The overall goal of the procedures presented here is to identify stages of spore germination so that gene transcript levels can be assessed. Analysis of these changes will provide further knowledge of a critical stage in disease dispersal. This method can help answer key questions about transitions during teliospore germination that are linked to changes in gene transcript levels and thus reflect shifts in gene expression control.
The main advantages of this technique are that it identifies transitions in respiration level that don't correspond to visibile morphological switches and allows for physical isolation of teliospores at distinct stages of germination. This technique has implications for the control of pathogen dissemination, because it allows us to identify key transitions in spore germination, that, if altered, could block disease spread. Begin this procedure with corncob infection, teliospore harvesting, as well as teliospore viability and germination test, as described in the text protocol.
To induce germination, first weigh an equal amount of teliospores for each respiration experiment. In a biosafety cabinet, add the teliospores to an autoclaved respiration chamber. Fill the chamber with potato dextrose broth, supplemented with streptomycin sulfate and kanamycin sulfate.
Pipette up and down to create a teliospore suspension. Place the chamber lid in the chamber to create an air-tight seal. To obtain the OCR measurement, first place the chamber in the chamber rack inside a water bath.
Then, place the oxygen probe inside the opening of the chamber. Monitor the data points appearing in real time on the sensor trace rate program. Let the probe stabilize for approximately three minutes after the probe is placed in the chamber.
Click Measure to measure oxygen levels continuously for six hours with measurements recorded at two-second intervals. Stop the measurement and repeat these steps for each sample to be analyzed before exporting the data to Microsoft Excel and performing data analysis as described in the text protocol. Place approximately 10 milligrams of umatus teliospores into a 1.5 milliliter micro centrifuge tube.
Suspend the teliospores in 500 microliters of PDB supplemented with streptomycin sulfate. Gently pipette to mix until there are no clumps of teliospores in the medium. Transfer the teliospore suspension to an auto-claved 250 milliliter Erlin Meyer flask, containing PDB, supplemented with streptomycin sulfate.
Incubate the flask overnight at 28 degrees Celsius, shaking at 90 RPM. Prepare a Petri dish by pipetting four to five five-microliter drops of distilled water across the top of the Petri dish. Then, pipette three two-microlitre droplets of RNA stabilization solution on the Petri dish to be used for sample collection.
Also pipette 30 five-microliter droplets of germinating teliospores on the Petri dish. Add 15 milliliters of mineral oil to the Petri dish. Ensure that all droplets are covered by oil before proceeding.
Next, prepare a microcapillary with a 15-micron inter diameter, one-millimeter flange, 55-milliliter length, and a 20 degree tip angle. Place it in the microcapillary holder, and submerge 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. Using the controls of the micromanipulator, move the prepared microcapillary to one of the germination droplets, and 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.
Slowly aspirate in order to capture the germinating teliospore. Stop aspirating once the teliospore has entered the microcapillary. Repeat until there are approximately five teliospores in the microcapillary.
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. Repeat these steps until approximately 1, 000 teliospores have been captured.
To recover the collection droplet, pipette up the collection droplet, and transfer it to the lid of an RNA's DNA's free 2.0 milliliter microcenterfuge tube. Carefully the mineral oil with the pipette, without disturbing the collection droplet. The teliospores can now be used for downstream applications such as RNA isolation.
This plot illustrates a typical result from assessing oxygen level changes in suspensions of teliospores induced to germinate. Relative to un-induced teliospores, the separation indicating the start of respiration occurs at about 30 minutes. Considering that it takes about 15 minutes to set up and transport the sample to the oxygen probe, this time is actually 45 minutes.
Shown here, are the five stages of teliospore germination. In stage one, teliospores are induced to germinate but show no morphological sign of germination. Stage two teliospores show an emerged promycelium with a length less than the diameter of the teliospore.
In stage three the promycelium length is greater than the diameter of the teliospore. Stage four is characterized by promycelia with attached basidiospores. And in stage five, there are budding basidiospores.
Here, the asynchroniously germinating teliospores are shown and the microcapillary is positioned near a stage three teliospore. The stage three teliospore is then aspirated into the microcapillary and transferred to a drop of collection solution. Once mastered, this microdissection technique can be used to isolate 1, 000 stage specific teliospores in six to seven hours.
While attempting this procedure, it's important to remember to take frequent breaks. After watching this video, you should have a good understanding of how to physically isolate spores at morphologically distinct stages of germination. And identify the timing of respiration initiation.
Generally, individuals new to this method will struggle, because of the need to maintain sterility. And the need for patience and isolating cell types by microdissectioning. The development of this technique paves the way for fungal plant pathologists to explore the morphological and molecular aspects of germination in devastating smut and rust fungi that threaten global food security.
Although this technique applies to us ligomatists, it could also be used in many other devastating agriculture crop pathogens that result in billions of dollars in losses annually. Following this procedure, other methods like RNA isolation can be performed in order to answer additional questions, like, what is the profile and timing of transcript level change?
