Mar 11, 2026
Protoplast and PEG-mediated transformation of Zymoseptoria tritici
- Michael Choi1,
- Megan C McDonald1
- 1University of Birmingham, School of Biosciences, Institute of Microbiology and Infection
- Zymoseptoria community protocols (STBnet)
Protocol Citation: Michael Choi, Megan C McDonald 2026. Protoplast and PEG-mediated transformation of Zymoseptoria tritici. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrbenblmk/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: March 10, 2026
Last Modified: March 11, 2026
Protocol Integer ID: 305182
Keywords: Zymoseptoria, protoplast, extralyse, PEG-mediated transformation, fungal transformation, fungal wheat pathogen zymoseptoria tritici, transformation of zymoseptoria tritici, generating competent protoplast, zymoseptoria tritici, linear dna, affordable commercial enzyme, mediated transformation, genome
Abstract
This is a protocol for generating competent protoplasts from the fungal wheat pathogen Zymoseptoria tritici using an affordable commercial enzymes, Extralyse/Vinotaste. This protocol uses PEG-mediated transformation to ectopically integrate linear DNA (PCR-products) into the genome.
Materials
- 100 ml YSB (250 ml flask)
- Centrifuge
- Extralyse powder
- Enzyme solution
- 50 ml sterile tubes
- 1 M sorbitol-tris solution
- 1 ml STC buffer
- 1.5 ml Eppendorf tube
- P1000 pipet
- 60% PEG solution
- CzV8 plates
- Hygromycin (Roche) 50 mg/ml
- V8 Juice
- Yeast extract
- Sugar
- Czapek-Dox broth
- Agar
- NaOH
**Additional Materials from Current Pages:**
- 15 ml falcons
- Centrifuged V8 Juice
- Yeast Sucrose Broth
- Czapek V8-agar
- Czapek V8-top agar
- Stock solutions (MgSO₄, Sorbitol, Tris, CaCl₂)
- Wash solution
- Protoplast overlay solution
- STC buffer
- 60% PEG solution
- Czapek V8-top agar**
- 2.27 g Czapek-Dox liquid
- 0.375 g agar
- 9.1 g sorbitol
- 10 ml centrifuged V8 juice
- Adjust to pH 6.0 with NaOH
- Stock solutions**
- 50 ml 1.2 M MgSO₄.7H₂O
- 20 ml 1.2M Sorbitol
- 100ml 1 M Tris pH 7.5
- 1 ml 1M CaCl₂
- Wash solution**
- 600 mM MgSO₄
- Enzyme solution**
- 1.2 M MgSO₄, 10 mM phosphate buffer pH 5.8
- Protoplast overlay solution**
- 600 mM Sorbitol, 10 Tris pH 7.5
- 1 M Sorbitol-tris solution**
- 1 M Sorbitol, 10 Tris pH 7.5
- STC buffer**
- 1.2 M Sorbitol, 10 mM CaCl₂, 10 Tris pH 7.5
- 60% PEG solution**
- 60% PEG-4000, 10 mM CaCl₂, 10 Tris pH 7.5
Troubleshooting
Fungal Growth Media pre-prepared
Yeast Sucrose Broth - Liquid media for flask culture of Z. tritici
Prepare 100 ml per 250 ml flask for each overnight culture
for 100 ml:
1g Yeast extract
1g Sucrose
Autoclave
Centrifuged V8 Juice
Centrifuge V8 juice for 15 min at 10,000 rpm
prepare in advance and store at −20ºC
Czapekz V8-agar
for 500 ml
22.7 g Czapek-Dox broth + 5g agar or Czapek-dox agar (follow manufactures instructions)
91.1 g sorbitol
100 ml centrifuged V8 juice
Add 350 mL of dd
adjust to pH 6.0 with NaOH
Top up with ddH₂O to 500 ml
Wash and protoplast digestion solutions (prepared in advance)
Concentrated Stock Solutions
1.2 M MgSO₄.7H₂O (246.47 g/mol)
Need approximately 50 mL per protoplast prep.
