Aug 02, 2024
Agrobacterium-mediated transformation of Diplodia sapinea V.1
Peer-reviewed method
- Anne Geertje Oostlander1,
- Laura Brodde2,3,
- Miriam von Bargen1,
- Bernard Slippers4,
- Yvonne Becker5,
- Ulrike Brandt1,
- Frank Klawonn6,
- Christiaan Grobler4,
- Lucas Well1,
- Jan Stenlid3,
- Jonàs Oliva3,7,
- Malin Elfstrand3,
- André Fleißner1
- 1Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany;
- 2SCA Skog, NorrPlant, Sundsvall, Sweden;
- 3Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden;
- 4Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa;
- 5Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn Institute (JKI)—Federal Research Centre for Cultivated Plants, Braunschweig, Germany;
- 6Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany;
- 7Department of Agricultural and Forest Sciences and Engineering, University of Lleida, Lleida, Spain
- PLOS ONE Lab ProtocolsTech. support email: plosone@plos.org
Protocol Citation: Anne Geertje Oostlander, Laura Brodde, Miriam von Bargen, Bernard Slippers, Yvonne Becker, Ulrike Brandt, Frank Klawonn, Christiaan Grobler, Lucas Well, Jan Stenlid, Jonàs Oliva, Malin Elfstrand, André Fleißner 2024. Agrobacterium-mediated transformation of Diplodia sapinea. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok7ozl4o/v1
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: July 23, 2024
Last Modified: August 02, 2024
Protocol Integer ID: 103916
Keywords: Diplodia sapinea, Diplodia tip blight, Sphaeropsis sapinea, Agrobacterium-mediated-transformation, homologous integration, genetic transformation, ATMT
Funders Acknowledgements:
Marie Skłodowska-Curie grant
Grant ID: 101008129
Ellen and Tage Westins foundation
Grant ID: .
Abstract
This protocol details an Agrobacterium-mediated genetic transformation
method for the fungal plant pathogen Diplodia sapinea. The technique
results in high rates of homologous integration, enabling both targeted
mutagenesis and heterologous gene expression.
Guidelines
Materials
Material.pdf Potassium Buffer
Reference: Michielse et al. 2008
Ingredients:
- 1.25 M K₂HPO₄: 217.7 g K₂HPO₄
- 1.25 M KH₂PO₄: 170.1 g KH₂PO₄
Instructions:
- Fill each component up to 1 liter with water and autoclave.
- Add K₂HPO₄ solution to KH₂PO₄ solution until pH 4.8 is reached.
Magnesium-Sodium Solution
Reference: Michielse et al. 2008
Ingredients:
- 0.12 M MgSO₄·7H₂O: 30 g MgSO₄·7H₂O
- 0.25 M NaCl: 15 g NaCl
Instructions:
- Fill up to 1 liter with water and autoclave.
Calcium Chloride Solution
Reference: Michielse et al. 2008
Ingredients:
- 1% CaCl₂ (wt/vol): 10 g CaCl₂·2H₂O
Instructions:
- Fill up to 1 liter with water and autoclave.
Glucose Solution
Reference: Michielse et al. 2008
Ingredients:
- 20% (wt/vol) C₆H₁₂O₆: 200 g C₆H₁₂O₆·H₂O
Instructions:
- Fill up to 1 liter with water and autoclave.
Ferric Sulfate Solution
Reference: Michielse et al. 2008
Ingredients:
- 0.01% FeSO₄: 0.1 g FeSO₄·7H₂O
Instructions:
- Fill up to 1 liter with water and sterile filtrate.
Trace Elements
Reference: Michielse et al. 2008
Ingredients:
- 0.01% ZnSO₄·7H₂O: 0.1 g ZnSO₄·7H₂O
- 0.01% CuSO₄·5H₂O: 0.1 g CuSO₄·5H₂O
- 0.01% H₃BO₃: 0.1 g H₃BO₃
- 0.01% MnSO₄·H₂O: 0.1 g MnSO₄·H₂O
- 0.01% Na₂MoO₄·2H₂O: 0.1 g Na₂MoO₄·2H₂O
Instructions:
- Fill up to 1 liter with water and autoclave.
Ammonium Nitrate Solution
Reference: Michielse et al. 2008
Ingredients:
- 2.5 M NH₄NO₃: 200 g NH₄NO₃
Instructions:
- Fill up to 1 liter with water and sterile filtrate.
Solid Lysogeny Broth Medium (LB)
Reference: Bertani 1951
Ingredients:
- 5 g Yeast Extract
- 10 g Peptone
- 10 g Sodium Chloride
- 15 g Agar
Instructions:
- Fill up to 1 liter with water and autoclave.
Liquid Lysogeny Broth Medium (LB)
Reference: Bertani 1951
Ingredients:
- 5 g Yeast Extract
- 10 g Peptone
- 10 g Sodium Chloride
Instructions:
- Fill up to 1 liter with water and autoclave.
