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

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.

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.

GUIDELINES
The Modular Cloning (MoClo) strategy implemented for pIV101 is based on the publication by Weber et al. 2011.For this protocol we use E. coli strain ST18 for transformation of our cloned construct and subsequent biparental mating to transfer the construct to R. rhizogenes.Another E. coli strain could be substituted but it must contain the RP4 conjugative machinery for mobilisation of pIV101 and we would recommend that it also contains the lacZΔM15 mutation for blue white/screening as well as an auxotrophy for efficicient conterselection (E.g.ΔdapA).
Ensure you wash the cells before and after biparental mating.This ensures adequate removal of both the antibiotics for plasmid selection, and the supplements required by the auxotrophic E. coli.
For standard molecular biology techniques (E.g.PCR, plasmid preparation etc.) please refer to Molecular Cloning: A Laboratory Manual, 4th edition.

SAFETY WARNINGS
Please refer to the manufacturers safety warnings for the individual reagents and chemicals used throughout.

BEFORE START INSTRUCTIONS
Ensure you have an appropriate E. coli cloning/donor strain that contains the RP4 conjugative machinery to enable conjugal transfer of pIV101.Some commonly used strains for this include: E. coli S17/ST18, E. coli MFDpir, E. coli WM3064.
If using an auxotrophic E. coli strain such as ST18 or MFDpir make sure you have the required supplement (e.g.5-aminolevulinic acid (ALA) for ST18 or diaminopimelic acid (DAP) for MFDpir).
Ensure you have a pBR322-derived R. rhizogenes acceptor strain e.g.R. rhizogenes AR1193. 1 Add the following components to a Golden Gate reaction: 12.1 If using X-gal for selection avoid blue colonies as pIV101 contains the lacZα fragment in the GGA cloning site

12.2
In parallel with the next step (step 13), select colonies from the transformation (step 12) and confirm the construct from the Golden Gate assembly.This can be carried out by several standard approaches: Perform colony PCR to amplify the region of the plasmid that contains the GGA cloning site to ensure that the cloning site contains the expected insert size This product can be sent for further confirmation by Sanger sequencing Additionally, perform a plasmid preparation (miniprep) from the E. coli clone which can then be used as template for PCR amplification or for whole plasmid sequencing

Note
If your insert is large then we recommended sending the construct for whole plasmid sequencing as an alternative to confirmation by standard Sanger sequencing

Note
Start liquid culture of the wild-type Agrobacterium rhizogenes (now Rhizobium rhizogenes) from a single colony in LB media with 100 μg/ml rifampicin (a 5 ml broth is sufficient) This section and the next section can be carried out in parallel to save time Incubate for 48:00:00 at 28 °C with shaking Inoculate an LB broth containing 100 μg/ml ampicillin, 150 μg/ml spectinomycin, and 50 μg/ml ALA with a single colony for an ST18 clone carrying the construct of interest (from the previous section) Incubate the LB broth from step 15 Overnight at 37 °C with shaking.
Buffer NEB T4 Ligase, 2,000,000 cohesive end units/ml Nuclease-free water Chemicals for E. coli chemical transformation Filter sterilised diaminopimelic acid (DAP) stock at 30 mM in water (For ΔdapA mutant E. coli strains).Previously prepared chemically competent aliquots of E. coli donor strain.KCM buffer (5X) 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) 1sterilise through 0.2 uM filter.Store at -20. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal): 20 mg/ml in dimethylformamide (2% w/v) Auxotrophic E. coli supplement 5-aminolevulinic acid (ALA): 50 mg/ml dissolved in water and filter sterlised Diaminopemelic acid (DAP): 30 mM dissolved in water and filter sterlised General materials Thermocycler suitable for Golden Gate reactions Tabletop microcentrifuge Spectrophotometer 1.5 ml nuclease-free microcentrifuge tubes Round and square (11 cm 2 ) petri dishes Gate reaction is failing to incorporate your desired insert, an additional step that may help: Following completion of the reaction cycle from step 1.1 -add 1 μl of the restriction enzyme used in the reaction (step 1) to the mix again and incubate for a further 1 -2hrs at 37C Because the ligase has already been deactivated, additional active restriction enzyme can help to deplete any remaining plasmids that do not contain the desired insert fragments Mix in a 1.5 ml tube: 20 µL 5X KCM buffer 5 µL Golden Gate cloning mix (from step 1.1 following completion of the program) of chemically competent E. coli ST18 cells on ice Mix together the chemical competent E. coli cells and the reaction mixture from step 2 of the overnight broth for the E. coli ST18 carrying the construct of interest at 14000 rcf, 00:02:00 Resuspend the E. coli pellet in 1 mL sterile dH 2 O and repeat the previous step.This is to wash away the broth culture containing antibiotics Resuspend the E. coli pellet in 50 µL sterile dH 2 O Centrifuge 1 ml of the broth culture of R. rhizogenes from step 13-14 at 8000 rcf, 00:02:00 Resuspend the R. rhizogenes pellet in 1 mL sterile dH 2 O and repeat the previous step.This is to wash away the broth culture containing antibiotics Resuspend the R. rhizogenes pellet in 50 µL sterile dH 2 O Perform a biparental mating by mixing the resuspended E.coli ST18 (step 19) and R. rhizogenes (step 22) in 100 µL total volume and spot onto plates of LB media supplemented with 50 μg/ml ALA (but no antibiotics) and then wait until the spot is dry Grow the biparental mating spot 00:02:00 to pellet, and resuspend in 1 mL sterile dH 2 O.This step should wash away any residual supplement from the mating plates that enables E. coli growth Centrifuge again at 14000 rcf, 00:02:00 to pellet, and resuspend in 100 µL sterile dH 2 O (The total volume will be more due to the pellet) Transfer the resuspended mix from the previous step onto LB media plates supplemented with 100 μg/ml ampicillin, 50 μg/ml spectinomycin, and 100 μg/ml rifampicin.(No ALA).Plate out for single colonies Note For step 28: if the bacterial suspension is too thick then try diluting the resuspended mix by 1:10 in sterile water and plate this out in parallel Incubate the plate(s) for 48:00:00 at 28 °C Re-streak the R. rhizogenes strains carrying the construct of interest on LB agar containing 100 μg/ml ampicillin, 50 μg/ml spectinomycin, and 100 μg/ml rifampicin to ensure single colonies Incubate the plate(sfor hairy root transformation (for Lotus j…