Institut für Genetik und Mikrobiologie, Maria-Ward-Str. 1a, 80638 München, Germany

Background and objectives
The phytopathogenic fungus Ustilago maydis is a facultative pathogen and certain steps of its life cycle strictly depend on its host. U. maydis exists in two morphological stages. Haploid sporidia grow yeast-like and are non-pathogenic, while the fusion product of compatible sporidia, the filamentous dikaryon, is able to infect maize plants. In contrast to sporidia, the dikaryotic form is not able to proliferate on artificial media. Mating and filamentous growth are regulated by the two mating-type loci a and b. After cell fusion only the b locus is required for pathogenic development. During growth in planta, nuclear fusion and spore formation take place. The life cycle is completed when spores germinate, undergo meiosis and generate haploid sporidia. We propose the existence of plant signals which are required for growth of the dikaryon in planta. To identify such plant signals we have set up a system to isolate fungal genes which are plant induced.

Materials and methods
To identify genes which are induced during fungal growth in planta we used a GFP (green fluorescence protein) reporter gene [1] construct in a REMI (restriction enzyme-mediated integration) approach [2] to generate tagged insertion mutants. The promoter which drives expression of GFP is not active but requires fusion to enhancer elements for activity. Plants were infected with REMI transformants that do not show GFP expression on different solid media. To circumvent the problem of knocking out genes required for pathogenic developement, we use a diploid solo-pathogenic U. maydis strain for mutagenesis. Mutants were identified where GFP is only expressed during fungal growth in planta.

Results and conclusions
We generated 1600 REMI mutants of which 40% showed GFP activity on different media. We were able to isolate 15 transformants showing fluorescence only during growth in planta. One gene isolated was the U. maydis pheromone gene which is known to be induced in planta. In addition we isolated a gene showing high homology to a transcription factor from yeast (LRG1) which is induced during mating and spore formation.

1. Spellig T, Kahmann R, 1996. MGG 252 503-509.
2. Bölker M, Genin S, Lehmler C, Kahmann R, 1995a. Canadian Journal of Botany 248, 547-552.