Abstract
The corn smut pathogen, Ustilago maydis (U. maydis) (DC.) Corda, is a semi-obligate plant pathogenic fungus in the phylum Basidiomycota (Alexopoulos et al., 1996). The fungus can be easily cultured in its haploid yeast phase on common laboratory media. However, to complete its sexual cycle U. maydis strictly requires its specific plant host, maize (Zea mays). The fungus is an interesting and important model organism for the study of the interactions of fungal biotrophic pathogens with plants. In this protocol, we describe the process of plant inoculation, teliospore recovery, germination, progeny isolation and initial mating type analysis. The primary purpose of this protocol is to identify individual progeny strains of U. maydis that can be used for downstream genetic analyses. Generation of targeted mutants to study various processes is a common approach with this and many plant pathogenic fungi. The ability to generate combinations of mutations is facilitated by sexual crossing without the need for additional selectable markers.
Materials and Reagents
Equipment
Procedure
U. maydis teliospores can potentially be produced in any above ground, actively growing tissues. However, for research purposes two plant stages will be described, each with its particular advantages. These commonly used stages are:
Representative data
Figure 1. U. maydis haploid colonies. Typical cream colored colonies of U. maydis grown on PDA medium. Size bar is 2 mm. Figure 2. Inoculation of maize seedlings with a mixture of compatible U. maydis haploid cells. Insert the syringe’s needle half the diameter into the maize seedling pseudo-stem at about 1 cm above the soil line. Inject enough cell mixture to fill the seedling pseudo-stem cavity; observe for liquid expression from top of leaf whorl. Figure 3. Gall production and teliospore development in maize seedlings and ears inoculated with compatible U. maydis strains. a. Large galls have developed on the lower portion of the seedling stems (arrowheads), close to the soil. b. Series of small galls formed at the distal portion of seedling stem and leaf ligule (arrowhead). c. Young ear galls cut transversely to show teliospore production; note small black areas corresponding to mature teliospores (arrows). d. Mature ear galls; teliospore production increases at later stages of gall development (arrows) and galls become darker in color due to the abundant melanized teliospores. Figure 4. Plate mating assay. a). Representation of suggested mating plate organization. Each isolated strain is co-spotted with tester strains of known mating type to determine mating type of progeny. b). Mating plate results with progeny of previously determined mating type. Four autophagy deletion mutant strains (Nadal and Gold, 2010) (MN8.1; MN8.4; MN8.11 and MN8.12) were tested for their ability to mate. White fuzzy growth indicates positive mating events. ɸ indicates mock (water) inoculation for the corresponding row showing the morphology of unmated strains.
Notes
Recipes
Note: Chemical reagents were obtained from Sigma-Aldrich.
Acknowledgments
We are grateful to our funding sources including the US Department of Agriculture and National Science Foundation competitive grant programs. The methods described here for teliospore harvesting and germination and progeny isolation were adapted from standard protocols (Holliday, 1974).
References
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