Mar 04, 2022
Agrobacterium-mediated transformation of Zymoseptoria tritici
- Harry Child1,
- Nicolas Helmstetter2
- 1University of Exeter;
- 2University of Exeter, UK
Protocol Citation: Harry Child, Nicolas Helmstetter 2022. Agrobacterium-mediated transformation of Zymoseptoria tritici. protocols.io https://dx.doi.org/10.17504/protocols.io.b5ukq6uw
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it’s working
Created: March 03, 2022
Last Modified: March 04, 2022
Protocol Integer ID: 58988
Keywords: Zymoseptoria tritici, transformation, Agrobacterium tumefaciens, fungal transformation, pathogenic fungus zymoseptoria tritici by agrobacterium tumefacien, agrobacterium tumefacien, plasmids into the plant, pathogenic fungus zymoseptoria tritici, transforming plasmid, transformation of zymoseptoria tritici, agrobacterium, competent cells with plasmid vector, mediated transformation, zymoseptoria tritici, competent cell, cell, plasmid vector
Abstract
This protocol describes the procedure for transforming plasmids into the plant pathogenic fungus Zymoseptoria tritici by Agrobacterium tumefaciens-mediated transformation. It is based on the protocol first described by Zwiers and De Waard (2001; doi: 10.1007/s002940100216) and utilises A.tumefaciens ternary vectors developed by Sidhu et al. (2015a; doi: 10.1016/j.fgb.2015.04.015), Sidhu et al. (2015b; doi: 10.1016/j.fgb.2015.03.021) and Kilaru et al. (2015; doi: 10.1016/j.fgb.2015.03.018).
Section 1 details the recipes for media used in the protocol
Section 2 describes how to make competent A. tumefaciens cells
Section 3 details the process of transforming A. tumefaciens competent cells with plasmid vectors
Section 4 describes the process of A. tumefaciens-mediated Z. tritici transformation
Attachments
Materials
Chemicals for media preparation (see section 1 of protocol)
Selective antibiotics (see section 1 of protocol)
Zymoseptoria tritici strain for transformation
Agrobacterium tumefaciens strain EHA105
Plasmid DNA from E. coli miniprep
Liquid nitrogen
Nitrocellulose discs (Product number '325 P cellulose film' from AA packaging limited, Preston, UK used here)
Tweazers
Troubleshooting
1. Media Recipes
Induction Media (IM)
- Make stock solutions in deionised water (diH20) according to Table 1, with K2HPO4/KH2PO4/NaCl combined to make ‘Stock A’, which can be autoclaved and used in a sterile environment to make IM for mutliple transformations.
- To prepare fresh for each transformation, dissolve the required amount of MES in 100 ml of deionised water before adding appropriate amount of each stock solution (Table 1).
Table 1 Induction medium components
| A | B | C | D | E | |
| Reagent | Stock Solution | Concentration factor | Volume stock for 1l | Final conc. | |
| K2HPO4 | 200mM (34.8 g/l) | 20x (Stock A) | 50 ml | 10 mM | |
| KH2PO4 | 200mM (27.2 g/l) | 10 mM | |||
| NaCl | 50mM (2.92 g/l) | 2.5 mM | |||
| MgSO4.7H20 | 1M (246.47 g/l) | x500 | 2 ml | 2 mM | |
| CaCl2 | 100 mM (11.1 g/l) | x142.85 | 7 ml | 0.7 mM | |
| FeSO4 | 10 mM (2.78 g/l) | x1000 | 1 ml | 10 μM | |
| (NH4)2SO4 | 1M (132.1 g/l) | x250 | 4 ml | 4 mM | |
| Glucose | 1M (180.16 g/l) | x100 | 10 ml | 10 mM | |
| Glycerol | 20% (v/v) | x40 | 25 ml | 0.5% | |
| MES | - | - | 7.81 g/l (solid) | 40 mM |
- Add diH20 to give the final volume
- Adjust the pH to 5.6 using 1M NaOH
- Decant 1/5 of the prepared IM for liquid medium (IM broth)
- Add 2% Agar (w/v) for solid media (IM agar)
- Autoclave both and place IM broth in fridge until use
- Once cooled, amend IM agar with kanamycin (100 μg/ml) and acetosyringone (40 µg/ml) and pour plates. Store in the fridge once plates are set.
Aspergillus Minimal Medium (AMM) agar
- Prepare 1l stock of 20x Salts solution (120 g/l NaNO3, 10.4 g/l KCl, 10.4 g/l MgSO4.7H2O, 30.4 g/l KH2PO4), autoclave and store at 4ºC
- To prepare 1000x Trace elements stock solution, add reagents in Table 2 in order to 80 ml diH2O, allowing each to dissolve before adding the next.
