Background

Sclerotinia sclerotiorum (Helotiales: Sclerotiniaceae), a phytopathogenic pathogen, infects more than 400 plants, including vegetables and ornamental plants (Kabbage et al., 2015). As the complete genome assembly of S. sclerotiorum is available, many genes encoding effector proteins have been preliminarily validated (Derbyshire et al., 2017). Preparation and transformation of protoplast are important measures to obtain mutants and research gene function. We introduce a protocol based on a standard protocol with minor modifications (Rollins, 2003). This method redounds to production of complete protoplasts for observation and transformation. Using this method, we successfully acquired SsVPS51, SsDCTN1, and SsSAC1 knockout mutants, respectively (Qiao et al., 2021). Therefore, polyethylene glycol–mediated transformation is suitable for protoplast transformation of S. sclerotiorum, providing a convenient method for studying its genetics.

Materials and Reagents

1. 1.5 mL microfuge tube (Axygen)

2. 2.0 mL microfuge tube (Axygen)

3. Petri dish (9 cm diameter)

4. 40 µm cell strainer (Xiyan Co., Ltd., catalog number: 15-1040)

5. Syringe filter (Pall Corporation, catalog number: 4612-0.2 µm)

6. Sterile blade (ShangHaiLianHui Medical Instrument co., Ltd.)

7. 50 mL centrifuge tube

8. 50 mL conical flask

9. 100 mL conical flask

10. 250 mL conical flask

11. KCl (BIODEE, catalog number: DE-0395A-250 g)

12. CaCl₂ (BIODEE, catalog number: DE-0556A-500 g)

13. Agar A (BBI Life Science, catalog number: A600010-500 g)

14. Potato dextrose broth (Solarbio Life Sciences, catalog number: P9240)

15. D-sorbitol (Sigma-Aldrich, catalog number: S3889-500 g)

16. Tris-HCl (Sigma-Aldrich, catalog number: T6666-500 g)

17. Sucrose (BBI Life Science, catalog number: A100335-250 g)

18. Yeast extract (BBI Life Science, catalog number: A515245-500 g)

19. Hygromycin B (Thermo Fisher Scientific, Gibco^TM , catalog number: 10687010-20 mL)

20. Glucanex (Sigma-Aldrich, catalog number: L1412-5 g)

21. Polyethylene glycol 3500 (Sigma-Aldrich, catalog number: 25322-68-3-250 g)

22. Spermidine (Sigma-Aldrich, catalog number: 05292-1 mL)

23. Heparin (Sigma-Aldrich, catalog number: H3149-500 KU)

24. Casamino acid (Sangon Biotech, catalog number: A603060-100 g)

25. KCl buffer (pH = 6–6.5) (see Recipes)

26. 0.5% Glucanex (see Recipes)

27. Phanta Super-Fidelity DNA Polymerase (Vazyme, catalog number: P501-d2)

28. STC solution (see Recipes)

29. SPTC solution (see Recipes)

30. PDA medium (see Recipes)

31. PDB medium (see Recipes)

32. RM medium (see Recipes)

33. RMA medium (see Recipes)

Equipment

1. 1,000 µL micropipette (Eppendorf)

2. 200 µL micropipette (Eppendorf)

3. 10 µL micropipette (Eppendorf)

4. Centrifuge (Eppendorf, model: 5424R)

5. Centrifuge (Eppendorf, model: 5810R)

6. Hemocytometer (Devan Scientific Co., Ltd., catalog number: AP-0650010)

7. Bacterial shaker incubator (Crystal Technology & Industries, model: IS-AX-190L)

8. Biochemical incubator (Shanghai Yuejin Medical Device Co., Ltd, model: HPX-B400)

9. Zeiss Axio Observer (Zeiss)

Procedure