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Peer-reviewed

Lab Protocol

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

  • Shaun Ferguson ,

    Contributed equally to this work with: Shaun Ferguson, Nikolaj B. Abel

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing

    ferguson@mbg.au.dk (SF); nikolaj.abel@mbg.au.dk (NBA)

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯
  • Nikolaj B. Abel ,

    Contributed equally to this work with: Shaun Ferguson, Nikolaj B. Abel

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing

    ferguson@mbg.au.dk (SF); nikolaj.abel@mbg.au.dk (NBA)

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯
  • Dugald Reid,

    Roles Conceptualization, Formal analysis, Funding acquisition, Resources, Supervision, Writing – review & editing

    Affiliations Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, Department of Animal, Plant and Soil Sciences, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Australia

    ⨯
  • Lene H. Madsen,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯
  • Thi-Bich Luu,

    Roles Investigation, Methodology, Writing – review & editing

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯
  • Kasper R. Andersen,

    Roles Funding acquisition, Resources, Supervision, Writing – review & editing

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯
  • Jens Stougaard,

    Roles Funding acquisition, Resources, Supervision, Writing – review & editing

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯
  • Simona Radutoiu

    Roles Funding acquisition, Resources, Supervision, Writing – review & editing

    Affiliation Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    ⨯

Abstract

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.

Citation: Ferguson S, Abel NB, Reid D, Madsen LH, Luu T-B, Andersen KR, et al. (2023) A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes. PLoS ONE 18(11): e0291680. https://doi.org/10.1371/journal.pone.0291680

Editor: Mojtaba Kordrostami, Nuclear Science and Technology Research Institute, ISLAMIC REPUBLIC OF IRAN

Received: March 6, 2023; Accepted: September 1, 2023; Published: November 1, 2023

Copyright: © 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.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

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/).

Competing interests: The authors have declared that no competing interests exist.

Introduction

Hairy root disease occurs when a plant becomes infected by Rhizobium rhizogenes (previously known as Agrobacterium rhizogenes [1]), which causes neoplastic growth at the site of infection, resulting in highly branched roots [2]. R. rhizogenes can induce hairy roots on a diverse range of monocotyledonous and dicotyledonous plants, which become susceptible when wounded [3–5]. A distinct phenolic compound profile is produced by damaged plant cells which activates a set of virulence (vir) genes resulting in the transfer of segments of DNA, known as transfer-DNA (T-DNA), from the root-inducing plasmid (pRi) to the plant host [5]. The T-DNA segment is demarcated by 25 bp repeat sequences, the only cis-acting requirement for transfer [6]. Following the discovery of this natural interkingdom DNA transfer mechanism, various strategies have emerged for generating transgenic hairy roots [7–10] for a plethora of applications, including, but not limited to: molecular breeding, metabolic engineering, recombinant protein production, rhizosphere physiology, and root physiology (e.g. nitrogen fixation) [11, 12].

One hairy root transformation approach uses ‘binary vectors’ that encode a segment of T-DNA carrying a gene of interest to be transferred to the plant. A range of binary vector options are available [13], and that system provides a simple method for constructing R. rhizogenes transconjugants, often directly via electroporation. Generally, those transconjugants maintain the binary vector alongside pRi, enabling co-transformation of both the native pRi T-DNA and the foreign DNA within the binary vector T-DNA. The binary vector system is appealing due to its simplicity and convenience, yet it is subject to considerable variation in co-transformation frequency, which can often result in poor numbers of transgenic hairy roots [10, 14, 15].

An alternative strategy relies on the introduction of suicide vectors into R. rhizogenes and two variants of this strategy can be implemented: integration vectors that encode a restriction fragment of the pRi TL-DNA, or pBR322-derived plasmids that can be used with previously constructed R. rhizogenes pBR322 acceptor strains. R. rhizogenes pBR322 acceptor strains were constructed by integrating pBR322-derived plasmids into the pRi15834 TL-DNA region by replacement mutagenesis [10, 16]. Both variants function by integrating the suicide vector into the pRi TL-DNA of R. rhizogenes through homologous recombination. The R. rhizogenes strain AR1193 is a pBR322 acceptor strain [10] that was developed for hairy root production in Lotus corniculatus [17] and its diploid relative Lotus japonicus. AR1193 has since successfully generated hairy roots in a range of plants, including significant species such as Pisum sativum (pea), Brassica napus (rapeseed), Phtheirospermum japonicum (Phtheirospermum), and Medicago truncatula (Barrel medic) [18–21]. Despite this, the main problem with the integration strategies stems from the extended workload and resources necessary to construct transconjugant R. rhizogenes. The major advantage, however, is the consistent and high rate of transgenic hairy roots [10].

Until now, the pBR322-derived vector pIV10 was utilised to transform R. rhizogenes pBR322 acceptor strains [14]. The use of pIV10 is reliant on E. coli strain GJ23, which contains two helper plasmids, R64drdl1 and pGJ28, that encode transfer (tra) and ColE1 mobilisation functions, respectively [22]. This out-dated transfer system requiring helper strain GJ23 for transfer of pIV10 is a major contributing factor to the unnecessary workload of this method.

