植物病原体の遺伝子操作

The overall goal of this protocol is to genetically modify the smut fungus, ustilago maydis. This strain generation method allows functional characterization of genes in a variety of ways. For example, we can delete the genes, we can exchange the promotus, or we can generate fusion constructs.

The main advantage of this method is that we can precisely verify the modification, so that any phenotype can be directly linked to the change in the gene of interest. Visual demonstration of this method is essential, because knowing when to stop the protoplasting reaction is critical for successful transformation. In preparation, cool down two milliliter round bottom tubes at minus 20 degrees Celsius.

Next, inoculate a three milliliter preculture in YEPS-Light using a fresh plate. Incubate it for 24 hours on a rotating wheel at 28 degrees Celsius. The next day, add 25 microliters of the preculture to 50 milliliters of YEPS-Light in a baffled flask.

Then, grow up the culture on an orbital shaker until it reaches the exponential phase. An optical density of 1.0 corresponds to one to two times 10 to the seventh cells in ustilago maydis. Check the optical density at 600 nanometers.

A value of 0.6 to 1.0 is acceptable. Next, check the cells for contamination under 40x magnification. Bacterial contaminations can be identified as small and often moving cells.

Fungal contaminations can be identified based on their shape or size. Always discard contaminated cultures and only work from clean ones. After pelleting the cells for five minutes at 1, 500 G, discard the supernantant.

Then, re-suspend the pellet in 25 milliliters of SCS. Repeat the centrifugation. And meanwhile, prepare three milliliters of protoplasting solution per cell pellet.

Pass this solution through a 22-micron filter. Now discard the supernatant and re-suspend the pellet in two milliliters of protoplasting solution. Then let the cells rest at room temperature until 30 to 40%of them are round or pinhead shaped.

The reaction can take five to 20 minutes. Start observing it under the microscope after five minutes. To obtain the maximum transformation efficiency, the protoplastic reaction has to be stopped at the right moment.

And then, the protoplasts have to be handled very carefully as if they were soap bubbles. Proceed with the protocol, always keeping the protoplasts on ice. The next step is to wash them with 10 milliliters of cold SCS, and then collect them with a five minute spin at 1, 000 G.Repeat this wash step a total of three times.

After the washes, re-suspend the pellet in 10 milliliters of cold STC. Then collect the cells again with another five minute spin at 1, 000 G, followed by re-suspending them in 1 milliliter of cold STC. Now make 100 microliter aliquots in the cold tubes and store these aliquots at minus 80 degrees Celsius.

To transform ustilago maydis protoplasts, prepare two selections per transformation. First, melt RegLight agar and cool it to 60 degrees Celsius. Then, to some of the melted agar, add the selective antibiotic at double the working concentration, and add 12 milliliters to each plate to form the bottom layer.

The sample should be mixed carefully. Avoid making bubbles. Keep the remaining RegLight without antibiotics at 60 degrees Celsius.

Next, thaw one tube of protoplasts on ice for each transformation. Once thawed, add one microliter of heparin solution and one microgram of linearized construct DNA. Then keep them on ice for 10 minutes.

While waiting, spread 12 milliliters of RegLight without antibiotics over the RegLight with antibiotics. Next, add 500 microliters of STC/PEG to each transformation tube and carefully mix them with inversions. Then continue the incubation on ice for 15 more minutes.

Now distribute each transformation reaction onto two separate selective RegLight plates. Make sure all the liquid has soaked into the plates before proceeding. Then, incubate the plates upright at 28 degrees Celsius for five to seven days to get 100 to 200 colonies.

If only a few colonies grow, the protoplasts might contain too much cell wall, and this can be tested by transforming them with a cell-fabricating plasma or they might not be able to regenerate and this can be tested by planting them on plates without antibiotics. Later, select 24 transformants for testing. Re-purify them on selective YEPS-Light to obtain single colonies.

On each plate, also streak out some of the original strain to make sure the antibiotics are selecting against non-transformants. It is critical to verify the mutations using a three step process that involves two PCRs and a Southern Blot analysis. These are detailed in the text protocol.

This section explains how to prepare the genomic DNA needed for these analyses. First, inoculate five milliliters of YEPS-Light with the candidate transformant, and incubate it on a rotating wheel for 24 hours at 28 degrees Celsius. The next day, drop two microliters of the culture on a CM plate.

Keep the culture sterile. Then prepare a two milliliter tube with about 200 microliters of hydrochloric acid-washed glass beads and two milliliters of the 24-hour culture. After spinning the tube at 12, 000 G for five minutes, aspirate and discard the supernatant.

To the pellet, which may be frozen for storage, add 500 microliters of usti-lysis buffer two. Then shake the tube at 1, 000 RPM on a Vibrax for five to 15 minutes. Make sure that the cells are completely re-suspended, then incubate the tube at 65 degrees Celsius for 15 to 20 minutes.

Next, place the tube on ice for five minutes. Once cooled down, add 100 microliters of 8 molar potassium acetate, and apply a brief vortex, or ten inversions. Next, spin the tube at 12, 000 G for 15 minutes at room temperature.

Then, aliquot 500 microliters of the supernatant to a 1.5 milliliter tube, and add 400 microliters of isopropanol to the aliquot. After mixing, spin down the tube for five minutes at 12, 000 G.Discard the supernatant and wash the pelleted DNA with 500 microliters of 70%ethanol. Now apply a five minute spin and completely remove the supernatant.

After another brief spin, let the DNA pellet air dry for a few minutes, and then add 50 microliters of RNase NTE. After adding the RNase, incubate with shaking, and then proceed to use the DNA for the analyses. Single and double mutants of the chintinases cts3 delta and multiple mutants with up to the three other chitinase genes were generated sequentially with the same constructs in two different genetic backgrounds.

Transformants were confirmed by PCR and then by Southern Blot analysis. For the cts3 gene, the homologous recombination rate was 70%in the SG200 strain, suggesting cts3 has a non-essential function. Some of the mutations lead to obvious growth defects.

For instance, the cts1/2 double mutant had a pronounced cell separation defect. Staining the septa showed that cytokinesis would complete, but the cells would remain connected. To test the contribution of chitinases to virulence, seedling infection assays were carried out using the SG200 strain mutants.

While mutation of a controlled gene, khd4, reduced virulence, deletion of all the chitinases did not affect the infection of maize seedlings. After watching this video, you should have a good understanding of how to genetically modify smut fungi like ustilago maydis. Once mastered, strains can easily be generated in about four weeks.

When using this procedure, it is really important to plan the strain verification detail by using future cloning programs. Do this before you start to work with fungal cultures. With slight modifications to this procedure, other smut fungi like sporsorium reilianum or ustilago esculenta or even filamentous fungi, like piriformospora indica, can be genetically modified.

Don't forget that working with genetically modified organisms need to be carried out according to regulations in your country.