Abstract
Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly use this spore material to infect Brachypodium seeds, in many cases it is more useful to isolate individual strains of U. bromivora for a genetically homogenous population. This protocol describes how to collect and germinate the spores of U. bromivora on plate in order to obtain strains derived from a single cell.
Keywords: Brachypodium distachyon, Ustilago bromivora, Biotrophic interaction, Plant pathogen, Filamentous fungus, Head smut
Background
Ustilago maydis infecting maize (Zea mays) has long been established as a model system for studying biotrophic pathogens (Brefort et al., 2009). This has led to many discoveries concerning the nature of biotrophic interactions but has limitations due to the practical difficulties of working with maize in the laboratory. The same is true for the model fungus Ustilago hordei infecting barley (Hordeum vulgare) (Laurie et al., 2012). In contrast to these crop plants, the model grass Brachypodium distachyon has a small genome, undemanding growth conditions and is amenable to genetic manipulation (Draper et al., 2001). B. distachyon has also been used to study non-host resistance to Puccinia striiformis f. sp. tritici due to the genetic complexity of its usual host, wheat (An et al., 2016). Recently, we have described Ustilago bromivora, a smut fungus related to U. maydis, which is able to infect Brachypodium sp. and proposed this as a new model system for studying biotrophic interactions (Rabe et al., 2016). During infection of Brachypodium sp. by U. bromivora, no visible symptoms can be detected for most of the infection. The only visible symptom of infection occurs during flowering when the plant produces spikelets that are filled with black, fungal spores. These spores can be used to directly infect new seeds but contain genetically disparate fungal strains. For most purposes, a pure culture from a single cell is preferable as it can be cultured axenically, characterized and genetically manipulated before being used to infect further seeds. This protocol describes the process of germinating the U. bromivora spores in vitro. It bears similarities to spore germination protocols of other smut fungi, for example, U. maydis (Heinze, 2009; Nadal et al., 2016), but has been modified to account for differences in the host species and infected organs. Please be aware that U. bromivora shows a mating type bias leading to the survival of only one mating type (mat a) on plate after spore germination (Rabe et al., 2016). This means that it will require additional effort to generate the second mating type or the use of the strain which we have previously isolated.
Materials and Reagents
Equipment
Procedure
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
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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