Structure of Ustilago maydis killer toxin KP6 alpha-subunit. A multimeric assembly with a central pore.
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Ustilago maydis is a fungal pathogen of maize, some strains of which secrete killer toxins. The toxins are encoded by double-stranded RNA viruses in the cell cytoplasm. The U. maydis killer toxin KP6 contains two polypeptide chains, alpha and beta, having 79 and 81 amino acids, respectively, both of which are necessary for its killer activity. The crystal structure of the alpha-subunit of KP6 (KP6alpha) has been determined at 1.80-A resolution. KP6alpha forms a single domain structure that has an overall shape of an ellipsoid with dimensions 40 A x 26 A x 21 A and belongs to the alpha/beta-sandwich family. The tertiary structure consists of a four-stranded antiparallel beta-sheet, a pair of antiparallel alpha-helices, a short strand along one edge of the sheet, and a short N-terminal helix. Although the fold is reminiscent of toxins of similar size, the topology of KP6alpha is distinctly different in that the alpha/beta-sandwich motif has two right-handed betaalphabeta split crossovers. Monomers of KP6alpha assemble through crystallographic symmetries, forming a hexamer with a central pore lined by hydrophobic N-terminal helices. The central pore could play an important role in the mechanism of the killing action of the toxin. (+info)
Environmental signals controlling sexual development of the corn Smut fungus Ustilago maydis through the transcriptional regulator Prf1.
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Environmental signals induce and coordinate discrete morphological transitions during sexual development of Ustilago maydis. In this fungus, mating of two compatible haploid sporidia is a prerequisite for plant infection. Cell fusion is governed by the action of pheromones and receptors, whereas the subsequent pathogenicity program is controlled by the combinatorial interaction of homeodomain proteins. The U. maydis pheromone response factor (Prf1) is a central regulator of both processes. We have analyzed the regulation of the prf1 gene and demonstrate that pheromone and cAMP signaling regulate prf1 post-transcriptionally. Transcriptional activation of prf1 was observed in the presence of carbon sources, such as glucose and fructose, allowing us to define the cis-acting element in the prf1 promoter that mediates these effects. The same element provides for negative control of prf1 gene transcription at high cAMP levels. A protein that specifically binds to this element was purified and analyzed for its role in prf1 gene regulation. On the basis of these results, we present a model in which prf1 integrates different environmental signals to control development in U. maydis. (+info)
Characterization of a Ustilago maydis gene specifically induced during the biotrophic phase: evidence for negative as well as positive regulation.
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The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle. To investigate the molecular events during infection, we used differential display to identify plant-induced U. maydis genes. We describe the U. maydis gene mig1 (for "maize-induced gene"), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection. mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development. Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication. mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP. Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation. Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation. During fungal growth in planta, sequence elements between positions -148 and -519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control. We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes. (+info)
The mating-type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region.
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The fungal pathogen Ustilago hordei causes the covered smut disease of barley and oats. Mating and pathogenicity in this fungus are controlled by the MAT locus, which contains two distinct gene complexes, a and b. In this study, we tagged the a and b regions with the recognition sequence for the restriction enzyme I-SceI and determined that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain. Characterization of the organization of the known genes within the a and b gene complexes provided evidence for nonhomology and sequence inversion between MAT-1 and MAT-2. Antibiotic-resistance markers also were used to tag the a gene complex in MAT-1 strains (phleomycin) and the b gene complex in MAT-2 strains (hygromycin). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a very low frequency, suggesting that recombination is suppressed within the MAT region. Overall, the chromosome homologues carrying the MAT locus of U. hordei share features with primitive sex chromosomes, with the added twist that the MAT locus also controls pathogenicity. (+info)
Inheritance of killer phenotypes and double-stranded RNA in Ustilago maydis.
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Three different killer specificities in U. maydis are inherited cytoplasmically and transmitted by cell fusion. Each killer generates low frequencies of specifically immune forms in crosses with sensitive strains. The properties of immunity to each killer are also inherited cytoplasmically and transmitted by cell fusion. Killer strains carry virus-like particles about 41 nm in diameter. Each killer possesses distinct double-stranded RNA components that range in molecular weight from 0.46 X 10(6) to 2.9 X 10(6). Two components are shared by all three killers. Immune strains possess new forms. Crosses and heterokaryons between different killers revealed unilateral or mutual restrictions that prevent inclusion of two killer specificities in the same cell. (+info)
A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.
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We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis. (+info)
The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth.
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Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis. (+info)
Reversible and permanent effects of the carbon sources and various antibiotics on the morphology and metabolic properties of Ustilago cynodontis cells.
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The effects of various carbon sources and of antibiotics on the morphology of hypha cells of the fungus Ustilago cynodontis is described. Nonfermentable substrates promote readily reversible yeastlike colonies from hypha cells: all the hypha cells spread on these substrates give rise to yeastlike colonies that revert to the mycelial phenotype when transferred to glucose medium. Among the antibiotics tested, chloramphenicol (CAP) is found to promote, under certain circumstances, a long-lasting, even permanent modification on the morphology of the colonies: the colonies developed on CAP-glucose media are yeastlike, and a percentage of them give rise to colonies whose morphology remains yeastlike even on drug-free media: this effect is also obtained with cells cultivated in liquid medium. This permanent morphological modification is accompanied by a change of metabolic properties. Similar permanent effects are obtained with ethidium bromide, suggesting that mitochondrial functioning is involved in these modifications. (+info)