The a2 mating-type-locus gene lga2 of Ustilago maydis interferes with mitochondrial dynamics and fusion, partially in dependence on a Dnm1-like fission component.

The a2 mating-type-locus gene lga2 of the basidiomycete Ustilago maydis encodes a mitochondrial protein that interferes with mitochondrial morphology and integrity, and that plays a role in uniparental inheritance of mitochondrial DNA. To address the mode of action of Lga2, we investigated its Dnm1 (a dynamin-related protein)-dependent effects. Here, we demonstrate that Dnm1 functions as a mitochondrial fission component in U. maydis and mediates Lga2-induced mitochondrial fragmentation. Mitochondrial fusion occurred very inefficiently in matings of U. maydis wild-type strains, but was strongly stimulated in the absence of dnm1 and highest in either wild-type or Deltadnm1 combinations when the a2 partner was deleted in lga2. This indicates that Dnm1 plays a central role in opposing mitochondrial fusion in response to endogenous lga2 expression and that Lga2 additionally inhibits fusion in a dnm1-independent manner. Our results further show that Lga2 does not stimulate increased turnover of the putative fusion protein Fzo1 and causes mitochondrial branching, loss of mitochondrial DNA and fitness reduction independently of dnm1. We conclude that Lga2 acts upstream of Dnm1, but controls mitochondrial integrity independently of Dnm1-mediated fission. In addition, we demonstrate a role of dnm1 in fungal virulence.

Selective activation by the guanine nucleotide exchange factor Don1 is a main determinant of Cdc42 signalling specificity in Ustilago maydis.

The 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 Delta cdc42 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.

The Ustilago maydis Cys2His2-type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stage.

The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys(2)His(2)-type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2-like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b-dependently regulated Cys(2)His(2)-type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings.

Rac1 and Cdc42 regulate hyphal growth and cytokinesis in the dimorphic fungus Ustilago maydis.

Small 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 Rho-specific 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.

The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis.

The phytopathogenic basidiomycete Ustilago maydis displays a dimorphic switch between budding growth of haploid cells and filamentous growth of the dikaryon. In a screen for mutants affected in morphogenesis and cytokinesis, we identified the serine/threonine protein kinase Cla4, a member of the family of p21-activated kinases (PAKs). Cells, in which cla4 has been deleted, are viable but they are unable to bud properly. Instead, cla4 mutant cells grow as branched septate hyphae and divide by contraction and fission at septal cross walls. Delocalized deposition of chitinous cell wall material along the cell surface is observed in cla4 mutant cells. Deletion of the Cdc42/Rac1 interaction domain (CRIB) results in a constitutive active Cla4 kinase, whose expression is lethal for the cell. cla4 mutant cells are unable to induce pathogenic development in plants and to display filamentous growth in a mating reaction, although they are still able to secrete pheromone and to undergo cell fusion with wild-type cells. We propose that Cla4 is involved in the regulation of cell polarity during budding and filamentation.