Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis

Aichinger, Christian; Hansson, Karen; Eichhorn, Heiko; Lessing, Franziska; Mannhaupt, Gertrud; Mewes, Hans‐Werner; Kahmann, Regine

doi:10.1007/s00438-003-0926-z

Cited by 70 publications

(52 citation statements)

References 49 publications

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“…DNA fragments were fused to the EGFP gene in plasmid p123 (1), where the fusion constructs were expressed under the control of the constitutive etef promoter.…”

Section: Methodsmentioning

confidence: 99%

Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

Zameitat

Freymark

Dietz

et al. 2007

Appl Environ Microbiol

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Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q 6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.

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“…DNA fragments were fused to the EGFP gene in plasmid p123 (1), where the fusion constructs were expressed under the control of the constitutive etef promoter.…”

Section: Methodsmentioning

confidence: 99%

Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

Zameitat

Freymark

Dietz

et al. 2007

Appl Environ Microbiol

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“…For complementation analysis we made use of the genome integrative p123 plasmid (Aichinger et al, 2003). To generate the plasmid for complementation of the opt triple mutant, plasmid pPH19 was constructed.…”

Section: Plasmid and Strain Constructionmentioning

confidence: 99%

The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis

et al. 2018

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The corn smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis we focused on fungal metabolism, nutritional strategies, secreted effectors and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.

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“…The PCR product was used for one tube BP/ LR Gateway cloning into the destination vector p123bb-GW (M. Vranes, unpublished data). p123bb-GW is a p123 derivative (Aichinger et al, 2003) in which the P otef :GFP cassette has been replaced with a chloramphenicol resistance cassette that is flanked by attR1 and attR2 recombination sites for Gateway cloning.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

Crosstalk between the Unfolded Protein Response and Pathways That Regulate Pathogenic Development inUstilago maydis

et al. 2013

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The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy.

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Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis

Cited by 70 publications

References 49 publications

Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis

Crosstalk between the Unfolded Protein Response and Pathways That Regulate Pathogenic Development inUstilago maydis

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