Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Podsumowanie

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Streszczenie

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Wprowadzenie

The smut fungi (Ustilaginales) consist of over 1,600 species that infect grasses including the important cereal crops of corn, barley, and wheat, causing billions of dollars in crop losses annually¹. These fungi are characterized by the production of teliospores, which have darkly pigmented cell walls and are the dispersal agents. Teliospores function to shield genetic material during the stresses of dispersal between host plants, and can persist in a dormant state for years². As such, teliospores are an essential component of disease spread.

In order to study teliospore biology, our laboratory utilizes the model smut fungus Ustilago maydis (U. maydis), which is the causal agent of the disease 'common smut of corn'. Mature U. maydis teliospores are characterized by growth arrest, reduced cellular metabolism, and low levels of cellular respiration³. In favorable environmental conditions (e.g., the presence of specific sugars), U. maydis teliospores germinate and complete meiosis, producing basidiospores which can initiate new rounds of infection. Germination is characterized by increased respiration, the return to metabolic activity, and the progression through observable morphological stages of germination⁴.

The initial stage of germination includes increased respiration and metabolic function, however, there are no morphological indications of change. The original measurements of respiratory change in U. maydis were carried out over 50 years ago, measuring oxygen consumption manometrically with a Warburg flask apparatus⁵. We have developed a new, simple method of studying precise changes in respiration during teliospore germination by measuring oxygen consumption over a time course of germination using a Clark-type microrespirometer. We previously used this method to study changes in respiratory rate between wild-type U. maydis haploid cells and mutants with defective mitochondria⁶, and have adapted the protocol here to study changes in teliospore respiration during germination. This provides a means of accurately identifying the timing of respiration change so that we can target teliospores at the appropriate time after the initiation of germination to investigate early molecular events. The progression of germination can be followed microscopically once the promycelia emerges from the teliospore, but the asynchronous nature inhibited the isolation of enough teliospores at a given stage for investigation. We developed a microdissection technique similar to those used for in vitro fertilization to physically collect teliospores at distinct morphological stages of germination.

Protokół

1. Corn Cob Infection

1. Grow Zea mays (cv. Golden Bantam) until cobs are formed and have started to silk (approximately 60 days).
2. Culture compatible haploid U. maydis strains using standard protocols as previously described⁷.
3. Infect corn cobs using standard protocols as previously described⁷.

2. Teliospore Harvesting

1. Autoclave equipment (Büchner funnels, Büchner flasks, blenders, 250 mL centrifuge bottles, flat spatulas, and water) using a standard dry cycle with at least 30 min sterilization at 121 °C (standard liquid cycle for water).
2. Remove infected cobs from plants (approximately 28–35 days post infection) using a razor and set the cobs on a tray covered in bench protector.
3. Remove the tumours from cobs with a razor blade and collect in a beaker.
4. Fill a 250 mL laboratory blender cup with tumours until approximately 1/3 full and add autoclaved dH₂O until the blender cup is approximately 3/4 full. Disrupt the tumours by pulsing the blender at low, until homogenized.
5. Connect the vacuum pump to a water trap and then to a 1 L Büchner flask.
6. Insert large Büchner funnel into the flask and line the bottom of the Büchner Funnel with four layers of cheesecloth.
7. Turn on the vacuum pump.
8. Pour a portion of the homogenized tumours through the cheesecloth and scrape with a spatula.
9. Pour some dH₂O into the cheesecloth in order to flush the teliospores through.
10. Repeat until the dH₂O coming through the cheesecloth is clear.
11. Wring out the cheesecloth containing the homogenized tumour material into the filter to ensure maximum teliospore recovery.
12. When the 1 L Büchner flask is getting close to full, empty it into a large Erlenmeyer flask and set it aside.
13. Put a new piece of cheesecloth into the filter and repeat the steps (2.7–2.12) until all of the tumours have been disrupted and filtered.
14. Pour the filtered teliospores into autoclaved 250 mL centrifuge bottles and centrifuge at 1,000 x g for 5 min, and decant the supernatant.
15. Repeat step 2.14 until all of the filtered teliospores are pelleted by centrifugation.
16. Suspend the pellets in a small amount of water and transfer to 50 mL centrifuge tubes.
17. Centrifuge the tubes at 1,000 x g for 5 min, and decant the supernatant.
18. Suspend the pellet in approximately 50 mL of dH₂O, centrifuge the tubes at 1,000 x g for 5 min, and decant the supernatant. Gently scrape off the gray top layer with a spatula and dispose of it. Repeat until there is no longer a gray layer on top.
19. Dry the samples overnight in a vacuum desiccator.
20. Store dried teliospores at 4 °C until use.
21. If desired, treat the teliospores with copper sulphate⁸ before inducing to germinate. If not treated, then perform thorough microscopic analysis of the teliospores to confirm the sample represents pure teliospores and is devoid of bacterial or other contaminations.
    1. Weigh out approximately 50 mg of teliospores in a 1.5 mL microcentrifuge tube.
    2. Add approximately 1.0 mL of 0.75% CuSO₄ to the 1.5 mL microcentrifuge tube containing the teliospores. Pipette up and down to suspend the teliospores in the CuSO₄ solution followed by agitating the sample for 3 h.
    3. Centrifuge the sample at 2,500 x g for 5 min and remove the supernatant. Resuspend the teliospore pellet with sterile water, repeat the centrifugation, and remove the supernatant.
    4. Repeat step 2.21.3 two more times.

