Our protocol is significant because it allows researchers working on non-model fungi the opportunity to establish cutting-edge genome editing technologies in their labs. As it does not rely on existing techniques, such as expression systems, this protocol has the advantage of being easier to establish in non-model systems. This method can be used across different fungus species and can be used to elucidate functions of genes involved in pathway mating, growth, and pathogenicity.
So when performing this procedure, one must be sure to have enough consecutive days in order to compete the protocol as there are only a few points in the experiment at which it can be paused. To harvest the conidia, filter the liquid culture through a layer of sterile laboratory cloth into 50 milliliter centrifuge tubes for centrifugation. Resuspend the conidia pellet in five milliliters of water and view a 10 microliter aliquot of the conidia solution under a light microscope at a 40X magnification to confirm that only conidia have been recovered.
Next, add 200 milliliters of fresh 1%malt extract broth to a 500 milliliter flask and transfer the entire volume of conidia to the flask. Then incubate the liquid culture for up to 12 hours in a 25 degree Celsius shaking incubator at 120 revolutions per minute. To harvest the germlings, transfer the culture to 50 milliliter centrifuge tubes for centrifugation and resuspend the germlings in up to 10 milliliters of one molar sorbitol.
Add one milliliter of germling solution to various concentrations of enzyme solution and incubate the spore enzyme solution for two to three hours in the shaking incubator at 80 revolutions per minute. To harvest the protoplasts, filter the enzyme solution through a layer of sterile laboratory cloth and collect the protoplasts by centrifugation. Then carefully resuspend the protoplast pellet in 200 microliters of STC buffer and check a 10 microliter aliquot of the solution under a microscope to confirm that only protoplasts have been recovered.
To begin the transformation, combine approximately five times 10 to the six protoplasts with a single volume of ribonucleoprotein solution and approximately six micrograms of the donor DNA fragment. Next, use a pipette to slowly and evenly drip one milliliter of freshly prepared 30%PTC solution onto the protoplast suspension to create a hydrophobic layer over the protoplasts and incubate the solution for 20 minutes at room temperature. At the end of the incubation, add five milliliters of osmotic control medium to the protoplast suspension, pipetting slowly and gently to thoroughly mix the solution.
After mixing, incubate the protoplast solution in the shaking incubator at 80 revolutions per minute overnight. The next morning, divide the solution between five 60 millimeter culture plates. Add 10 milliliters of osmotic control medium agar supplemented with 30 micrograms per milliliter of Hygromycin B to each plate and slowly rotate each plate to mix.
Allow the first layer of agar to set before adding 10 milliliters of osmotic control medium agar supplemented with 40 micrograms per milliliter of Hygromycin B two each plate. After allowing the second layer of agar to set, incubate the cultures at 25 degrees Celsius until single isolates can be observed growing through both layers of agar. To recover the successfully transformed isolates, transfer the individual growth-capable isolates to fresh malt extract agar plates supplemented with 50 micrograms per milliliter of Hygromycin B.To assess the effects of the targeted gene disruption on the heterothallic capabilities of the fungus, co-inoculate fresh malt extract agar medium with one mutant strain as well as a strain of the opposite mating type.
When working with H.omanensis, cover but do not seal the plates. Place the plates at room temperature for seven days. At the end of the incubation, visually assess for the production of sexual structures.
To test the homothallic capabilities of the mutant strain, inoculate fresh malt extract agar medium with the mutant strain of interest and incubate the plate at room temperature for one week as demonstrated. To assess the effects of the disruption on the growth rate of the fungus being studied, insert the backside of a large sterile pipette tip into the actively growing edges of the culture of each mutant and wild-type strain of interest to create mycelial-covered agar plugs and inoculate fresh malt extract agar medium. At least three replicates should be done per culture type.
After three days of growth at 20 degrees Celsius, measure the growth in each plate on two perpendicular diameters. Conidia used as the starting material for the protocol are allowed to germinate and grow until they become young germlings. Note that mature mycelial strands such as these are too mature for degradation and should not be used.
When the cells no longer have cell walls, they become very sensitive to mechanical disruption and release round protoplasts that can be harvested for transformation. The success of the protocol can be confirmed upon a phenotypic analysis of the mutant strains. For this mutant MAT 127 isolate, the vegetative radial growth rate was significantly reduced, suggesting a pleiotropic effect for the novel mating gene.
Furthermore, the mutant isolate was incapable of completing a sexual cycle producing only immature sexual structures that did not produce sexual spores compared to the wild-type isolate which completed the entire sexual cycle within a few days of incubation. RNA is very sensitive and degrades very easily. Therefore, a clean work environment and working quickly on ice are both essential to the success of this experiment.
Once mutant isolate have been successfully generated, they can be subjected to phenotypic or RNA-seq analysis as appropriate for the gene being characterized.
