Identification of genes in the bW/bE regulatory cascade in Ustilago maydis

Abstract

In the phytopathogenic fungus Ustilago maydis, the switch to filamentous growth and pathogenic development is controlled by a heterodimeric transcription factor consisting of the bW and bE homeodomain proteins. To identify genes in the regulatory cascade triggered by the bW/bE heterodimer, we have constructed strains in which transcription of the b genes is inducible by either arabinose or nitrate. At different time-points after induction, genes that are switched on or off were identified through a modified, non-radioactive RNA fingerprint procedure. From 348 gene fragments isolated initially, 48 fragments representing 34 different genes were characterized in more detail. After eliminating known genes, false positives and genes influenced in their expression profile by media conditions, 10 new b-regulated genes were identified. Of these, five are upregulated and five are downregulated in presence of the b heterodimer. Two do not share significant similarity to database entries, whereas the other eight show similarity to disulphide isomerases, exochitinases, cation antiporters, plasma membrane (H⁺)-ATPases, acyl transferases, a capsular associated protein of Cryptococcus neoformans, DNA polymerases X, as well as to a potential protein of Neurospora crassa. We demonstrate that in one of the early upregulated genes, the promoter can be bound by a bW/bE fusion protein in vitro. Interestingly, three out of the four genes that are downregulated by the b heterodimer appear upregulated after pheromone stimulation, suggesting a connection to the mating process.