3. Teliospore Viability and Germination Test

1. Weigh out approximately 10 mg of U. maydis teliospores in a 1.5 mL microcentrifuge tube to assess their viability and the timing of germination.
2. In a biosafety cabinet, prepare potato dextrose broth (PDB, 24 g/L) supplemented with streptomycin sulfate (160 µg/mL).
3. Suspend the teliospores in 500 µL of PDB. Gently pipette to mix and break up all clumps of teliospores.
4. Transfer the teliospore suspension to an autoclaved 250 mL Erlenmeyer flask containing 10 mL of the PDB.
5. Incubate the flask at 28 °C shaking at 90 rpm for 12–16 h.
6. In a biosafety cabinet, remove a 20 µL sample of the teliospores induced to germinate and prepare a microscope slide.
7. Using a microscope, visually assess stages of germination that are present and the presence of bacterial contamination.
   1. Count the number of teliospores at stages I through V using a hemocytometer and determine the percent that have germinated.
   2. If only stage I teliospores are present, continue to incubate the flask for a total of 24 h before assessing teliospore germination. Continue incubation for a maximum of 48 h before deeming the sample non-viable.
   3. If bacterial contamination is present, supplement the PDB with kanamycin sulfate (50 µg/mL) as well as streptomycin sulfate (160 µg/mL) and then repeat steps 3.1 to 3.7. If bacterial contamination persists, treat teliospores with copper sulfate and repeat steps 3.1 to 3.7.

4. Induction of Germination for Respiration Monitoring

1. Weigh equal amount (e.g., 50 mg) of teliospores for each respiration experiment.
2. In a biosafety cabinet, add teliospores to an autoclaved respiration chamber.
3. Fill the chamber with PDB (24 g/L) supplemented with streptomycin sulfate (160 µg/mL) and kanamycin sulfate (50 µg/mL).
4. Pipette up and down to create a teliospore suspension.
5. Place the chamber lid in the chamber to create air tight seal.

5. Obtaining Oxygen Consumption Rate (OCR) Measurements

1. Place the chamber in the chamber rack inside a water bath (preheated to 28 °C).
2. Place the O₂ probe inside the opening of the chamber.
3. Monitor the data points appearing in real time on the "SensorTrace Rate" program, and let the probe stabilize (~3 min after the probe is placed in the chamber).
4. Click "Measure" to measure O₂ levels continuously for 6 h with measurements recorded at 2 s intervals.
5. Stop the measurement, and repeat steps 4.1–5.4 for each sample to be analyzed.
6. Export the data to Microsoft Excel by clicking "File | Export | Save as .xls".

6. Data Analysis

1. Calculate OCR
   1. In the exported Excel file under the "Within_Rates" tab, record the "Rate" for each chamber measurement (nmol/h).
   2. For each experimental sample, subtract the "Rate" of the blank chamber from the "Rate" of the experimental sample chamber to obtain a corrected OCR value, and take the absolute value of this number.
   3. Calculate the total OCR per mg of teliospores by dividing the corrected absolute OCR value by the cellular mass used.
   4. Average replicate "OCR per mg of teliospores" values for each strain.
2. Analyze the data using appropriate statistical method (e.g., student's t-test, analysis of variance) using Microsoft Excel of other statistical software.
3. To graph raw data, calculate the percentage of oxygen remaining for each time-point you wish to graph. Divide first reading by itself and multiply by 100 (100% oxygen remaining), then divide each subsequent reading by the first reading, and multiply by 100 to obtain the percent of oxygen remaining in the chamber.

