Materials and Reagents

Paper bag (HERA, catalog number: 716P50 )
1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
Petri dish (SARSTEDT, catalog number: 82.1473.001 )
Micro-homogenizer (Carl Roth, catalog number: K994.1 )
Pipetman Diamond tips, D200 (Gilson, catalog number: F161931 )
Pipetman Diamond tips, D1000 (Gilson, catalog number: F161671 )
Glass beads (Sigma-Aldrich, catalog number: 18406 )
Copper sulfate (CuSO₄) (AppliChem, catalog number: 131270 )
Ampicillin (Carl Roth, catalog number: K029.2 )
Tetracycline (Duchefa Biochemie, catalog number: T0150 )
Chloramphenicol (AppliChem, catalog number: A1806 )
Potato dextrose broth (BD, catalog number: 254920 )
Agar (BD, catalog number: 214040 )
PD_{Amp, Tet, ChlA}plates (see Recipes)

Equipment

Scissors
Cooled incubator (ST) ST 1 (Pol-Eko Aparatura, catalog number: ST 1 )
Pipetman P1000 (Gilson, catalog number: F123602 )
Pipetman P200 (Gilson, catalog number: F123601 )
Vortex
Heraeus^TM Pico^TM 17 Microcentrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraeus^TM Pico^TM 17 , catalog number: 75002410)

Procedure

Harvest the infected spikelets from the plant by cutting them off using a pair of scissors. Figure 1 shows the appearance of the infected spikelets. Spikelets should be stored in a paper bag for ~10 days at 28 °C to dry out. They can then be stored at room temperature until use.

Figure 1. Spikelets of B. distachyon infected by U. bromivora. The black masses of fungal spores are clearly visible in both the hydrated (right) and dehydrated (left) spikelet. The scale bar represents approximately 1 cm. The image background of the spikelets was digitally removed.
Carefully grind the spikelets filled with spore material with a micro-homogenizer in 1.5 ml microcentrifuge tubes to break up the spikelet. The spores are very robust to survive harsh environments and will not be damaged by the grinding.
Add 500 µl ddH₂O and continue to grind softly. It is best to use a rotating motion to grind the spikelet as pushing into the microcentrifuge tube will cause the liquid to splash out. The liquid will turn black as it is ground, indicating that the spores have been released and are properly suspended.
Incubate the ground spores in ddH₂O for 1 h at room temperature to allow faster-germinating, contaminating, fungal spores to germinate. This will leave them more susceptible to the sterilisation treatment and reduce overall contamination levels.
Add 500 µl 3% CuSO₄ and mix the solution either by pipetting up and down or by using a vortex. This will kill most, if not all, of the contaminating spores that have germinated but not the U. bromivora spores which can take up to 17 h to germinate.
Note: CuSO₄ is a heavy metal and should be handled and discarded according to its MSDS.
Incubate the spore/CuSO₄ mixture for 15 min at room temperature.
Centrifuge the spore mixture at 1,200 x g for 5 min. This will cause the spores to pellet. Carefully pour out the liquid and re-suspend the spores in 1 ml ddH₂O.
Repeat step 7 three times but, on the third time, instead of re-suspending the spores in ddH₂O, proceed to step 9.
Re-suspend the spores in 300 µl ddH₂O with antibiotics (ampicillin, tetracycline and chloramphenicol). These will kill any non-fungal cells that might have survived the CuSO₄ treatment.
Make a dilution series of the fungal spore suspension (10⁰-10^-4) in ddH₂O with the three antibiotics.
Plate 100 µl of each dilution on a PD_{Amp, Tet, ChlA} plate (see Recipes), spread using glass beads and incubate at 21 °C for several days to obtain colonies.
As U. bromivora spores contain tetrads, the original colonies should be singled out on PD plates (with or without antibiotics) to obtain colonies derived from a single cell.

Notes

While we have provided information on the specific equipment and reagents used, we have no reason to believe that it is essential to use them exactly. The equivalent equipment or reagents from other manufacturers should be just as suitable.

Recipes

PD_{Amp, Tet, ChlA} plates
2.4% (weight/volume) potato dextrose broth
2.0% (weight/volume) agar
Note: Measure out the appropriate amount of potato dextrose broth and agar for the intended volume. Dissolve them in ddH₂O then autoclave the mixture at 121 °C for 15 min. Once it has cooled to approximately 50 °C, add ampicillin, tetracycline and chloramphenicol to their final concentrations. Pour the mixture into Petri dishes (20 ml per 9 cm Petri dish) and wait ~20 min for it to set.

Acknowledgments

This protocol was developed in the Djamei laboratory and has had aspects contributed and/or changed by multiple members of the Djamei group. Due to this, the people listed as authors are not the only ones who have been involved in developing the protocol (for this see Rabe et al., 2016) but are merely the ones who have prepared it for publication. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement No. [EUP0012 ‘Effectomics’], the Austrian Science Fund (FWF): [P27429-B22, P27818-B22, I 3033-B22], and the Austrian Academy of Science (OEAW).

References

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Rabe, F., Bosch, J., Stirnberg, A., Guse, T., Bauer, L., Seitner, D., Rabanal, F. A., Czedik-Eysenberg, A., Uhse, S., Bindics, J., Genenncher, B., Navarrete, F., Kellner, R., Ekker, H., Kumlehn, J., Vogel, J. P., Gordon, S. P., Marcel, T. C., Munsterkotter, M., Walter, M. C., Sieber, C. M., Mannhaupt, G., Guldener, U., Kahmann, R. and Djamei, A. (2016). A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem. Elife 5.