TITLE: Agrobacterium-mediated transformation of Zymoseptoria tritici
AUTHORS: Harry T Child, Nicolas  Helmstetter

[DESCRIPTION]
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




[STEPS]
SECTION: 1.	Media Recipes
1. 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
     Reagent      Stock      Conc. Factor      Vol. 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.
SECTION: 1.	Media Recipes
2. 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
     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
SECTION: 1.	Media Recipes
3. 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 
SECTION: 1.	Media Recipes
4. 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.
SECTION: 1.	Media Recipes
5. 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
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
6. 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.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
7. Incubate overnight at 28°C with shaking at 250 rpm.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
8. 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.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
9. Incubate flask at 28°C with shaking at 250 rpm for ~8 hours until it reaches OD600 of 0.6.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
10. Chill the flask on ice for 5 minutes.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
11. Pellet cells in a 50 ml Falcon tube at 3000 rpm at 4°C for 5 min.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
12. Resuspend cells in 1 ml of 20 mM CaCl2.
SECTION: 2.	Making competent Agrobacterium tumefaciens cells
13. Make aliquots and store at –80°C (50 µl of cells used per transformation).
SECTION: 3.	Transformation of A. tumefaciens
14. Thaw A. tumefaciens competent cells on ice (takes 1-2 hours).
SECTION: 3.	Transformation of A. tumefaciens
15. 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.
SECTION: 3.	Transformation of A. tumefaciens
16. Freeze tubes in liquid nitrogen. Wait until bubbling subsides then remove the tube.
SECTION: 3.	Transformation of A. tumefaciens
17. Thaw frozen tubes in 37°C water bath for 5 minutes. 
SECTION: 3.	Transformation of A. tumefaciens
18. Add 0.5 ml LB Broth without antibiotics.
SECTION: 3.	Transformation of A. tumefaciens
19. Incubate cells at 28°C with 200 rpm shaking for 2-3 hours.
SECTION: 3.	Transformation of A. tumefaciens
20. 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).
SECTION: 3.	Transformation of A. tumefaciens
21. Incubate plates at 28°C for 2 days.
SECTION: 3.	Transformation of A. tumefaciens
22. Pick one transformant colony and inoculate 5 ml LB Broth amended with Kanamycin (100 μg/ml) and Rifampicin (100 μg/ml).
SECTION: 3.	Transformation of A. tumefaciens
23. Incubate overnight 28°C with 250 rpm shaking.
SECTION: 3.	Transformation of A. tumefaciens
24. Make glycerol stock of transformed A. tumefaciens cells (850 µl of cells + 150 µl sterile glycerol).
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
25. 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.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
26. 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. 
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
27. 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. 
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
28. The day before transformation, prepare IMbroth and IM agarplates amended with Kanamycin (100 μg/ml) and acetosyringone (40 µg/ml).
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
29. 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).
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
30. 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.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
31. 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).
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
32. 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.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
33. (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).
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
34. 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.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
35. Pass Z. tritici cell suspension through a 100 µm sterile cell strainer/miracloth to remove clumped cells.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
36. Measure the concentration of yeast-like cells on a haemocytometer, diluting where necessary.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
37. Dilute Z. tritici yeast-like cells to 5x106 cells/ml in sterile H2O.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
38. Equal volumes of the A. tumefaciens culture and Z. tritici cells are mixed together. 
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
39. 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.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
40. Plate 150 μl aliquots of the above mixtures onto IM agar plates with nitrocellulose discs and spread with L-shaped spreader (or glass beads). 
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
41. 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).
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
42. Incubate the plates for 48 h at 19 °C.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
43. 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
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
44. 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.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
45. When the plates are set, transfer the nitrocellulose discs using sterile tweazers from IM plates onto selective plates.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
46. 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. 
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
47. 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
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
48. Plates are incubated at 19 °C for 5-7 days and any growing colonies are then verified by PCR screening.
SECTION: 4.	Agrobacterium-mediated transformation of Z. tritici
49. Confirmed transformants are subcultured on selection media a second time before being stored in 50% glycerol at -80oC.