Therefore, the purpose of this study was to create a simplified and streamlined protocol for generating transconjugant R. rhizogenes via the pBR322 acceptor-based strategy. We constructed a modified version of pIV10, resulting in the new vector pIV101. Crucially, pIV101 now contains an IncP oirT compatible with RP4 transfer machinery and, additionally, the lacZα fragment situated within a Golden Gate Assembly (GGA) compatible integration site [23, 24]. The addition of RP4 oriT renders pIV101 compatible with commonly used E. coli strains that contain the IncP RP4 transfer machinery integrated into their chromosome [25]. In addition, these strains often carry an auxotrophy for counterselection, e.g. E. coli ST18 and MFDpir [26, 27]. Together, these features provide efficient transfer of compatible mobilisable plasmids to recipient bacteria.

This system was originally developed for Lotus, as it is a model legume from the Leguminosae family. Many legumes are capable of forming a symbiotic partnership with bacteria known as rhizobia, where they become housed in structures on the roots, called nodules, and they fix atmospheric nitrogen for the plant to utilise as a nitrogen source [28]. Lotus has provided extensive understanding of legume–rhizobia symbiosis, in part due to the potential for generating transgenic plants through transformation with R. rhizogenes [29, 30]. Accordingly, to test the efficacy of pIV101, we investigated hairy root transformation of Lotus japonicus using R. rhizogenes AR1193 and our new plasmid.

We directly compared the original pIV10-based method with the new protocol utilising vector pIV101, to construct transconjugant R. rhizogenes AR1193. The time and labour required was markedly reduced when using pIV101, and critically, when we infected Lotus with transconjugants generated by the parallel methodologies, we observed equal efficiencies for both transgenic root transformation and the ability to form infected nodules when subsequently inoculated with rhizobia.

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io, (DOI: dx.doi.org/10.17504/protocols.io.261ge3xkjl47/v1) and is included for printing as S1 File with this article.

Results

Construction of pIV101, an improved pBR322-derived Golden Gate Assembly destination plasmid for transformation of R. rhizogenes

The original4.5 kb pIV10 was a composite of several plasmids, including the pBR322 sequence encoding the ColE1 origin of replication, the ColE1 basis of motility (bom), and ampicillin resistance from plasmid pHC79, the spectinomycin/streptomycin resistance from plasmid R702, and the pUC19 EcoRI/HindIII polylinker [10, 14, 31]. The pBR322 bom site enables in trans mobilisation of the plasmid by the ColE1 mobilisation functions, and the ColE1 oriV capacitates replication in E. coli but not in R. rhizogenes, making it a suicide plasmid in that species [32]. Transfer of exogenous DNA via conjugation into R. rhizogenes with pIV10 requires E. coli strain GJ23, which contains two helper plasmids, R64drdl1 and pGJ28, that encode transfer (tra) and ColE1 mobilisation functions, respectively [22].

We made two important modifications to improve pIV10, resulting in the new vector pIV101. First, the pUC19 polylinker sequence was replaced with a GGA compatible cloning site and domesticated for BpiI and BsaI recognition sequences to implement the Modular Cloning (MoClo) system developed by Weber et al. [33]. Secondly, we included a new IncP origin of transfer. Within pIV10, there is a section of 2,297 bp of pBR322 sequence (Fig 1A) that must remain unmodified, as it is required for homologous recombination with pBR322 sequence integrated within the TL-DNA of the R. rhizogenes acceptor strains [16]. Therefore, in pIV101 the original ColE1 bom remains and the additional IncP oriT was inserted between the GGA cloning site and the sp/sm resistance genes to enable mobilisation with RP4 tra functions (Fig 1A) without affecting recombination capability.

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Fig 1. pBR322-derived R. rhizogenes transformation vector pIV101.

A) Schematic representation of pIV101 indicating: the cis acting ColE1 basis of mobility (bom), pBR322 vegetative origin of replication (oriV), ampicillin resistance (ampR), spectinomycin/streptomycin resistance (sm/spR), the cis acting IncP origin of transfer (oriT), the Golden Gate Assembly cloning site (cyan box), and 2,297 bp of the original pBR322 plasmid sequence (highlighted in grey). B) Sequence of the Golden Gate Assembly cloning site with important features bolded: the bpiI recognition sequences (red) with corresponding fusion sites (red/underlined), the bsaI recognition sequences (green) with corresponding fusion sites (green/underlined), the lacZα fragment (blue), the CAP binding site (black), the lac promoter (black/underlined), and the lac operator (grey).

https://doi.org/10.1371/journal.pone.0291680.g001

We designed pIV101 to be a level 2 destination vector (pL2-1) based on the MoClo GGA strategy [33]; as such, the GGA cloning site contains TGCC and GGGA fusion sites generated by digestion with BpiI. Additionally, the GGA cloning site contains nested BsaI recognition sequences, which upon digestion will result in GGAG and CGCT fusion sites compatible with the insertion of level 1 fragments (Fig 1B). The lacZα fragment was incorporated within the nested restriction sites of the GGA cloning site to permit blue/white screening (Fig 1B).

