Protoplast Transformation of Rice Blast Fungus

1. Generate liquid (Complete Media) mycelial culture
  -Cut the growing edge of the M. oryzae mycelium from 3 CM plates. (5 days old colonies)
  -Blend the mycelium in 250mL liquid CM. Incubate at 25°C, with shaking (200 rpm) for 48 h. Note 1
2. Transform mycelia into protoplast
  -Harvest mycelia by filtration through sterile Miracloth, rinse with sterile water and pat dry with paper towel.
  -Split and transfer the mycelia into two 50mL conical tubes.
  -Add 40 mL OM buffer and incubate for 3 hr at 29°C with gentle shaking (75rpm)
  -Transfer the OM-mycelia solution to sterile clear Oakridge tubes ~20ml for each tube.
  -Gently overlay the OM-mycelia solution with equal volume of cold ST buffer. Note 2
  -Spin at 5000 rpm, 4°C, 15 mins in a swinging bucket rotor without breaks engaged.
  -Recover the protoplasts from the OM/ST interface. Transfer into new sterile Oakridge tubes. Keep the protoplast on ice the whole time.
  -Wash the protoplast with cold STC buffer and spin at 3000 rpm, 4°C, 10 mins in a swinging bucket rotor with break half engaged. Repeat this step three times. Completely resuspend the pellet each time and fill the Oakridge tube to its neck with cold STC buffer.
  -In the last wash, combine the protoplast from all tubes and resuspend them 1 mL STC, depending to the size of the pellet and the number of transformations. Check protoplast concentration by counting microscopically. Yield should be 2-5 X 10⁸
  -Keep the protoplasts on ice until the transformation.
3. Fungal transformations
  -In a 1.5 mL Eppendorf, combine 100 μL protoplasts and DNA (2-5 μg resuspended into 50 μL STC). Incubate at room temperature (RT) for 20 mins.
  -Add 1 mL of PTC. Mix gently by inversion and incubate at RT for 15 mins.
  -After incubation, add the protoplast to 100 mL molten (45-50°C) bottom medium, mix and pour into four 100mm plates. Incubate for at least 16 hrs at RT and overlay with approx. equal volume of top media containing the selection marker.
-Incubate at 25°C until colonies appear. Note 3

Notes:
1. Antibiotic solution can be added to the culture to avoid any bacterial contamination.
2. If overlayed too quickly, the OM/ST will mix, and harvesting will be poor.
3. Select transformants and plate onto CM selection plates.

Sterile Ware
Plastic Funnels, blenders, polycarbonate or polysulfonate Oakridge tubes, Miracloth, pipette tips, Eppendorf tubes

Transformation supplies and reagents:
Penicillin/Streptomycin/Neomycin Mix (Fisher, BP296150) (Antibiotic solution)
Miracloth 22-25UM (Fisher, NC9147303)
Lysing Enzymes from Trichoderma Harzianum (Sigma, L1412)
PEG 4000 Resin Cargowax (Fisher, NC9756220)

Buffers
Osmotic (OM) Buffer (100 mL)
MgSO4·7H2O 29.0 g
1M NaPO4 (pH 5.8) 1 mL
Lysing Enzymes from Trichoderma (Sigma) 0.75 g
pH to 5.5 with 1M Na2HPO4
Bring up volume to 100 mL and filter sterilize into sterile 50 mL centrifuge tubes using a 0.45μm syringe filter.

ST Buffer (500 mL)
Sorbitol 54.66 g
1M Tris-HCl pH 7.0 50 mL
Sterilize by autoclaving and then store in the cold room.

STC Buffer (500 mL)
Sorbitol 109.32 g
1M Tris-HCl pH 7.5 5.0 mL
1M CaCl2 5.0 mL
Sterilize by autoclaving and then store in the cold room.

PTC Buffer (10 mL)
PEG 4000 6.0 g
1M Tris-HCl pH 7.5 100 μL
1M CaCl2 100 μL
In a glass beaker add the PEG to 4 mL sterile ddiH2O, microwave on high 10 seconds, and then add the Tris and CaCl2. Swirl to mix and filter sterilize into a sterile 50 mL centrifuge tube using a 0.45μm syringe filter.

Transformation media based on selection
BDCM (Sulphonyl urea)          Bottom   Top
Yeast Base w/o Amino Acids   1.7 g       1.7 g
Ammonium Nitrate                 2.0 g       2.0 g
Asparagine                              1.0 g       1.0 g
Dextrose                                  10.0 g     10.0 g
Sucrose                                    273.83 g  -----
pH each to 6.0 with Na2HPO4 Bring each up to 1 L
Divide each into 100mL / bottle. Add the corresponding amount of agar to each bottle
CM (Hygromycin)                    Bottom   Top
20X(+)NO3 Salts                      50.0 mL  50.0 mL
Trace Elements                         1.0 mL     1.0 mL
Vitamin Solution                      1.0 mL     1.0 mL
Peptone                                    2.0 g        2.0 g
Yeast Extract                             1.0 g        1.0 g
Cassamino Acids                      1.0 g        1.0 g
Dextrose                                   10.0 g      10.0 g
Sucrose                                     273.83 g  -----
pH each to 6.5 with NaOH Bring each up to 1 L
Divide each into 100mL / bottle. Add the corresponding amount of agar to each bottle
Bottom bottles (Agar 1.5 g)  Top bottles (Agar 1.0 g)