MES Buffer
Reference: Michielse et al. 2008
Ingredients:
- 1 M 2-(N-Morpholino) ethanesulfonic acid (MES): 195.24 g MES
Instructions:
- Adjust pH to 5.5 with NaOH.
- Fill up to 1 liter with water and sterile filtrate.
- Aliquot (10 ml) and store at -20°C in darkness.
Glycerol Solution
Reference: Michielse et al. 2008
Ingredients:
- 50% Glycerol (v/v): 500 ml Glycerol
Instructions:
- Fill up to 1 liter with water and autoclave.
Acetosyringone (AS) Solution
Reference: Michielse et al. 2008
Ingredients:
- 0.2 M Acetosyringone: 785 mg Acetosyringone
Instructions:
- Fill up to 20 ml with DMSO and sterile filtrate.
- Aliquot and store at –20°C in darkness.
- Concentration of work: 200 µM.
Hygromycin B Stock Solution
Ingredients:
- Hygromycin B (100 mg/ml): 1 g
Instructions:
- Fill up to 10 ml with dH₂O and sterile filtrate.
- Aliquot and store at –20°C in darkness.
Cefotaxime Stock Solution
Reference: Michielse et al. 2008
Ingredients:
- 0.2 M Cefotaxime: 955 mg Cefotaxime
Instructions:
- Fill up to 10 ml with dH₂O and sterile filtrate.
- Aliquot and store at –20°C in darkness.
Solid Induction Medium (IM)
Reference: Michielse et al. 2008
Ingredients:
- 15 g Agar
- 905.7 ml Water
Instructions:
- Autoclave.
- Directly before use, add for 1 l of medium:
- 800 µl Potassium buffer
- 20 ml Magnesium-sodium solution
- 1 ml Calcium chloride solution
- 5 ml Glucose solution
- 10 ml Ferric sulfate solution
- 5 ml Trace elements
- 2.5 ml Ammonium nitrate solution
- 10 ml Glycerol solution
- 200 µM Acetosyringone (AS) solution: 250 µl
- 40 ml MES buffer: 250 µl
Liquid Induction Medium (IM)
Reference: Michielse et al. 2008
Ingredients:
- 800 µl Potassium buffer
- 20 ml Magnesium-sodium solution
- 1 ml Calcium chloride solution
- 10 ml Glucose solution
- 10 ml Ferric sulfate solution
- 5 ml Trace elements
- 2.5 ml Ammonium nitrate solution
- 10 ml Glycerol solution
- 900.7 ml autoclaved water
Instructions:
- Directly before use, add for 1 l of medium:
- 200 µM Acetosyringone (AS): 1 ml AS
- 40 ml MES buffer
Vogels Trace Element Solution
Reference: Vogel 1956
Ingredients:
- 238 mM Citric acid (monohydrate): 5 g Citric acid (monohydrate)
- 174 mM ZnSO₄·7H₂O: 5 g ZnSO₄·7H₂O
- 25 mM (NH₄)₂Fe(SO₄)₂·6H₂O: 1 g (NH₄)₂Fe(SO₄)₂·6H₂O
- 10 mM CuSO₄·5H₂O: 0.25 g CuSO₄·5H₂O
- 3 mM MnSO₄·H₂O: 0.05 g MnSO₄·H₂O
- 8 mM H₃BO₃: 0.05 g H₃BO₃
- 2 mM Na₂MoO₄·2H₂O: 0.05 g Na₂MoO₄·2H₂O
Instructions:
- Dissolve all components successively in 95 ml distilled water while stirring at room temperature.
Vogels Biotin Solution
Reference: Vogel 1956
Ingredients:
- 5 mg Biotin
Instructions:
- Dissolve in 100 ml 50% ethanol.
Vogels Salts Solution
Reference: Vogel 1956
Ingredients:
- 125 g Na₃Citrate·2H₂O
- 250 g KH₂PO₄
- 100 g NH₄NO₃
- 10 g MgSO₄·7H₂O
- 5 g CaCl₂·2H₂O
- 5 ml Vogels Trace Element Solution
- 2.5 ml Vogels Biotin Solution
Instructions:
- Dissolve all components successively while stirring.
- Fill up to 1 liter with water.
Vogels Minimal Medium (VMM)
Reference: Vogel 1956
Ingredients:
- 20 ml Vogels Salts Solution
- 20 g Sucrose
- 15 g Agar
Instructions:
- Fill up to 1 liter with water and autoclave.
Transformation of electrocompetent Agrobacterium sp. AGL-1cells with plasmid DNA by electroporation
Transformation of electrocompetent Agrobacterium sp. AGL-1cells with plasmid DNA by electroporation
Thaw electrocompetent cells on ice.
Add 1 - 1.5 µl of plasmid DNA to 50 µl of cells.
Incubate on ice for 2 min.