Table 2 Trace elements components
| A | B | |
| Reagent | Mass for 100ml | |
| ZnSO4.7H2O | 2.2g | |
| H3BO3 | 1.1g | |
| MnCl2.4H2O | 0.5g | |
| FeSO4.7H2O | 0.5g | |
| CoCl2.5H2O | 0.16g | |
| CuSO4.5H2O | 0.16g | |
| (NH4)6Mo7O24.4H2O | 0.11g | |
| Na4EDTA | 5g |
- Heat Trace elements stock solution to boiling, cool to 60º, adjust pH to 6.5 – 6.8 with 5M KOH and adjust the volume to 100 ml.
- Autoclave Trace elements stock solution, then store in the dark at 4º. (Note: stored in the fridge, the FeSO4 may preciptitate out of solution but solution will still work)
- Prepare 1l of Aspergillus MM agar by combining the following:
- 10 ml of 20x Salts
- 1 ml of 1000x Trace elements
- 10g Glucose
- Add to 800ml diH2O and adjust the pH to 6.5 with 5M KOH, then adjust volume to 1l.
- For solid medium add 10 g/l Agar No. 1 and autoclave.
- Once cool add antibiotics to remove A. tumefaciens and select for transformant Z. tritici colonies
Basal Medium (BM) agar
- Prepare basal medium (1.7 g/l Yeast nitrogen base w/o amino acids or NH4SO4, 2 g/l Asparagine, 1 g/l NH4NO3, 10 g/l Glucose) in diH2O
- Adjust the pH to 6 with 1M Na2HPO4
- Add 2 g/l agar and autoclave
- Once cool add antibiotics to remove A. tumefaciens and select for transformant Z. tritici colonies
YPD Agar Media
- Prepare YPD (10 g/l Yeast extract, 20 g/l Peptone, 20 g/l Glucose, 20 g/l Agar) in diH2O and autoclave.
Prepared the following antibiotics stock solutions, before being filter sterilised and stored at –20°C. These are added to cooled media when required.
- Rifampicin: 100 mg/ml in DMSO
- Kanamycin: 100 mg/ml in H2O.
- Cefotaxime: 250 mg/ml in H2O.
- Streptomycin: 100 mg/ml in H2O.
- Ampicillin: 100 mg/ml in H2O.
- Hygromycin B: 50 mg/ml in H2O.
- Sulfonylurea: 10 mg/ml in DMF (Dimethylformamide)
- Glufosinate Ammonium (BAR selection): 100 mg/ml in H2O
- Carboxin: 40 mg/ml in Ethanol
- Geneticin (G418 selection): 200 mg/ml in H2O
- Acetosyringone: 40 mg/ml in DMSO
2. Making competent Agrobacterium tumefaciens cells
Inoculate 5 ml LB broth amended with Rifamipicin (100 μg/ml) with a colony of A. tumefaciens or 15 µl of A. tumefaciens glycerol stock.
Incubate overnight at 28°C with shaking at 250 rpm.
The next day, inoculate 50 ml LB broth amended with Rifamipicin (100 μg/ml) in a sterile 250 ml conical flask with 2 ml of the above overnight culture.
Incubate flask at 28°C with shaking at 250 rpm for ~8 hours until it reaches OD600 of 0.6.
Chill the flask on ice for 5 minutes.
Pellet cells in a 50 ml Falcon tube at 3000 rpm at 4°C for 5 min.
Resuspend cells in 1 ml of 20 mM CaCl2.
Make aliquots and store at –80°C (50 µl of cells used per transformation).
3. Transformation of A. tumefaciens
Thaw A. tumefaciens competent cells on ice (takes 1-2 hours).
Add 10 µl of plasmid miniprep DNA to 50 µl of A. tumefaciens competent cells in a 1.5 ml Eppendorf tube. Mix gently by flicking tube.
Freeze tubes in liquid nitrogen. Wait until bubbling subsides then remove the tube.
Thaw frozen tubes in 37°C water bath for 5 minutes.
Add 0.5 ml LB Broth without antibiotics.
Incubate cells at 28°C with 200 rpm shaking for 2-3 hours.
Pellet cells and resuspend with 150 µl LB broth before plating onto LB agar plates amended with Kanamycin (100 μg/ml) and Rifampicin (100 μg/ml).
Incubate plates at 28°C for 2 days.
Pick one transformant colony and inoculate 5 ml LB Broth amended with Kanamycin (100 μg/ml) and Rifampicin (100 μg/ml).
Incubate overnight 28°C with 250 rpm shaking.
Make glycerol stock of transformed A. tumefaciens cells (850 µl of cells + 150 µl sterile glycerol).
4. Agrobacterium-mediated transformation of Z. tritici
Before transformation day:
Five days before the transformation, inoculate a YPD agar plate with the Z. tritici strain to be used in transformation and incubate at 19oC.
