Comparative studies on lipid components of Ustilago bullata and Ustilago maydis spores.
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Basidiomycota↗
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Anaerobic disinfection of seed against Ustilago nuda (Jens.) Rostr. and Ustilago tritici (Pers.) Rostr. According to a new method and with a new equipment.
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Basidiomycota↗
Haem ligands of the ferricytochrome c of Ustilago sphaerogena.
The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH-cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14-21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N(2)-acetylhistidine and monocarboxymethyl derivatives of N(2)-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N(2)-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6-8.4.
Acetates↗
Mutation in a heat-regulated hsp70 gene of Ustilago maydis.
Four genes (ums1, ums2, ums3 and ums4) representing an hsp70-related gene family were isolated from a genomic library of Ustilago maydis. All four genes are transcriptionally active during normal growth. Following a heat shock, the mRNA levels of ums1 and ums2 increase by approximately 5-fold, whereas the ums3 transcript becomes less abundant. The amount of ums4 mRNA remains relatively unchanged after heat treatment. The nucleotide sequence of the 5' non-coding and a portion of the ums2 coding region was determined. The sequence encoding the first 90 amino acids is 73% identical to corresponding regions of the Drosophila and yeast (SSA1) hsp70 genes. To investigate the effect of a mutation in ums2, a plasmid was constructed in which most of the transcriptional unit of ums2 was deleted and substituted with the Escherichia coli hygromycin B (hygB) phosphotransferase gene. Transcription of this gene is controlled by the ums2 promoter, allowing the expression of hygB resistance in Ustilago. The marker was introduced into diploid cells as a linear sequence with termini homologous to the 5' and 3' flanking regions of ums2. In approximately 50% of transformants examined, one of the two wild-type ums2 alleles had been replaced by the mutated sequence, demonstrating the feasibility of using one-step gene disruption to create heterozygous diploids in Ustilago. The ums2/ums2::hygBr heterozygote produced teliospores after injection into corn plants, but only cells carrying functional ums2 were found among their meiotic progeny. Therefore ums2::hygBr segregates as a recessive lethal, which strongly suggests that ums2 is essential for growth in Ustilago.
Amino Acid Sequence↗
Characterization of Ustilago maydis DNA binding protein one (UBP1).
The DNA binding properties of a protein from the lower eukaryote Ustilago maydis have been characterized. Using both filter binding and gel retention assays, we demonstrate that this protein, termed UBP1 (Ustilago binding protein one), binds preferentially to DNA molecules lacking chain interruptions. The introduction of DNA breaks by a restriction enzyme or a purified nuclease, from Ustilago maydis, causes the dissociation of protein-DNA complexes. UBP1 stimulates the relaxation of negatively supercoiled DNA, mediated by Ustilago type I topoisomerase, through a mechanism most likely involving the association of UBP1 with the DNA rather than with the topoisomerase. The prebinding of UBP1 to DNA templates, subsequently assembled into minichromosomes, results in the development of a disorganized nucleosomal array. Possible roles for UBP1 in processes that involve changes in DNA topology, such as chromatin assembly, are discussed.
DNA↗
The isolation of a Dol-P-Man synthase from Ustilago maydis that functions in Saccharomyces cerevisiae.
Genomic DNAs from several fungi were screened for a homologous sequence to Saccharomyces cerevisiae DPM1, an essential gene which encodes dolichyl phosphoryl mannose synthase. The fungi examined included Aspergillus nidulans, Neurospora crassa, Schizophyllum commune and Ustilago maydis. Only U. maydis gave a significant signal after Southern hybridization using DPM1 as a probe. The Ustilago homolog was subsequently cloned and sequenced. The predicted protein of 294 amino acids has 60% identity to the S. cerevisiae protein, but lacks the putative "dolichol recognition sequence'. RNA of ca. 900 bp is transcribed in both yeast and filamentous cells of Ustilago. In Escherichia coli, the U. maydis sequence expressed a 35 kDa protein exhibiting dolichyl phosphoryl mannose synthase activity. The sequence was also shown to complement a haploid strain of S. cerevisiae containing a deletion of the DPM1 gene. The U. maydis sequence therefore, encodes a dolichyl phosphoryl mannose synthase that can support normal vegetative growth in S. cerevisiae.
Amino Acid Sequence↗
DNA relaxation mediated by Ustilago maydis type I topoisomerase; modulation by chromatin associated proteins.
