Analysis of genes involved in developmental pathways in two basidomycetous fungi, Ustilago maydis and Sclerotium rolfsii
Takach, Johanna Elise
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Fungi rely on complex molecular pathways to orchestrate the physical changes necessary to survive and reproduce. In pathogenic fungi, these pathways can also mediate host infection. Identification of the factors that coordinate these changes in morphology is critical for improved management of fungal pathogens. To this end, genetic factors involved in developmental and morphological change in Ustilago maydis and Sclerotium rolfsii were investigated. In the model system U. maydis, the well-characterized cAMP/PKA signaling pathways mediates filamentation in response to environmental cues. Genome analysis identified two potential additional components of the signal cascade. An adenylate cyclase-associated protein (CAP) homolog was detected. Yeast 2-hybrid analysis indicates that the U. maydis Cap1 interacts with adenylate cyclase (Uac1). Deletion of cap1 results in abnormal colony morphology and filamentous cells that revert to budding when treated with exogenous cAMP, similar to U. maydis strains with low cAMP production. Δcap1 strains are reduced in mating ability and pathogenicity. These results suggest that Cap1 positively regulates cAMP production by adenylate cyclase and is required for budding growth. A genome search also revealed the presence of a second cAMP binding domain, in addition to Ubc1, the only previously identified target of cAMP in U. maydis. The gene encoding the domain, cab1, was deleted, but no morphological, mating, or pathogenicity phenotypes were observed. Δcab1 strains had a severe growth defect in medium enriched with yeast extract. These results indicate that cab1 has little to no role in the cAMP signaling cascade. S. rolfsii produces small sclerotia that serve as overwintering structures and inocula. Little is known about the genetic factors involved in sclerotial development. To this end, two cDNA libraries were created using Suppressive Subtractive Hybridization PCR (SSHP) and screened to identify genes differentially expressed during sclerotium formation and development. Of the 53 unique sequences identified, 31 were homologous to expressed sequences in fungal systems, including a lectin, cytochrome P450, and two ribosomal proteins. Relative gene expression was measured using quantitative real-time PCR, with a subset further verified by northern blot analysis. The results from this study will provide a foundation for future genetic studies in S. rolfsii.