TITLE: A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes
AUTHORS: Shaun Ferguson, Nikolaj B. Abel, Dugald Reid, Lene H. Madsen, Thi-Bich Luu, Kasper R. Andersen, Jens Stougaard, Simona Radutoiu

[DESCRIPTION]
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.

[BEFORE_START]
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).

pIV101 is available on Addgene and can be found here: https://www.addgene.org/196671/.

Ensure you have a pBR322-derived R. rhizogenes acceptor strain e.g. R. rhizogenes AR1193.

[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.


[STEPS]
SECTION: Transformation of chemical competent E. coli ST18
2. 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)
 75 µL ddH2O

SECTION: Transformation of chemical competent E. coli ST18
3. Incubate for 2 min on ice
SECTION: Transformation of chemical competent E. coli ST18
4. Thaw a 100 µL aliquot of chemically competent E. coli ST18 cells on ice

SECTION: R. rhizogenes transformation
13. 
 

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)
SECTION: R. rhizogenes transformation
14. Incubate for 2880 min at 28 °C with shaking

SECTION: R. rhizogenes transformation
15. 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)
SECTION: Preparation of seeds for hairy root transformation (for Lotus japonicus)
32. To scarify the seeds, transfer the required number of seeds to a mortar and rub them with sand paper until they become white on the ends

38. Transfer germinated seeds to a square petri dish that contains solid Gamborg B5 media including vitamins
36. Using sterile forceps, transfer seeds to square petri dishes containing sterile filter paper soaked in sterile dH2O (approximately 15 min per plate)
33. Transfer the seeds to a sterile tube (at least 15 ml capacity) and sterilise the seeds by immersing them in a 1% hypochlorite solution, and incubate in this solution for 15 min at Room temperature 

SECTION: Hairy root transformation of Lotus seedlings with R. rhizogenes
41. Wound seedlings with syringe needle (0.4mm) at the hypocotyl
SECTION: Hairy root transformation of Lotus seedlings with R. rhizogenes
42. Add one large drop (~ 100 µL) of the thick suspension of R. rhizogenes (from step 40) on top of the wound 

SECTION: Hairy root transformation of Lotus seedlings with R. rhizogenes
43. Incubate the seedling for 60 min horizontally to let the R. rhizogenes infect the hypocotyl
SECTION: Hairy root transformation of Lotus seedlings with R. rhizogenes
44. Seal the plates containing the now infected seedlings with parafilm on the sides and bottom (prevents dehydration). Seal top with micropore tape on the top edge (Keeps the plates sealed but allows gas exchange)
SECTION: Selecting hairy roots
47. Hairy roots will emerge and develop from the infected wound sites
SECTION: Selecting hairy roots
48. Place the plates containing the transformed plants on a transilluminator. Remove the non-fluorescently labelled roots (untransformed roots) using a scalpel

 

SECTION: Selecting hairy roots
49. Transfer plants with transformed roots to new pots or plates and grow plants at 21 °C with  16H/8H light/dark cycle for one week 

SECTION: Preparation of R. rhizogenes for hairy root transformation
40. Resuspend the R. rhizogenes strain from the agar plate (step 30-31) into sterile dH2O as a thick suspension (OD600 >2). Approximately 100 µL is needed per plant, so make sure the volume of water for the suspension is sufficient for the number of plants that will be transformed

 


SECTION: Hairy root transformation of Lotus seedlings with R. rhizogenes
45. Grow the infected seedlings for 4320 min at 21 °C in the dark to enhance infection
SECTION: Hairy root transformation of Lotus seedlings with R. rhizogenes
46. Grow the infected seedlings for 3 weeks at 21 °C with 16H/8H light/dark cycle until transformed roots emerge

SECTION: R. rhizogenes transformation
16. Incubate the LB broth from step 15  1440 min at 37 °C  with shaking.

SECTION: R. rhizogenes transformation
17. Centrifuge 1 ml of the overnight broth for the E. coli ST18 culture carrying the construct of interest at 14000 rcf, 2 min

SECTION: R. rhizogenes transformation
18. Resuspend the E. coli pellet in 1 mL sterile dH2O and repeat the previous step. This is to wash away the broth culture containing antibiotics

SECTION: R. rhizogenes transformation
20. Centrifuge 1 ml of the broth culture of R. rhizogenes from step 13-14 at 8000 rcf, 2 min 
SECTION: R. rhizogenes transformation
22. Resuspend the R. rhizogenes pellet in 50 µL sterile dH2O
SECTION: R. rhizogenes transformation
23. Perform a biparental mating by mixing the resuspended E.coli ST18 (step 19) and R. rhizogenes (step 22) in 100 µL and spot onto plates of LB media supplemented with 50 μg/ml ALA (but no antibiotics) and then wait until the spot is dry
SECTION: R. rhizogenes transformation
24. Grow the biparental mating spot plates   at 28 °C 
SECTION: R. rhizogenes transformation
25. Scrape the biparental mating spot of the E. coli ST18 clone + R. rhizogenes and resuspend in 1 mL  sterile dH2O
SECTION: R. rhizogenes transformation
26. Centrifuge at 14000 rcf, 2 min to pellet, and resuspend in 1 mL  sterile dH2O. This step should wash away any residual supplement from the mating plates that enables E. coli growth