Ustilago maydis topoisomerase I relaxes superhelical DNA in the absence of any co-factors. The reaction reaches a defined end-point proportional to the amount of enzyme added and an analysis of the reaction by Hill plot transformation indicates that at least two molecules of topoisomerase must interact with the DNA to catalyze relaxation. The addition of purified Ustilago histone H1 reduces the stoichiometric amount of topoisomerase I required by 50%. H1 histone may function to enhance DNA relaxation through a cooperative mechanism. The purified HMG-like protein from Ustilago also enhances DNA relaxation mediated by the topoisomerase. Whereas H1 stimulates topo I-mediated DNA relaxation through a processive mode, the HMG-like protein enhances through a distributive mechanism. Taken together, these results demonstrate that the interaction of chromosomal proteins with topoisomerase can influence DNA topology, and mechanisms are proposed to explain this enhancement.
DNA Topoisomerases, Type I↗
The primary structure of cytochrome c from the rust fungus Ustilago sphaerogena.
1. The complete amino acid sequence of cytochrome c from the basidiomycete Ustilago sphaerogena was determined from the amino acid compositions and sequences of either tryptic or chymotryptic peptides, and in homology with at least thirty other established sequences of cytochrome c. 2. The primary structure of the molecule bears all of the characteristics of a mammalian-type cytochrome c, showing the typical clustered distribution of hydrophobic and basic residues with a single polypeptide chain of 107 residues. 3. Like all other fungal cytochromes c, it possesses a free N-terminus, and one less residue at the C-terminus than vertebrate cytochromes c. The region of residues 70-80 is strictly conserved, as is histidine at position 18. Position 26 is occupied by an asparagine residue, in contrast to histidine which occurs at this location in most of the known sequences of mammalian-type cytochromes c. 4. In contrast to some other fungal and plant cytochromes c of known primary structures, the Ustilago cytochrome c molecule does not contain trimethyl-lysine. 5. The sequence of Ustilago cytochrome c differs from the sequences of human, horse, chicken, tuna, wheat, and baker's yeast proteins at loci 47, 43, 44, 44 and 38 respectively.
Amino Acid Sequence↗
The killer phenomenon in Ustilago: electron microscopy of the dsRNA encapsidated in individual virus particles.
From earlier studies with the Ustilago maydis virus and other dsRNA viruses it is known that discrete dsRNA segments typical of each virus are obtained by extraction. A variation exists with respect to the encapsidation of these segments among different viruses. The encapsidation of the genome in individual particles of the Ustilago virus was examined by electron microscopy after disruption of virus particles. The study included the P6 wild-type and 2 mutants containing only part of the genome. The results indicate that most virus particles of the wild-type and the mutants contain up to 12-14 X 10(6) daltons of dsRNA. Since the largest extracted molecule is 3.2 X 10(6) D these findings suggest that an individual particle may contain more than one segment of dsRNA. Free linear molecules that exceed in size the extracted segments were also found following the disruption of each of the 3 virus types examined. Thus, the viral genome seen segmented after extraction is organized as a concatamer in the capsid and each virus particle can contain an entire viral genome consisting of each type of the segments seen after extraction, a repeat of a single segment or a random assortment. In each case the information may be organized as a concatamer.
Basidiomycota↗
Homologous pairing of DNA molecules promoted by a protein from Ustilago.
A protein from mitotic cells of Ustilago maydis was purified on the basis of its ability to reanneal complementary single strands of DNA. The protein catalyzed the uptake of linear single strands by super-helical DNA, but only in reactions with homologous combinations of single-strand fragments and super-helical DNA from phages phi X174 and fd. No reaction occurred with heterologous combinations. The protein also efficiently paired circular single strands and linear duplex DNA molecules. The product was a joint molecule in which the circular single strand displaced one strand of the duplex. Efficient pairing depended upon ATP, and ATPase activity was found associated with the purified protein. ATP-dependent reannealing of complementary single strands was not detectable in the rec1 mutant of Ustilago, which is deranged in meiotic recombination, as complete tetrads are rare, and is defective in radiation-induced mitotic gene conversion.
Adenosine Triphosphatases↗
Three selectable markers for transformation of Ustilago maydis.
Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.
Acetyltransferases↗
The effect of some organic substances on the mycelium of the fungus Ustilago nuda (Jens.) Rostr.
Research was performed for studying the effect of some organic compounds, considered by many authors as the products ob barley seed metabolism generated after anaerobic seed treatment, on the mycelium of the fungus Ustilago nuda (Jens.) Rostr. The author examined the effectiveness of ethylacohol, acetaldehyde, acetic acid, succinic acid, lactic acid, and hydroquinone in concentrations from 1 M to 10(-6) M, and the effectiveness of extracts from disinfected seeds in doses from 10 g to 0.001 g/l. The effect of the mentioned solutions was examined as exerted on the growth of dicaryotic mycelium and on the growth of the haploid promycelium of the fungus. The dicaryotic mycelium of Ustilago nuda (Jens.) Rostr. was cultivated on potato agar with benzoic acid. The presence of the acid prevents mitosis, and the chlamydospores germinate on the nutritive medium with two fibres having binuclear cells. The haploid promycelium was cultivated on potato agar; chlamydospores germinated with one four-cell fibre, and individual cells are mononuclear and haploid. Only later, a dicarytic mycelium is created in a complex process. In all the substances used, the concentration of 1 M was found to stop further growth of mycelium. The concentration of 10(-1) M of acetic acid and hydroquinone also stopped growth, the same concentration of acetaldehyde, lactic acid, succinic acid, ethylacohol stimulated mycelium growth in comparison with the control. The concentration of 10(-6) M stimulated mycelium growth in a majority of cases. Extracts from disinfected seeds did not influence mycelium growth significantly in all cases in comparison with the control. The results were similar in the two types of mycelium.
