1.10.2S
SIGNALLING VIA CAMP IS IMPORTANT FOR MORPHOGENESIS AND VIRULENCE INUSTILAGO MAYDIS

J KRONSTAD, F DORRENBERGER, N LEE and D LAIDLAW

Biotechnology Laboratory, Departments of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Background and objectives
Mating between haploid, budding cells of the corn smut fungus Ustilago maydis establishes a filamentous, dikaryotic cell type that is capable of infecting host tissue and inducing tumors. Two mating type loci, a and b, govern the interactions of mating partners. The a locus, with two alternate specificities called a1 and a2, encodes pheromone and pheromone receptors. The b locus encodes two proteins, bE and bW; these homoeodomain proteins are thought to control the expression of genes involved in mating, filamentous growth and pathogenicity. In addition to control by mating, the choice between budding and filamentous growth is influenced by the CAMP signal transduction pathway. In previous work, we have isolated genes encoding components of the CAMP pathway such as adenylate cyclase and the regulatory subunit of cAMP-dependent protein kinase (PKA [1]). Mutants defective in the gene for adenylate cyclase (uacl) have a constitutively filamentous phenotype and fail to cause disease symptoms. Mutants defective in the gene encoding the regulatory subunit (ubcl) display a multiple budding phenotype in a uacl- or a wild-type background; these mutants are capable of proliferating in host tissue, although they fail to induce tumors [2]. Overall, these results highlight the importance of the CAMP pathway in U. maydis morphogenesis and virulence. In general, there is a growing realization that signal transduction is an important aspect of fungal pathogenesis [3). Currently, our work is focused on identifying and characterizing components of the CAMP pathway, as well as important targets of phosphorylation by PKA. In addition, we are interested in defining points of integration between different signalling pathways.

Results and conclusions
We have identified several genes encoding kinases with sequence similarity to the catalytic subunit of PKA. The disruption of one of these genes, adrl, resulted in a constitutively filamentous cellular morphology and a non-pathogenic phenotype. In contrast, the ukal gene played a minor role in morphogenesis and pathogenesis. Assays of PKA activity indicated that the adrl-encoded enzyme accounted for approximately 90% of the activity and the ukal-encoded enzyme accounted for less than 10%. A large collection of suppressor mutants that restore budding growth to an adrl mutant has been obtained and genes identified by these mutants have been isolated by complementation. One of these genes, termed hgil, encodes a protein with little sequence similarity to known proteins. However, the predicted protein sequence contains several potential phosphorylation sites for PKA and the transcription of the gene appears to be regulated by the state of the b mating-type locus. In addition to suppressor analysis, we also employed subtractive hybridization to identify fungal genes that are differentially expressed as a result of defects in components of the CAMP pathway. The characterization of these genes is currently under way. Overall this will contribute to an understanding of the role of the CAMP pathway in morphogenesis and virulence in U. maydis.

References
1. Gold SE, Duncan GA, Barrett KJ, Kronstad JW, 1994. Genes & Development 8, 2805-2816.
2. Gold SE, Brogdon SM, Mayorga ME, Kronstad JW, 1997. Plant Cell 9 1585-1594.
3. Kronstad JW, 1997. Trends in Plant Science 2, 193-199.