TITLE: Agrobacterium-mediated transformation of Diplodia sapinea
AUTHORS: 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

[DESCRIPTION]
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.

[STEPS]
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
1. Thaw electrocompetent cells on ice.
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
2. Add 1 - 1.5 µl of plasmid DNA to 50 µl of cells.
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
3. Incubate on ice for 2 min.
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
4. 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.
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
5. Electroporate the mixture with the following settings: 

Voltage: 2500 V
Capacitance: 25 µF
Resistance: 400 Ω
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
6. Add 1 ml of LB medium to the cuvette immediately after pulsing and gently pipette up and down to resuspend the cells.
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
7. Transfer the cell suspension to a reagent tube and incubate the culture:

28 – 30°C
250 rpm
3 h
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
8. 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)
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
9. Incubate at 28 – 30°C. Transformed colonies are visible after 24-72 h.
SECTION: Transformation of electrocompetent Agrobacterium sp. AGL-1cells with  plasmid DNA by electroporation
10. Prepare a glycerin stock from several colonies and check via colony-PCR or plasmid preparation and PCR if your strains contain the expected fragment.
SECTION: Plate Agrobacterium sp. AGL-1
11. 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)
SECTION: Pre-culture of Agrobacterium sp. AGL-1
12. 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)
SECTION: Pre-culture of Agrobacterium sp. AGL-1
13. Incubate until the cultures reached an OD600nm of 0.5 to 0.9:

200 rpm
28°C
~ 22 h
SECTION: Main-culture Agrobacterium sp. AGL-1
14.       
Transfer 12-15 ml of the Agrobacterium sp. AGL-1 suspension to a 50 ml centrifuge tube and centrifuge at: 

3500 rpm
10 min
SECTION: Main-culture Agrobacterium sp. AGL-1
15. 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
SECTION: Main-culture Agrobacterium sp. AGL-1
16. Resuspend the pellet in liquid IM to an OD600nm of about 0.3.
SECTION: Main-culture Agrobacterium sp. AGL-1
17. 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
SECTION: Preparation of fungal spore suspension
18. Harvest spores of D. sapinea by rinsing the plate with 0.01 % Tween (Incubated on VMM, 21 d, constant light, 5000 – 7000 Lux)
SECTION: Preparation of fungal spore suspension
19. Centrifuge spores for 10 s at 5000 rpm, discard supernatant.
SECTION: Preparation of fungal spore suspension
20. Wash spores twice with 1 - 2 ml liquid IM and centrifuge at:

5000 rpm
10 s 
SECTION: Preparation of fungal spore suspension
21. Resuspend cells in IM to a concentration of 2 · 106 spores/ml. 50 µl suspension is needed per transformation.
SECTION: Selection
26. 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)
SECTION: Selection
27. Incubate at:

28°C
1 - 2 weeks
darkness

SECTION: Harvest of Transformants
28. 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.

SECTION: Co-Cultivation
23. Mix 50 µl of the spore suspension and 50 µl of the Agrobacterium sp. AGL-1 culture and 20 µl IM per transformation.
SECTION: Co-Cultivation
22. 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.
SECTION: Co-Cultivation
24. 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.
SECTION: Co-Cultivation
25. Incubate at:

22°C
upside down
in darkness
3 days