Project description:This SuperSeries is composed of the following subset Series: GSE18750: Controlled expression of compatible and incompatible combinations of Ustilago maydis b-mating type locus genes bE and bW GSE18754: Effect of rbf1 deletion during controlled expression of of Ustilago maydis b-mating type locus genes bE1 and bW2 GSE18756: Rbf1 induced gene expression in Ustilago maydis Refer to individual Series
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. To identify genes regulated by the bE/bW transcription factor, changes in gene expression were monitored during a 12-h time course (0h, 1h, 2h, 3h, 5h, 12h) in strains AB31 and AB33 that harbor the active combination bE1 and bW2 under the control of the arabinose-inducible crg1 promoter and the nitrate-inducible nar1 promoter, respectively (Brachmann et al., 2001, Mol Microbiol 42, 1047-1063). Induction of bE1/bW2 in these strains results in a filament that resembles the infectious dikaryotic hypha formed after fusion of compatible sporidia. Strains AB32 and AB34, which harbor the incompatible bE2 and bW2 combination, were used as controls.
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. Rbf1 encodes a zinc-finger transcription factor that is expressed upon formation of an active bE/bW heterodimer. To identify gene regulated by rbf1 independently from b, the b mating type locus was replaced by a copy of rbf1 under control of the arabinose-inducible crg1 promoter (strain CP27). Samples were taken 5h after induction of rbf1 gene expression. Strain JB2, carrying a deletion of the b-locus, was used as control.
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. Rbf1 encodes a zinc-finger transcription factor that is expressed upon formation of an active bE/bW heterodimer. To analyse the dependency of b and rbf1, changes in gene expression were monitored in strain AB31 (Brachmann et al., 2001, Mol Microbiol 42, 1047-1063) and in the derivative AB31Δrbf1 in which rbf1 was deleted. AB31 harbours the active combination bE1 and bW2 under the control of the arabinose-inducible crg1 promoter. Samples were taken 3h, 5h and 12h after induction of bE/bW gene expression. Strain AB32, which harbors the incompatible bE2 and bW2 combination, was used as control.
Project description:In fungi, sexual compatibility is controlled by mating type loci that prevent self-fertilization. In the plant pathogenic fungus Ustilago maydis, the b mating type locus encodes a pair of unrelated homeodomain proteins, termed bE and bW. After fusion of two compatible, haploid cells, the bE and bW proteins form a heterodimeric complex, but only if they are derived from different, compatible alleles. The active bE/bW complex is required and sufficient to initiate pathogenic development of U. maydis, which is prerequisite for sexual reproduction of the fungus. However, the role of the b heterodimer during later stages of pathogenic development was unclear. To analyze b function during in planta development, we generated a temperature-sensitive bE allele (bEts) encoding a protein with a single amino acid alteration at the border of the homeodomain. This mutation leads to a stop in pathogenic development at the restrictive temperature in planta, while bEts strains show normal development at permissive temperature. At restrictive temperature, hyphae develop enlarged, bulbous cells at their tips that contain multiple nuclei, indicating a severe defect in cell division. DNA array analysis of bEts mutant strains in planta revealed a b-dependent regulation of genes coding for secreted proteins that were shown to influence fungal virulence. Our data demonstrate that in U. maydis the b heterodimer is not only essential to establish the heterodikaryon after mating of two compatible sporidia and to initiate fungal pathogenicity, but also to sustain in planta proliferation and ensure sexual reproduction.
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. To identify genes regulated by the bE/bW transcription factor, changes in gene expression were monitored during a 12-h time course (0h, 1h, 2h, 3h, 5h, 12h) in strains AB31 and AB33 that harbor the active combination bE1 and bW2 under the control of the arabinose-inducible crg1 promoter and the nitrate-inducible nar1 promoter, respectively (Brachmann et al., 2001, Mol Microbiol 42, 1047-1063). Induction of bE1/bW2 in these strains results in a filament that resembles the infectious dikaryotic hypha formed after fusion of compatible sporidia. Strains AB32 and AB34, which harbor the incompatible bE2 and bW2 combination, were used as controls. Strains were grown to an OD600 of 0.4-0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter-sterilized). For induction of genes under control of the crg1 promoter (strains AB31 and AB32) cells were inoculated in liquid array medium containing 1% (w/v) arabinose instead of glucose as a carbon source. For induction of the nar1-promoter glutamine was exchanged by nitrate (3g KNO3/l) as the sole nitrogen source. For induction, cells were washed once with inducing medium; time point 0 h of the timecourse experiments corresponds to the first contact with inducing medium. Experiments were performed in two biological replicates for each time point.