For 50 mL: Add 14.8 g of MgSO₄.7H₂O to 30 mL of ddH2O and dissolve. Bring to final volume of 50 mL.
For 100 mL: Add 29,6 g of MgSO₄.7H₂O to 70 mL of ddH2O and dissolve. Bring to final volume of 100 mL.
Autoclave or filter sterilise for long-term storage.
1.2M Sorbitol (182.17 g/mol)
Need approximately 20 mL per protoplast prep.
For 20 mL: Add 4.37 g of sorbitol to 12 mL of ddH2O and dissolve. Bring to final volume of 20 mL.
For 50 mL: Add 10.9 g of sorbitol to 35 mL of ddH2O and dissolve. Bring to final volume of 50 mL.
Filter sterilise for long-term storage.
1 M Tris pH 7.5 (121.14 g/mol)
Need ~1 mL per protoplast prep.
For 10 mL: Add 1.2 g of Tris to 9 mL of ddH2O and dissolve. Adjust pH to 7.5 and bring to final volume of 10 mL.
Autoclave or filter sterilise for long-term storage.
1 ml 1M CaCl₂ * 2 H2O (147.02 g/mol)
Need ~ 1 mL per protoplast prep.
For 10 mL: Add 1.47 g to 9mL of of ddH2O and dissolve. Bring to final volume of 10 mL.
Filter sterilise for long-term storage.
Day 1 – Overnight liquid culture of Z. tritici
Prepare an overnight culture of Z. tritici by harvesting 1 plate containing 5-day old yeast like growth of Z. tritici. Yeast like growth should be dense and pink and cover most of the surface of the plate. Harvest spores by adding 2-3 mL of sterile H2O and gently suspend spores in with a sterile tip or L-spreader. Inoculate 100 mL YSB (in 250 mL Erlenmeyer flask) with 2 mL of this concentrated spore suspension.
Incubate culture at 20ºC with shaking at 100 rpm.
Day 1 - Prep Wash and protoplast digestion solutions (day or week before use)
Spore Wash Solution
600 mM MgSO₄ buffer
50 mL required per protoplast prep
Add 25 mL 1.2 M MgSO₄.7H₂O to 25 mM ddH2O.
Autoclave or filter sterilise before use.
Enzyme buffer (1.2 M MgSO₄, 10 mM phosphate buffer pH 5.8)
25 mL needed per protoplast prep
Add 0.04g NaH₂PO₄ to 20 mL 1.2 M MgSO₄.7H₂O and adjust pH to 5.8 using NaOH.
Add the NaOH drop by drop. A precipitate will form after each drop but will go back into solution after a couple of minutes stirring. Wait until precipitate has dissolved before recording pH and re-adjusting if required. Once pH is correct, make up volume to 25 ml with remaining 1.2 M MgSO₄.7H₂O. (Autoclave)
On the morning of the protoplast prep, add 3% w/v of Extralyse (Laffort) (0.75g per 25ml) to the autoclaved Enzyme solution prepared above and shake well to re-suspend.
NOTE: Some protocols recommend filter sterilising this mixture, however we found no benefit to filter sterilising and saw a high level of loss of the enzyme. Always make fresh on day of use and do not store once enzyme has been added.
