Agrobacterium-mediated transformation of Diplodia sapinea

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.

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)

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 OD_600nm of 0.5 to 0.9:

200 rpm

28°C

~ 22 h

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 OD_600nm 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

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.

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

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

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.