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A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes

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posted on 2023-11-21, 04:26 authored by S Ferguson, NB Abel, Dugald ReidDugald Reid, LH Madsen, TB Luu, KR Andersen, J Stougaard, S Radutoiu

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.

Funding

SF, NBA, DR, LHM, KRA, JS, and SR were funded by the project Molecular Mechanisms and Dynamics of Plant–microbe interactions at the Root–Soil Interface (InRoot), supported by the Novo Nordisk Foundation grant [no. NNF19SA0059362] (https://ccrp.vcl.ncsu.edu/ content/inroot). NBA, DR, TBL, KRA, JS, and SR were funded by the Bill and Melinda Gates Foundation and the UK government’s Foreign, Commonwealth and Development Office (FCDO) through the Engineering Nitrogen Symbiosis for Africa project [ENSA; OPP11772165] (https:// www.ensa.ac.uk/).

History

Publication Date

2023-11-01

Journal

PLoS One

Volume

18

Issue

11

Article Number

0291680

Pagination

9p.

Publisher

PLOS

ISSN

1932-6203

Rights Statement

© 2023 Ferguson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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