TITLE: A small scale of Fusarium oxysporum protoplast generation
AUTHORS: Jun-Ze Zheng, Tao-Ho Chang

[DESCRIPTION]
The generation of fungal protoplasts is crucial for advancing fungal gene editing methods. We aim to implement the ribonucleoprotein (RNP) method for gene editing (Wang et al., 2018). Generating protoplasts is a demanding task that requires substantial resources to obtain an adequate amount for a single transformation reaction. The cost of cell wall degrading enzymes is the most significant expense in the protoplast generation process. In this study, we have developed a process for small-scale protoplast generation that not only reduces resource usage but also significantly cuts down the amount of cell wall degrading enzyme required.

[BEFORE_START]
The protocol for generating protoplasts on a small scale is for Fusarium oxysporum and is in development. We are open to other fungal species attempting the task and providing a report on their success or any modifications to the methods.

[STEPS]
SECTION: Fungus single spore isolation
1. Rinse Fusarium oxysporum fungal spores from the two-week-old culture plate with 7 mL distilled water, and filter the spore suspension with sterilized filter paper or 4 layers of Miracloth. 

SECTION: Mycelium collection
5. The 700 µL of germinated spores were added into the filter column.

SECTION: Enzymes digestion
9. Add the digestion buffer (10 mg/mL Driselase and 15 mg/mL ß-glocanase dissolved in 0.8 M NaCl solution ) 700 µL 

SECTION: Protoplast isolation
12. 5000 x g, 4 °C 
Centrifuge the mixture, pelleting the protoplast to the centrifuge tubes.
SECTION: Observation and following applications
18. The protoplasts are able to apply in CRISPR/Cas gene editing system or subject to cell staining for microscopy.
SECTION: Fungus single spore isolation
2. Count the number of spores in spore suspension with hemocytometer.


SECTION: Fungus single spore isolation
3. The 106 / mL spore suspenstion  were added into the culture medium 50 mL half-strength PDB, and grow the culture in 28 ºC for 20-24 hours.
SECTION: Mycelium collection
6. 1000 x g, 28 °C 
Centrifuge to remove the ungerminated spores
SECTION: Enzymes digestion
10. Mix   180 min in room temperature

SECTION: Protoplast isolation
13. Remaining the pellet of protoplast, remove the digestion buffer carefully.
SECTION: Protoplast isolation
14. Add 1000 µL of SuTC stabilizer buffer (20% sucrose, 10 mM CaCl2 ,10 mM Tris-HCl, pH 7.5) for washing pellet.

SECTION: Mycelium collection
7. Wash the mycelium with 700 µL of distilled water, and continue pipetting until the mycelium is suspended in the distilled water.
SECTION: Mycelium collection
8. Centrifuge again to remove water and change new collection tubes.
SECTION: Protoplast isolation
11. Change collection tube to 2 mL tube for collecting protoplast.
SECTION: Mycelium collection
4. Assemble sterilized collection tube and tissue filter column (Lot.: CDC25049A, FAVORGEN®Biotech Corp., Pingtung, Taiwan).
SECTION: Protoplast isolation
15. centrifuge at 1000 xg for 5 min, and repeat wash process twice.
SECTION: Protoplast isolation
16. dissolve pellet carefully in 500 µL SuTC buffer, and count protoplast concentration with hemocytometer. 
SECTION: Protoplast isolation
17. Generally, the concentration of protoplast per column will be fall around 105 to 106 / mL.