SECTION: R. rhizogenes transformation
27. Centrifuge again at 14000 rcf, 2 min to pellet, and resuspend in 100 µL sterile dH2O (The total volume will be more due to the pellet)
SECTION: Transformation of chemical competent E. coli ST18
6. Incubate the mix for 20 min on ice

SECTION: Transformation of chemical competent E. coli ST18
7. Incubate for 10 min at Room temperature 

SECTION: Transformation of chemical competent E. coli ST18
8. Add 800 µL LB with 5 μg/ml 5-aminolevulinic acid (ALA) and grow for 60 min at 37 °C with shaking

11. Plate out the  100 µL of resuspended pellet onto LB agar containing 100 μg/ml ampicillin, 150 μg/ml spectinomycin, and 50 μg/ml ALA

12. Grow at 37 °C    and check for colonies the following day



 

SECTION: Golden Gate cloning reaction with plasmid pIV101
1. Add the following components to a Golden Gate reaction:
Plasmid pIV101 DNA (100 ng) - X µL 
Insert(s) DNA (100 ng) - X µL 
Ligase buffer (10x) - 1 µL 
T4 DNA Ligase - 1 µL 
BpiI (BbsI) (or BsaI for level 1 constructs) - 1 µL 
MilliQ water - up to final volume of 10 µL 
37. Incubate the square plates containing the surface sterilised seeds sitting on damp filter paper
from the previous step for 4320 min at 21 °C
35. Fill the tube with sterile water and incubate for at least 120 min at Room temperature with shaking (alternatively incubate   at 4 °C )

34. Remove the hypochlorite solution and discard appropriately. Add sterile water and invert the tube several times. Repeat this 5 times to wash the seeds and remove any residual hypochlorite
39. Grow seedlings for 4320 min at 21 °C with 16H/8H light/dark cycle, until the root has attached itself to the media
SECTION: Golden Gate cloning reaction with plasmid pIV101
1.1. 

Incubate reaction in thermocycler with the following steps:
 

 

SECTION: Transformation of chemical competent E. coli ST18
9. Centrifuge to pellet the cells at 14000 rcf, 2 min, 25 °C 

10. Resuspend the pellet in 100 µL of LB containing 50 μg/ml ALA

SECTION: For rhizobium inoculation of hairy roots (optional)
50. Resuspend the rhizobium strain (or your bacteria of interest) from a freshly streaked agar plate into sterile dH2O
SECTION: Transformation of chemical competent E. coli ST18
5. Mix together the chemical competent E. coli cells and the reaction mixture from step 2 by pipetting
SECTION: For rhizobium inoculation of hairy roots (optional)
51. Adjust the OD600 of the suspension to between 0.01 - 0.05 in a volume that is sufficient to provide 100 µL per plant to be inoculated
SECTION: For rhizobium inoculation of hairy roots (optional)
51.1. If plants are on square agar plates: lay the square plate flat. Inoculate the roots of the plants by carefully applying 100 µL of the suspension from the previous step using a pipette. Ensure that you apply the inoculum evenly to as much of the root as possible
SECTION: For rhizobium inoculation of hairy roots (optional)
51.2. Leave inoculated plant plate(s) flat for a short period so that the bacterial suspension can spread evenly across the plate and ensure contact with the roots
SECTION: For rhizobium inoculation of hairy roots (optional)
51.3. If the plants are in pots: determine the volume that is equal to  100 µL X the number of plants present, and distribute this evenly to the plant pot substrate using a pipette
11.1. If using blue/white selection then also add 2% (w/v) 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) to the media
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

 

SECTION: R. rhizogenes transformation
19. Resuspend the E. coli pellet in 50 µL sterile dH2O
SECTION: R. rhizogenes transformation
21. Resuspend the R. rhizogenes pellet in 1 mL sterile dH2O and repeat the previous step. This is to wash away the broth culture containing antibiotics
SECTION: R. rhizogenes transformation
28. 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

 

SECTION: R. rhizogenes transformation
30. 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
SECTION: R. rhizogenes transformation
29. Incubate the plate(s) for 2880 min at 28 °C
SECTION: R. rhizogenes transformation
31. Incubate the plate(s) for 2880 min at 28 °C
12.1. If using X-gal for selection avoid blue colonies as pIV101 contains the lacZα fragment in the GGA cloning site 
SECTION: For rhizobium inoculation of hairy roots (optional)
52. Grow the inoculated plants for 3 weeks post inoculation at 21 °C  with 16H/8H light/dark cycle until nodules are formed