A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense

Background Soybean production around the globe faces significant annual yield losses due to pests and diseases. One of the most significant causes of soybean yield loss annually in the U.S. is sudden death syndrome (SDS), caused by soil-borne fungi in the Fusarium solani species complex. Two of these species, F. virguliforme and F. brasiliense, have been discovered in the U.S. The genetic mechanisms that these pathogens employ to induce root rot and SDS are largely unknown. Previous methods describing F. virguliforme protoplast generation and transformation have been used to study gene function, but these methods lack important details and controls. In addition, no reports of protoplast generation and genetic transformation have been made for F. brasiliense. Results We developed a new protocol for developing fungal protoplasts in these Fusarium species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of F. virguliforme and F. brasiliense are sensitive to the antibiotics hygromycin and nourseothricin, but strains transformed with resistance genes displayed resistance to these antibiotics. In addition, integration of fluorescent protein reporter genes demonstrates that the foreign DNA is expressed and results in a functional protein, providing fluorescence to both pathogens. Conclusions This protocol provides significant details for reproducibly producing protoplasts and transforming F. virguliforme and F. brasiliense. The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of F. virguliforme and F. brasiliense. Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species. Electronic supplementary material The online version of this article (10.1186/s40694-019-0070-0) contains supplementary material, which is available to authorized users.


2.
Flood the plate with 5 mL of sterile water (or 0.01% Triton X-100) and gently rub with a sterile spreader.
3. Re-collect as much of the solution as possible (typically ~ 4 mL), which now contains conidia. Use 100 µL to inoculate X number of 50 mL flasks of PDB.

5.
Filter PDB cultures through sterile Miracloth and a Buchner funnel into a waste container.
6. Use a sterile spatula to scrape the mycelia from the Miracloth back into the 250 mL flask from which it came from (or a new sterile flask).

7.
Add 30 mL of Protoplasting solution as quickly as possible to the mycelia, and incubate at 30°C for 3-5 hours, shaking at 75 rpm. Check them every 30 minutes to 1 hour. Incubate for up to 5 hours maximum.

8.
Gravity filter the digested protoplasts through sterile 30 µm Nylon mesh filter and a Buchner funnel into a 50 mL conical tube.

9.
Centrifuge tube at 3000 x g and 4°C for 5 minutes.

10.
Carefully pour off supernatant. Gently resuspend protoplasts in 10 mL chilled STC buffer using wide orifice pipet tips. (Fully resuspending the protoplasts can be difficult, but still be gentle!)

12.
Repeat steps 9 and 10 again, but gently resuspend in 1 mL chilled STC buffer using a wide orifice pipet tip.

13.
Quantify protoplasts using a hemocytometer. Make a 100X dilution (10 µL protoplasts in 990 µL STC). Take 10 µL of the dilution, inject it into a hemocytometer, and count cells under a microscope.

14.
Dilute the 1 mL protoplast suspension with STC buffer to obtain 10 7 protoplasts per mL.

Use protoplasts immediately for transformation.
Alternatively, aliquot 400 µL into as many tubes as possible, and add 30.1 µL DMSO to each (final concentration of 7% DMSO) and store directly in a -80°C freezer. At this point, you should have protoplasts (frozen or fresh) and a transformation construct. It is also recommended to make the Regeneration Media at this point and place it in a 45°C water bath to begin cooling, but not solidifying. 2. Obtain protoplasts. If using frozen protoplasts, continue with step 3. If using fresh protoplasts, skip to step 6. 3. Obtain protoplasts from -80°C freezer and thaw on ice. Once thawed, centrifuge at 3000 x g for 4 minutes at 4°C. 4. Pipet off all supernatant and gently resuspend the pellet in 200 µL chilled STC using a wide orifice pipet tip. 5. Repeat the centrifugation and resuspension 2 more times. 6. Create a "transformation reaction" in this order: -200µL of protoplasts in fresh STC buffer -5 µg of linearized plasmid -should use less than 10 µL for this -50 µL 30% PEG solution -about 250 µL total reaction volume (if >10 µL is needed when adding DNA construct, increase PEG volume so that PEG is 20% total reaction volume -Invert gently 2-3 times 7. Incubate the transformation reaction on ice for 1 hour. While waiting, add 2 mL of 30% PEG to a 15 mL conical tube, and 4 mL STC buffer to a different 15 mL conical tube. Keep both on ice. 8. Gently transfer the "transformation reaction" to the 2 mL of 30% PEG using a wide orifice pipet tip and incubate for 15 minutes on ice without mixing or inverting. 9. Gently pour the 4 mL of STC buffer into the transformation reaction and gently mix by inversion 2-3 times. 10. Immediately pour the protoplasts into 250 mL of regeneration media cooled to between 40-45°C.