Four days before transformation, streak LB agar amended with Kanamycin (100 μg/ml) and Rifampicin (100 μg/ml) with A. tumefaciens glycerol stock and incubate at 28°C for 2-3 days before use for fungal transformation.
The day before transformation, inoculate a single colony of A. tumefaciencs cells into 4 ml of LB media in a 50 ml falcon tube, amended with Kanamycin (100 μg/ml) and Rifampicin (100 μg/ml) and grow at 28°C with 250 rpm shaking overnight.
The day before transformation, prepare IMbroth and IM agarplates amended with Kanamycin (100 μg/ml) and acetosyringone (40 µg/ml).
Transformation day:
Measure the OD600 of the overnight culture.It can be used when it has reached an OD600 of at least 0.75 to around 1. (Ensure the blank for spectrophotometer readings is LB + Rifampicin).
Dilute A. tumefaciens overnight culture to an OD600 of 0.09 in IM (amended with Kanamycin 100 μg/ml and Acetosyringone 40 µg/ml) in a volume of 10 ml in a 50 ml sterile flask.
Grow cultures at 28°C/250 rpm. Measure OD600 every hour, until it reaches an OD600 of 0.21-0.24 (maximum 0.28). This usually takes 1.5-3 hours. (Ensure the blank for spectrophotometer readings is IM containing a similar dilution of LB + Rifampicin to A. tumefaciens cultures).
While A. tumefaciens cultures are incubating, place a single sterile nitrocellulose disc dipped in sterile water on the centre of each induction plate using sterile tweazers. Make sure to drain as much water as possible off the disc before laying it on the plate. Leave plates open to dry.
(Optional: Pour 5-10 sterile glass beads into each IM agar plate if plating out too many transformant plates for spreading with L-shaped spreader).
Harvest the Z. tritici yeast-like cells by adding 2 ml sterile H2O to the Z. tritici plate and spreading thoroughly using an L-shaped spreader and transfer to a 15 ml falcon tube.
Pass Z. tritici cell suspension through a 100 µm sterile cell strainer/miracloth to remove clumped cells.
Measure the concentration of yeast-like cells on a haemocytometer, diluting where necessary.
Dilute Z. tritici yeast-like cells to 5x106 cells/ml in sterile H2O.
Equal volumes of the A. tumefaciens culture and Z. tritici cells are mixed together.
Prepare the following controls
- ‘No Agrobacterium’ control (equal amounts of induction media and Z. tritici cells) as a control for background growth of Z. tritici on selective plates.
- ‘No selection’ control (equal amounts of Z. tritici and A. tumefaciens) to assess Z. tritici growth without selection.
Plate 150 μl aliquots of the above mixtures onto IM agar plates with nitrocellulose discs and spread with L-shaped spreader (or glass beads).
Allow plates to dry before placing in a plastic box containing silica gel beads to avoid build up of moisture in plates. (Note: no need to parafilm plates).
Incubate the plates for 48 h at 19 °C.
Two days after transformation:
Prepare one of the following media for selection depending on selective marker used. Remember to pour a plate for the ‘No selection’ control plate before adding the Z. tritici selective antibiotic.
- AMM agar with Hygromycin B (200 μg/ml) – HYGRselection
- AMM agar with glufosinate ammonium (200 μg/ml) – BARR selection
- AMM agar with Geneticin (200 μg/ml) - G418Rselection
- BM agar with Sulfonylurea (10 μg/ml) – SURRselection
- Czapek Dox agar with Carboxin (40ug/ml) – sdi1R selection
Add the following antibiotics to kill A. tumefaciens (including ‘No selection’ control plate): Cefotaxime 250 μg/ml OR Timentin 100μg/ml, AND Streptomycin 100 μg/ml AND Ampicillin 100 μg/ml.
When the plates are set, transfer the nitrocellulose discs using sterile tweazers from IM plates onto selective plates.
Incubate the plates at 19 °C. Colonies should be visible after around 10 days but can take up to 3 weeks to be ready for subculturing.
Putative Transformants:
After 2-3 weeks, any colonies which appear are patch subcultured onto the following media depending on selective marker used:
- HYG/G418/sdi1R: YPD plates amended with Hygromycin OR Geneticin (200 μg/ml) OR Carboxin (40 μg/ml) and antibiotics
- BAR: AMM plates with Glufosinate ammonium (200 μg/ml) and antibiotics
- SUR: BM plates with sulfonylurea (10 μg/ml) + antibiotics
Plates are incubated at 19 °C for 5-7 days and any growing colonies are then verified by PCR screening.
Confirmed transformants are subcultured on selection media a second time before being stored in 50% glycerol at -80oC.