Protoplast overlay solution (600 mM Sorbitol, 10 Tris pH 7.5)
5 ml needed per protoplast prep
Add 2.5 ml of 1.2M Sorbitol to 2.45 mL sterile water and add 50 µl 1 M Tris. (Filter sterilise, or prep sterile ingredients in biosafety cabinet)
1 M Sorbitol-tris solution (1 M Sorbitol, 10 Tris pH 7.5)
5 ml needed per protoplast prep
Add 4.17 mL 1.2M Sorbitol to 780 µl water and 50 µL 1 M Tris. (Filter sterilise, or prep sterile ingredients in biosafety cabinet)
STC buffer (1.2 M Sorbitol, 10 mM CaCl₂, 10 Tris pH 7.5)
10 mL needed per protoplast prep
Add 9.8 mL 1.2M Sorbitol to 100 µL 1 M Tris and 100 µL 1M CaCl₂. (Filter sterilise, or prep sterile ingredients in biosafety cabinet)
60% PEG solution (60% PEG-4000, 10 mM CaCl₂, 10 Tris pH 7.5)
5 ml generally enough for 3 protoplast preps
Add 6 g PEG 4000 to 5.3 ml water, 100 µl 1 M Tris and 100 µL 1M CaCl₂. (Autoclave)
Day 2 – Spore harvesting and cell-wall digestion
Make Digestion solution
Add 3% w/v of Extralyse (Laffort) (0.75g per 25ml) to the autoclaved Enzyme buffer prepared above and shake well to re-suspend.
NOTE: Some protocols recommend filter sterilising this mixture, however we found no benefit to filter sterilising and saw a high level of loss of the enzyme. Always make fresh on day of use and do not store once enzyme has been added.
Harvest overnight Z. tritici culture by pouring ~40 mL of culture into two sterile 50 mL tubes and centrifuge to form a spore pellet (4000rpm/5 min/4ºC).
After centrifuging pour off supernatant and add 25 mL of Spore Wash solution (600 mM MgSO₄) to each falcon tube. Gently invert the tube to wash the spores, pellet can remain at the bottom of the tube. Centrifuge for at ~ 4000 rpm for 5 min at 4ºC.
After centrifuging pour off wash and re-suspend spores in each falcon tube with ~half of the 25 mL Digestion solution by pipetting slowly (12.5 mL into each of two falcon tubes) . Once spores are fully re-suspended pour everything into a sterile 100 mL Erlenmeyer flask.
Incubate flask at 30 ºC for 4h with shaking (100rpm). During this incubation, prepare the top agar (Prevent the agar from solidifying, keep warm).
Day2- Top-agar (day of transformation only)
Czapek V8-top agar (prepare for day of antibiotic over-lay only!)
For 50 mL
2.27 g Czapek-Dox liquid (follow manufacture's instructions)
0.375 g agar
9.1 g sorbitol
10 ml centrifuged V8 juice
Dissolve in ~30 mL of ddH2O
adjust to final pH 6.0 with NaOH
add ddH₂O to 50 ml
Autoclave and keep warm (~50-55C) until needed
Day 2 – Harvesting protoplasts after digestion
After 4- hrs digestion prepare to filter protoplasts from undigested spores.
Fold 2 pieces of miracloth in half (to get 4 layers) and place the miracloth in alternate orientation. Pour the solution through the miracloth into a 50 mL sterile falcon tube. Then, gently pour 5 mL of protoplast overlay solution on top of this 25 mL.
Centrifuge at 4,000 rpm for 15 min at 4 ºC.
After centrifugation a cloudy layer will appear (where the protoplast is), carefully collect the cloudy layer using a 25ml pipette. Transfer the layer to a 50 ml sterile falcon tube and add 5ml of 1 M sorbitol-tris solution, mix gently and centrifuge at 4,000 rpm for 5 min at 4ºC.
Red brackets show cloudy layer to be taken than holds protoplasts
A white pellet should form at the bottom of the falcon tube (protoplasts). Without disturbing the pellet pour off the supernatant and wash the protoplasts with 1 ml STC buffer, transfer the solution to a 1.5 mL Eppendorf and spin at 10,000rpm for 1 min. Pour off the supernatant and repeat this step again and re-suspend the protoplasts (white pellet) in 500 uL of STC.
Note total STC suspension amount depends on the number of DNA constructs needed for transformation. Each construct requires 100 uL of concentrated protoplasts in STC. This amount should be enough for four transformations of 100 uL each plus one no template control. The number of protoplasts can vary from prep to prep if you need more conentrated protoplasts you can re-suspend in a smaller volume of STC.