Optimised workflow for construction of transconjugant R. rhizogenes clones

The generation of transconjugant R. rhizogenes previously required a minimum of 12 days with substantial hands-on time. By utilising the modified pBR322-derived level 2 destination vector (pIV101), it was possible to construct transconjugant R. rhizogenes in seven days, with less hands-on time, a substantial decrease in time and labour comparable to that required for a binary vector transformation system (Fig 2).

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Fig 2. Simplified overview comparing several workflows required to construct transconjugant R. rhizogenes for subsequent hairy root transformation.

The left column represents the protocol followed when using pBR322-derived vector pIV10; the middle column represents an optimised and simplified version of the pBR322-derived vector protocol using pIV101; the right column represents a protocol for transformation using a binary vector.

https://doi.org/10.1371/journal.pone.0291680.g002

Transgenic hairy root transformation of Lotus remains equally efficient with pIV101

To compare pIV101 to the original pIV10 vector, an insert was cloned into each vector and subsequently used for hairy root transformation of wild-type Lotus japonicus Gifu in parallel.

A separate GGA was performed for each destination vector in a reaction that included the preassembled level 1 vector (PMC-03820) (Table 1) encoding the Lotus ubiquitin promoter (pUBI) driving nuclear-localised triple YFP (tYFP) followed by a 35s terminator. This was combined with the level 1 end-linker with either lacZα (pL1M-ELB-1-49255) or the level 1 end-linker empty (pL1M-ELE-1-41722) to assemble the level 2 constructs, pIV10::pUBI-tYFP and pIV101::pUBI-tYFP (Table 1), respectively. For pIV10, the GGA reaction included the end-linker containing lacZα for blue/white selection. For pIV101, the empty end-linker was substituted, as lacZα is present within the GGA cloning site.

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Table 1. Strains and plasmids used in this study.

https://doi.org/10.1371/journal.pone.0291680.t001

Afterwards, pIV10::pUBI-tYFP and pIV101::pUBI-tYFP were transformed into E. coli MFDpir and conjugated into R. rhizogenes AR1193 as described in Fig 2 and Materials and Methods. Hairy root transformation was performed on 45 and 46 plants with pIV101::pUBI-tYFP and pIV10::pUBI-tYFP, respectively, divided into four replica. A similar percentage of plants showed the formation of hairy roots after three weeks (53% and 43%, respectively) (Fig 3A).

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Fig 3. R. rhizogenes mediated hairy root formation on L. japonicus Gifu was unaffected by the plasmid used for transformation.

(A) The number of hairy roots formed three weeks post infection by R. rhizogenes clones transformed by either pIV10 or pIV101, each containing pUBI-tYFP. Each symbol represents the average number of plants with hairy roots from one replicate, and the error bars represent ± the SEM. (B) The number of nodules formed on the same hairy roots from (A), three weeks post inoculation with Mesorhizobium japoniucm MAF303099. Each symbol represents a single plant, and the error bars represent ± the SEM. Unpaired t-tests for (A) and (B) revealed no significant difference between the two plasmid groups as displayed on the right y-axis of each graph, which represents the 95% CI. (C) and (D) Nodules formed by M. japonicum expressing DsRed on hairy roots induced by R. rhizogenes that was transformed by either pIV101 (C) or pIV10 (D). Both plasmids enabled similar expression of tYFP and resulted in infected nodules.

https://doi.org/10.1371/journal.pone.0291680.g003

To validate whether the hairy roots induced by R. rhizogenes transformed with pIV101 remain efficacious for nodule formation by symbiotic rhizobia, we inoculated plants with Mesorhizobium japonicum expressing the DsRed protein [34]. Hairy roots induced by R. rhizogenes transformed with either construct formed infected nodules to a similar level three weeks post inoculation (Fig 3B) and revealed similar morphology (Fig 3C and 3D).

Therefore, we successfully expedited the construction of transconjugant R. rhizogenes clones in order to make transgenic hairy roots, without affecting the effectiveness of hairy root formation, nodule infection by rhizobia, or morphology of the nodule. Furthermore, we have no reason to believe pIV101 would not work similarly in studies where other plant–microbe interactions are investigated, provided an appropriate R. rhizogenes pBR322-acceptor strain is available or could be constructed. Thus, we believe that this vector will be highly beneficial for plant studies across a range of fields.

Supporting information

S1 File. Step-by-step protocol, also available on protocols.io.

https://doi.org/10.1371/journal.pone.0291680.s001

(PDF)

Acknowledgments

T. G. FitzGerald for proofreading the manuscript.

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