Acetaldehyde↗
Castles and cuitlacoche: the first international Ustilago conference.
The first international Ustilago conference was held in Marburg, Germany from August 22 to 25, 2002. The meeting focused on molecular genetic and cell biology research with Ustilago maydis, the causative agent of common smut of maize. This fungus has emerged as a useful experimental organism for studying the biology of basidiomycete fungi, with a particular emphasis on the interaction of the fungus with the host plant. Thus presentations at the meeting covered the range of current research topics including DNA recombination and repair, mating and sexual development, phytopathology, cell biology, the cell cycle, signaling, and genomics. The meeting also highlighted historical aspects of U. maydis research with presentations by pioneers in the field including Robin Holiday (recombination), Yigal Koltin (killer phenomenon) and Peter Day (plant pathology).
Crosses, Genetic↗
Topoisomerase from Ustilago maydis forms a covalent complex with single-stranded DNA through a phosphodiester bond to tyrosine.
Highly purified topoisomerase from Ustilago breaks single-stranded DNA, forming a complex with protein covalently bound to the DNA. Methods used to detect the complexes include a nitrocellulose filter assay, electrophoresis of the DNA-protein complex in agarose gels containing alkali, and isolation of the complex after removal of all but a small oligonucleotide fragment bound to the protein. The linkage of the Ustilago topoisomerase is to the 3' end of the broken strand of DNA. The DNA-protein complex formed is through a phosphodiester bond to tyrosine.
Basidiomycota↗
A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence.
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. Previously, 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 cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.
Amino Acid Sequence↗
The ubc2 gene of Ustilago maydis encodes a putative novel adaptor protein required for filamentous growth, pheromone response and virulence.
The Basidiomycete fungus Ustilago maydis causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilago bypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated by complementation and is required for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and Src homology 3 (SH3) protein-protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone-responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone-responsive MAP kinase cascade and ubc2 is required for pheromone-responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus, ubc2 encodes a putative novel adaptor protein that may act directly upstream of the pheromone-responsive MAP kinase cascade in U. maydis.
Adaptor Proteins, Signal Transducing↗
A maize-specifically expressed gene cluster in Ustilago maydis.
The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual cycle. We have identified the fungal mig2-1 gene as being specifically expressed during this biotrophic stage. Intriguingly, mig2-1 is part of a gene cluster comprising five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. Deletion analysis of the mig2-1 promoter provides evidence for negative and positive regulation. The predicted polypeptides of all five genes lack significant homologies to known genes but have characteristic N-terminal secretion sequences. The secretion signals of mig2-1 and mig2-5 were shown to be functional, and secretion of a full length Mig2-1-eGFP fusion protein to the extracellular space was demonstrated. The central domains of the Mig2 proteins are highly variable whereas the C-termini are strongly conserved and share a characteristic pattern of eight cysteine residues. The mig2 gene cluster was conserved in a wide collection of U. maydis strains. Interestingly, some U. maydis isolates from South America had lost the mig2-4 gene as a result of a homologous recombination event. Furthermore, the related Ustilago scitaminea strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster. We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.
Amino Acid Sequence↗
The crk1 gene encodes an Ime2-related protein that is required for morphogenesis in the plant pathogen Ustilago maydis.
The fungal pathogen Ustilago maydis alternates between budding and filamentous growth during its life cycle. This dimorphic transition is regulated by environmental factors and mating. We cloned a new gene, crk1, which encodes a protein with sequence similarity to Ime2, a kinase involved in developmental choices in S. cerevisiae. Disruption of the crk1 gene in U. maydis resulted in cells that are unable to respond in an appropriate manner to environmental stimuli and show defects in morphogenesis and cell cycle adjustment to changing conditions. We have analysed the regulation of the crk1 gene and demonstrated that cAMP and MAPK pathways have opposite influences on the transcript levels for crk1. Furthermore, we have shown that alterations in the components of these pathways impair the ability of the cellular machinery to adapt to changing conditions. These results demonstrate an important role for the crk1- encoded protein in the morphogenesis and environmental adaptation in Ustilago maydis.
Amino Acid Sequence↗