Red arrow pointing to protoplast pellet to be harvested
View from haemocytometer showing mixture of protoplasts (spherical cells) with some undigested spores (elongaged cells) that should be visible after all concentration steps
Protoplast transformation with linear DNA
Aliquot 100 uL of protoplasts in STC into desired number of transformation tubes plus one no DNA template control. For 500 uL you should have enough for 4 transformations plus 1 no DNA control. An additional regeneration control can be made with a small volume of leftover/residual protoplasts.
Tube summary:
4 x DNA transformations- aliquots of 100 uL each
1x No DNA control- 100 uL aliquot with no template DNA added
1 x Regeneration control (optional): If desired set aside a small aliquot of protoplasts (50 uL) to plate out without selective antibiotics to check the viability and regeneration of the cells.
Purified DNA products should be resuspended in 100-125 uL of STC buffer. Typical amounts used for transformation are 3-10 ug for linear PCR product. For larger inserts 10 ug of DNA is recommended.
Mix protoplasts with DNA very gently using P1000 pipet with 1 mL filter tip. Incubate at room temperature for 15 min.
During the DNA protoplast incubation, prepare aliquots of warm top agar by dispensing 5 mL of Top Agar into pre-warmed 15 mL tubes. Return aliquots to 50C water bath to keep from solidifying.
Add 200 µl 60% PEG solution to the protoplasts-DNA solution and mix by inversion.
Add a further 200 µl 60% PEG solution and mix by inversion.
Add a final 800 µl 60% PEG solution and mix by inversion.
Incubate at room temperature for 15-20 min.
Transfer the DNA-protoplast-PEG mixture to 5 mL pre-warmed 50C top agar, mix by inversion and pour onto CzV8 plates in the center.
Note: This step must be performed quickly to prevent the top agar from solidifying. Pour the protoplast mix in the center of the plate an allow it to gently flow towards the edges of the plate. The top-agar might not reach the outer edges of the plate, this is OK.
Controls: No DNA-control plate, regeneration control (if desired).
Incubate plates in the dark at 18ºC for 72 hours.
Day 4-5 Antibiotic overlay prep
Stock solutions
Hygromycin (Roche, 10843555001) 50 mg/mL in 20 mL solution.
Required amount for Z. tritici (total concentration for the whole plate)
Hyg 100 ug/mL
Plate agar volume 20 mL
Protoplast top agar volume 5 mL
Antibiotic overlay volume 5 mL
Total 30 mL
Prepare antibiotic overlay top agar at 6x hyg concentration required for the whole plate.
Need 5 mL of antibiotic overlay to have a final concentration of 600 ug/mL, stock hygromycin is 50 mg/mL.
So add 60 uL of stock Hygromycin (50 mg/mL) to 5 mL of warm top-agar.
Day 5 Antibiotic overlay (72 hours after transformation)
Prepare 5 mL of top agar for each plate to be overlayed with excess to account for pipetting errors.
Regeneration control plate (optional)
Transfer 5 mL of top agar without antibiotic to pre-warmed 15 mL tube or immediately transfer 5 mL onto top of control protoplast plate. This is no-antibiotic control plate to show protoplasts successfully regenerate without selection.
Transformation plates and no DNA control plate
For remaining top agar volume transfer the appropriate amount of antibiotic to achieve a 6x concentrate of the final desired concentration. For the example above this is 60 uL of hygromycin (50 mg/mL) per 5 mL of media.
Pour 5 mL of top agar with antibiotics onto each transformation plate and no DNA control plate.
Incubate plates in the dark at 18ºC until colonies appear growing at the surface or just under the surface. Pick growing colonies and transfer to YSA plates + 100 ug/mL of hygromycin (concentration depends on isolate and species, we recommend this for Z. tritici IPO323).