations knocked out the transgene expressing the green fluorescent protein (GFP) in transgenic tomato plants which have a hairy roots phenotype, after being transformed with CRISPR Cas9 by Agrobacterium rhizogenes (Ron et al, 2014 sporelings were transformed with CRISPR Cas9 via Agrobacterium (Sugano et al, 2014), and in soybean plants (Glycine max) CRISPR Cas9 was used to successfully knock out a GFP and bialaphos resistance (bar) transgene, as well as the endogenous genes GmFEI2, GmSHR, and Glyma07g14530, and the homoeologous pair, Glyma01g38150 and Glyma11g07220, using multiplex CRISPR (Cai et al, 2015;Jacobs et al, 2015). …”