7. Induction of Teliospore Germination to Isolate Teliospores at Distinct Stages of Germination

1. Prepare PDB (24 g/L) supplemented with streptomycin sulfate (160 µg/mL) in a biosafety cabinet.
2. Place approximately 10 mg of U. maydis teliospores into a 1.5 mL microcentrifuge tube.
3. Suspend the teliospores in 500 µL of PDB. Gently pipette to mix until there are no clumps of teliospores in the medium.
4. Transfer the teliospore suspension to an autoclaved 250 mL Erlenmeyer flask containing PDB supplemented with streptomycin sulfate.
5. Incubate the flask overnight at 28 °C shaking at 90 rpm.

8. Preparation of Petri Dish and Micromanipulator

1. Prepare a Petri dish (57 cm²) by pipetting rows of droplets for microcapillary preparation, and sample collection.
   1. Pipette 5 µL (x4) dH₂O droplets across the top of the Petri dish.
   2. Pipette 2 µL (x3) of RNA stabilization solution on the Petri dish to be used for sample collection.
   3. Pipette 5 µL (x30) droplets of germinating teliospores on the Petri dish.
2. Add 15 mL of mineral oil to the petri dish. Ensure that all droplets are covered by oil before proceeding.
3. Prepare a microcapillary with a 15 µm inner diameter, 1 mm flange, 55 mm length, and a 20° tip angle by placing it in the microcapillary holder and submerging it in the mineral oil where capillary action will allow the mineral oil to enter the microcapillary. Release the pressure in the microcapillary before bringing it to the water droplet. Aspirate to prepare the microcapillary with water.

9. Isolation of Stage-specific Germinating Teliospores

1. Using the controls of the micromanipulator, move the prepared microcapillary to one of the germination droplets. Penetrate the droplet, lower the microcapillary, and bring the mouth of the microcapillary up to a germinating teliospore at the stage of germination of interest.
2. Slowly aspirate to capture the germinating teliospore. Stop aspirating once the teliospore has entered the microcapillary. Repeat until there are approximately five teliospores in the microcapillary.
3. Raise the microcapillary with the micromanipulator and bring it to the collection droplet of RNA stabilization solution. Penetrate the droplet and inject the teliospores into the droplet.
4. Repeat steps 8.1 to 8.3 until approximately 1,000 teliospores have been captured.

10. Recovery of Collection Droplet

1. Pipette up the collection droplet and transfer it to the lid of an RNase/DNase-free 2.0 mL microcentrifuge tube. Carefully remove the mineral oil with a pipette without disturbing the collection droplet.
2. Use the teliospores for downstream applications such as RNA isolation.

Wyniki

Using the Clark-type microrespirometer-based method of measuring changes in respiration during teliospore dormancy and germination, we confirmed that dormant teliospores exhibit a low level of respiration (~1,075 µmol/h/mg) compared to germinating teliospores (~2,614 µmol/h/mg; Figure 1A). This represents a ~2.4-fold change in average rate of respiration between dormant teliospores and teliospores that have been induced to germinate. In addition, we...

Dyskusje

Basidiomycete biotrophic plant pathogens cause billions of dollars in crop losses annually. The vast majority of these pathogens produce teliospores that are integral to fungal dispersal and sexual reproduction. Gaining knowledge of the development and germination of teliospores is critical to understanding the spread of the devastating diseases caused by these fungi. In order to identify molecular changes at key control points we have devised a method to identify the timing of physiological shifts and another to isolate...

Materiały

Name	Company	Catalog Number	Comments
Streptomycin Sulfate	BioShop	STP101
Kanamycin Sulfate	BioShop	KAN201
Potato Dextrose Broth	BD Difco	254920
1 L Waring Laboratory blender	Waring	7011S
Cheesecloth	VWR	470150-438
Nalgene Polypropylene Desiccator with Stopcock	ThermoFisher Scientific	5310-0250
Unisense MicroRespiration system
MicroRespiration Sensor (O2)	Unisense	OX10
MicroOptode Meter Amplifier	Unisense	N/A
MR-Ch Small	Unisense	MR-Ch
SensorTrace Rate Software	Unisense	N/A
MicroRespiration Rack	Unisense	MR2-Rack
MicroRespiration Stirrer	Unisense	MR2-Co
Microdissection system
Axio Vert.A1 Inverted Light Microscope	Zeiss
Coarse Manipulator	Narishige	MMN-1
Three-axis Hanging Joystick Oil Hydraulic Micromanipulator	Narishige	MMO-202ND
Pneumatic Microinjector	Narishige	IM-11-2
TransferTip (ES)	Eppendorf	5175107004