Functional characterization of a seven-WD40 repeat protein Rak1 in Ustilago maydis

In the phytopathogenic smut fungus Ustilago maydis cell fusion of compatible haploid cells is controlled by a pheromone/receptor system. The pheromone signal is transmitted via a conserved MAP kinase module that activates Prf1, an essential regulator of sexual and pathogenic development. To find additional components in MAP kinase signaling, U. maydis Rak1, a seven-WD40 repeat motif protein that is orthologous to mammalian RACK1 was studied. rak1 gene was constitutively expressed and Rak1 protein localized in the cytoplasm as well as in the membrane fraction. In U. maydis Rak1 was found to affect cell wall synthesis and cell growth and could partially complement the growth phenotype of Saccharomyces cerevisiae asc1 mutant at elevated temperature. Deletion of rak1 strongly attenuated conjugation tube formation in haploid cells resulting in poor mating ability. This defect could be traced back to reduced expression of the pheromone and pheromone-receptor genes. With the genetic activation of the MAP kinase module, the formation of conjugation tubes of FB1rak1 could be rescued. Furthermore, the defect of FB1Drak1 in conjugation tube formation could be restored by the constitutive expression of the pheromone receptor gene pra1 or the pheromone response transcription factor prf1 upon pheromone stimulation. In solopathogenic strain the deletion of rak1 led to attenuated filamentation and pathogenicity, which could be rescued by the constitutive expression of an active bE/bW heterodimer. This analysis made it likely that rak1 controls the expression of prf1. By microarray analysis, 201 genes were identified to be differentially regulated in the rak1 deletion strain. 163 up-regulated genes showed a significant enrichment in the functional categories like metabolism, energy, virulence and stress and toxin resistance. 38 down-regulated genes showed a significant enrichment in lipid metabolism, fermentation and a MAPK signaling-dependent pathway. Among the down-regulated genes in FB1Δrak1, rop1, a direct positive transcriptional regulator of prf1, was detected. The constitutive expression of rop1 in FB1Δrak1 could induce the expression of mfa1 as well as conjugation tube formation in response to pheromone stimulation. Collectively, rak1 functions as a novel regulator of rop1 and consequently of prf1 gene expression during mating and pathogenic development.