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. Rbf1 encodes a zinc-finger transcription factor that is expressed upon formation of an active bE/bW heterodimer. To analyse the dependency of b and rbf1, changes in gene expression were monitored in strain AB31 (Brachmann et al., 2001, Mol Microbiol 42, 1047-1063) and in the derivative AB31Δrbf1 in which rbf1 was deleted. AB31 harbours the active combination bE1 and bW2 under the control of the arabinose-inducible crg1 promoter. Samples were taken 3h, 5h and 12h after induction of bE/bW gene expression. Strain AB32, which harbors the incompatible bE2 and bW2 combination, was used as control. Strains were grown to an OD600 of 0.4-0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter-sterilized). For induction of the bE and bW genes, cells were inoculated in liquid array medium containing 1% (w/v) arabinose instead of glucose as a carbon source. For induction, cells were washed once with inducing medium; time point 0 h of the time course experiments corresponds to the first contact with inducing medium. Experiments were performed in two biological replicates for each time point.
Project description:In this project, we conducted a quantitative analysis of secreted proteins during the initial stages of the pathogenic program in Ustilago maydis. We utilized the AB33 strain, which contains the compatible bE2/bW1 genes controlled by the nitrate-inducible nar1 promoter. Consequently, in nitrogen-rich media, the virulence program is initiated, leading to the development of filaments. Filamentation was induced for 5 hours in minimal medium supplemented with NO3 (MM-NO3) in both the AB33 wild-type and Δrow1 strains. Subsequently, samples were collected, precipitated, processed, and analyzed.
Project description:In fungi, sexual compatibility is controlled by mating type loci that prevent self-fertilization. In the plant pathogenic fungus Ustilago maydis, the b mating type locus encodes a pair of unrelated homeodomain proteins, termed bE and bW. After fusion of two compatible, haploid cells, the bE and bW proteins form a heterodimeric complex, but only if they are derived from different, compatible alleles. The active bE/bW complex is required and sufficient to initiate pathogenic development of U. maydis, which is prerequisite for sexual reproduction of the fungus. However, the role of the b heterodimer during later stages of pathogenic development was unclear. To analyze b function during in planta development, we generated a temperature-sensitive bE allele (bEts) encoding a protein with a single amino acid alteration at the border of the homeodomain. This mutation leads to a stop in pathogenic development at the restrictive temperature in planta, while bEts strains show normal development at permissive temperature. At restrictive temperature, hyphae develop enlarged, bulbous cells at their tips that contain multiple nuclei, indicating a severe defect in cell division. DNA array analysis of bEts mutant strains in planta revealed a b-dependent regulation of genes coding for secreted proteins that were shown to influence fungal virulence. Our data demonstrate that in U. maydis the b heterodimer is not only essential to establish the heterodikaryon after mating of two compatible sporidia and to initiate fungal pathogenicity, but also to sustain in planta proliferation and ensure sexual reproduction. Maize plants were infected with a mixture of either FB1 and FB2 (wild-type), or RAb1ts and RAb2ts (temperature-sensitive b heterodimer) and kept at 22°C (permissive conditions). Control samples of FB1/FB2 and RAb1ts and RAb2ts were taken 5 days post inoculation at 22 °C (1 replicate each), to see if strains expressing the bts heterodimer show wildtype-like biotrophic development. To measure the impact of the b heterodimer on pathogenic development, infected plants were shifted for 9 hours to resrictive conditions (31°C) 111 hours post infection. After the temperature shift FB1/FB2 (3 replicates) infections developed normal, whereas RAb1ts/RAb2ts (3 replicates) infections were not able to further proliferate in planta because of a none-functional b heterodimer.