SummarySmall GTP-binding proteins of the highly conserved Rho family act as molecular switches regulating cell signalling, cytoskeletal organization and vesicle trafficking in eukaryotic cells. Here we show that in the dimorphic plant pathogenic fungus Ustilago maydis deletion of either cdc42 or rac1 results in loss of virulence but does not interfere with viability. Cells deleted for cdc42 display a cell separation defect during budding. We have previously shown that the Rhospecific guanine nucleotide exchange factor (GEF) Don1 is required for cell separation in U. maydis . Expression of constitutive active Cdc42 rescues the phenotype of don1 mutant cells indicating that Don1 triggers cell separation by activating Cdc42. Deletion of rac1 affects cellular morphology and interferes with hyphal growth, whereas overexpression of wild-type Rac1 induces filament formation in haploid cells. This indicates that Rac1 is both necessary and sufficient for the dimorphic switch from budding to hyphal growth. Cdc42 and Rac1 share at least one common essential function because depletion of both Rac1 and Cdc42 is lethal. Expression of constitutively active Rac1 Q61L is lethal and results in swollen cells with a large vacuole. The morphological phenotype, but not lethality is suppressed in cla4 mutant cells suggesting that the PAK family kinase Cla4 acts as a downstream effector of Rac1.

SummaryThe highly conserved GTP-binding proteins Cdc42 and Rac1 regulate cytokinesis, establishment of cell polarity and vesicular trafficking. In the dimorphic fungus Ustilago maydis, Rac1 is required for cell polarity and budding, while Cdc42 is essential for cell separation during cytokinesis. The same cell separation defect is also observed in mutants that lack Don1, a guanine nucleotide exchange factor (GEF) of the Dbl family. We have generated a series of chimeric GTP-binding proteins consisting of different portions of Cdc42 and Rac1. In vivo complementation analysis revealed that a short region encompassing amino acids 41-56 determines signalling specificity. Remarkably, substitution of a single amino acid at position 56 within this specificity domain is sufficient to confer Cdc42 function to Rac1 in vivo. Expression of Rac1 W56F in Dcdc42 mutant cells resulted in complementation of the cell separation defect. In vitro GDP/ GTP exchange assays demonstrated that the Dbl family GEF Don1 is highly specific for Cdc42 and cannot activate Rac1. However, if Rac1 W56F is used as a substrate, Don1 is able to stimulate GDP/GTP exchange. Together these data indicate that activation by the GEF Don1 is an important determinant of Cdc42-specific signalling in vivo.

In the dimorphic fungus Ustilago maydis, Rac1 and its activator Cdc24 are essential for hyphal tip growth. Rac1 is shown to stimulate Cla4 kinase, which in turn triggers destruction of Cdc24. Expression of stabilized Cdc24 interferes with cell polarization, indicating that negative feedback regulation of Cdc24 is critical for tip growth.