Transfer the cell-DNA mixture to a chilled electroporation cuvette (2 mm) without introducing bubbles. Flick the cuvette downward quickly to distribute cells across the bottom of the well.
Electroporate the mixture with the following settings:
Voltage: 2500 V
Capacitance: 25 µF
Resistance: 400 Ω
Add 1 ml of LB medium to the cuvette immediately after pulsing and gently pipette up and down to resuspend the cells.
Transfer the cell suspension to a reagent tube and incubate the culture:
28 – 30°C
250 rpm
3 h
Spread the cells (10 µl, 100 µl, rest) onto selective plates with following selection markers:
50 µg/ml Kanamycin (10 µl stock / 10 ml medium)
25 µg/ml Rifampicin (5 µl stock / 10 ml medium)
100 µg/ml Carbenicillin (10 µl stock / 10 ml medium)
Incubate at 28 – 30°C. Transformed colonies are visible after 24-72 h.
Prepare a glycerin stock from several colonies and check via colony-PCR or plasmid preparation and PCR if your strains contain the expected fragment.
Plate Agrobacterium sp. AGL-1
Plate Agrobacterium sp. AGL-1
Inoculate LB plates (10 ml) containing the following selection markers with Agrobacterium sp. AGL-1 from the glycerin stock. Use the untransformed strain as a negative control. Incubate at 28°C for about 2 days.
AGL1 transformed: 50 µg/ml Kanamycin (10 µl stock / 10 ml medium), 25 µg/ml Rifampicin (5 µl stock / 10 ml medium), 100 µg/ml Carbenicillin (10 µl stock / 10 ml medium)
AGL1 untransformed: 25 µg/ml Rifampicin (5 µl stock / 10 ml medium), 100 µg/ml Carbenicillin (10 µl stock / 10 ml medium)
Pre-culture of Agrobacterium sp. AGL-1
Pre-culture of Agrobacterium sp. AGL-1
Inoculate 25 ml of liquid LB with the following selection markers in a 250 ml flask with a colony from fresh plates:
AGL1 transformed: 50 µg/ml Kanamycin (25 µl stock / 25 ml medium)
AGL1 untransformed: 25 µg/ml Rifampicin (12,5 µl stock / 25 ml medium)
Incubate until the cultures reached an OD600nm of 0.5 to 0.9:
200 rpm
28°C
~ 22 h
Main-culture Agrobacterium sp. AGL-1
Main-culture Agrobacterium sp. AGL-1
Transfer 12-15 ml of the Agrobacterium sp. AGL-1 suspension to a 50 ml centrifuge tube and centrifuge at:
3500 rpm
10 min
Wash the pellet with 1 ml freshly made liquid IM (see table, 100 ml Medium + 100 µl AS + 4 ml MES buffer) and centrifuge at:
3500 rpm
10 min
Resuspend the pellet in liquid IM to an OD600nm of about 0.3.
Incubate ca. 25 ml until the OD600 is doubled (about 0.6 – 0.8) in a 250 ml Erlenmeyer flask.
28°C
200 rpm
8 - 10 h
Preparation of fungal spore suspension
Preparation of fungal spore suspension
Harvest spores of D. sapinea by rinsing the plate with 0.01 % Tween (Incubated on VMM, 21 d, constant light, 5000 – 7000 Lux)
Centrifuge spores for 10 s at 5000 rpm, discard supernatant.
Wash spores twice with 1 - 2 ml liquid IM and centrifuge at:
5000 rpm
10 s
Resuspend cells in IM to a concentration of 2 · 106 spores/ml. 50 µl suspension is needed per transformation.
Co-Cultivation
Co-Cultivation
Onto 5.5 cm IM plates (freshly made or made the day before, ca. 5 ml medium per plate; stored in darkness at 4°C) place a nitrocellulose filter (MF-Millipore™ HAWP03700) with sterile tweezers.
Mix 50 µl of the spore suspension and 50 µl of the Agrobacterium sp. AGL-1 culture and 20 µl IM per transformation.
Pipette 110 µl of the mixture onto the filter and spread by tilting the plate. Ensure that the suspension does not run off the filter.
Incubate at:
22°C
upside down
in darkness
3 days
Selection
Selection
Add ca. 5 ml selection medium per plate (freshly made or made a few days before).
Selection medium:
VMM + 300 µM Cefotaxim (150 µl in 50 ml) +10 µg/ml Hygromycin B (10 µl in 100 ml)
Incubate at:
28°C
1 - 2 weeks
darkness
Harvest of Transformants
Harvest of Transformants
Pick fungal colonies that grow through the selection medium and transfer them onto new selection plates (VMM + 10 µg/ml Hygromycin B (10 µl in 100 ml)). Verify successful transformation by PCR.
Protocol references
Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc 2008; 3(10):1671–8.
Vogel HJ. A convenient growth medium for Neurospora crassa (Medium N). Microbial genetics bulletin 1956; (13):42–3.