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M Bölker

Publications and source records attributed to M Bölker.

At least 19 recordsLinked to original sources
PubMed2004

A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (< C4) and produces H2O2. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.

Alcohol Oxidoreductases↗
PubMed2000

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.

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.

Amino Acid Sequence↗
PubMed1998

Sex and crime: heterotrimeric G proteins in fungal mating and pathogenesis.

Heterotrimeric G proteins act as signal transducers that couple cell-surface receptors to cytoplasmic effector proteins. In fungi, G proteins play essential roles during sexual and pathogenic development. They are part of the pheromone signaling cascade in both ascomycetes and basidiomycetes, which is crucial for the recognition and fusion of cells of opposite mating type. In addition, G proteins affect a number of developmental and morphogenetic processes which determine the virulence of plant and human fungal pathogens. Cloning and targeted disruption of genes encoding alpha subunits of G proteins allowed the attribution of specific functions to these signal transducing molecules. Several lines of evidence indicate that many of the known fungal G proteins influence the intracellular level of cAMP by either stimulating or inhibiting adenylyl cyclase.

Fungal Proteins↗
PubMed1998

Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration.

A gene encoding the heavy chain of conventional kinesin (kin2) has recently been identified in the dimorphic fungus Ustilago maydis (Lehmler et al., 1997). From the phenotype of kin2 null-mutants it was concluded that Kin2 might be involved in vesicle traffic towards the tip. However, this model did not explain why kin2-null mutant hyphae were unable to create empty cell compartments that are normally left behind the growing tip cell. Here we present a re-investigation of the function of Kin2 in hyphae and sporidia. We provide evidence that suggests a different and unexpected role of this kinesin motor in hyphal growth of Ustilago maydis. In addition, Kin2 was partially purified from U. maydis and in vitro properties were investigated. Isolated kinesin supported in vitro microtubule gliding at speeds of up to 1.8 micron/second, and showed motility properties and hydrodynamic behavior similar to those described for kinesin from N. crassa. It appears to be the product of the kin2 gene. Compared with wild-type sporidia, the kin2-null mutant sporidia grew normally but were defective in accumulation of Lucifer Yellow in their vacuoles, which were smaller than normal and often misplaced. The dikaryotic hyphae, produced by the fusion of two kin2-null sporidia, showed tip growth, but unlike wild-type hyphae, these structures lacked the large, basal vacuole and contain significantly more 200-400 nm vesicles scattered over the hole hypha. This defect was accompanied by a failure to generate regular empty cell compartments that are left behind in wild-type tip cells as the hyphae grow longer. These results suggest that Kin2 is a microtubule-dependent motor enzyme which is involved in the formation of vacuoles. The accumulation of these vacuoles at the basal end of the tip cell might be crucial for the formation of the empty sections and supports cytoplasmic migration during the growth of dikaryotic hyphae.

Cytoplasm↗
PubMed1997

Identification of a motor protein required for filamentous growth in Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; delta kin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In delta kin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)-Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.

Amino Acid Sequence↗
PubMed1997

G proteins in Ustilago maydis: transmission of multiple signals?

In the phytopathogenic fungus Ustilago maydis, cell fusion is governed by a pheromone signalling system. The pheromone receptors belong to the seven transmembrane class that are coupled to heterotrimeric G proteins. We have isolated four genes (gpa1 to gpa4) encoding alpha subunits of G proteins. Gpa1, Gpa2 and Gpa3 have homologues in other fungal species, while Gpa4 is novel. Null mutants in individual genes were viable and only disruption of gpa3 caused a discernible phenotype. gpa3 mutant strains were unable to respond to pheromone and thus were mating-deficient. A constitutively active allele of gpa3 (gpa3(Q206L)) was generated by site-directed mutagenesis. Haploid strains harbouring gpa3(Q206L) were able to mate without pheromone stimulation, indicating that Gpa3 plays an active role in transmission of the pheromone signal. Surprisingly, Gpa3 is also required for pathogenic development, although pheromone signalling is not essential for this process.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential.

Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and alpha-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.

Amino Acid Sequence↗
PubMed1996

A novel class of small amphipathic peptides affect aerial hyphal growth and surface hydrophobicity in Ustilago maydis.

Ustilago maydis, a fungal pathogen of corn, can alternate between yeast-like and filamentous growth. This dimorphic switch is governed by the mating-type loci. We have identified an abundant class of small SDS-insoluble cell wall proteins, designated repellents, specifically present in the filamentous form. Genetic analysis revealed that these peptides are processed from a single precursor protein, Rep1. Rep1 comprises 652 amino acids with a leader sequence for secretion. A characteristic feature of Rep1 is 12 repeats of a 37 amino acid consensus sequence; 10 of these repeats are separated by Kex2 protease cleavage sites. In (delta)rep1 mutants formation of aerial hyphae and surface hydrophobicity were reduced dramatically. This and the fact that expression of rep1 is regulated by the mating-type loci indicates that repellents play a structural role in the formation of aerial hyphae.

Amino Acid Sequence↗
PubMed1996

Identification of the pheromone response element in Ustilago maydis.

The a mating type locus of Ustilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. Binding of pheromone to its cognate receptor includes mating competence in haploid cells and stimulates filamentous growth of the dikaryon. We have analyzed transcription of genes located in the a locus and demonstrate that all genes are induced by pheromone. Transcriptional stimulation is mediated by a 9 bp DNA element (ACAAAGGGA) that occurs in multiple copies in both alleles of the a locus. By fusing multimers containing this 9 bp sequence to the pheromone gene promoter and to a heterologous promoter we demonstrate that this sequence acts as a pheromone response element. In addition, we show that expression of the b genes, which regulate pathogenic development of the dikaryon, is also stimulated by pheromone. Pheromone-inducible genes can be divided into three classes depending on whether their expression is reduced, maintained, or increased after cell fusion. These differences may suggest some regulatory cross-talk between the a and b loci.

Alleles↗
PubMed1996

The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis.

In Ustilago maydis, the a and b mating type loci regulate cell fusion, filamentous growth and pathogenicity. The a locus encodes a pheromone-based cell recognition system, and the b locus specifies two homeodomain proteins. The expression of all genes in the a and b loci is induced by pheromone. We have identified a HMG protein (Prf1) that binds sequence specifically to pheromone response elements present in the a and b loci. prf1 mutants do not express the a and b genes and are sterile. The disruption of prf1 in pathogenic haploid strains results in a loss of pathogenicity. The constitutive expression of the b genes restores pathogenicity and induces filamentous growth in the absence of the pheromone signal. These results provide evidence that pheromone signalling, filamentous growth and pathogenic development are linked through Prf1.

Amino Acid Sequence↗
PubMed1996

The biallelic a mating type locus of Ustilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor.

The a mating type locus of Ustilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termed a1 and a2. We have completed the analysis of the nucleotide sequences unique to a1 and a2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of the a1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in the a2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in the a2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.

Alleles↗
PubMed1995

Tagging pathogenicity genes in Ustilago maydis by restriction enzyme-mediated integration (REMI).

In the maize pathogenic fungus Ustilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a single BamHI site is integrated at chromosomal BamH1 sites, if BamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially at BamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1-2% of these mutants were unable to induce symptoms when tested in planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.

Base Sequence↗
PubMed1995

Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis.

In the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides. Only combinations of bE and bW of different allelic origin are active. We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon. By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles. Dimerization involves the N-terminal variable domains. Different point mutants of bE2 were isolated that function in combination with bW2. The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system. Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins. Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization.

Alleles↗
PubMed1995

Control of mating and development in Ustilago maydis.

In the fungus Ustilago maydis, the ability to distinguish between partners that are of the same or of different mating type is controlled by two mating-type loci. One locus allows extracellular recognition though a pheromone-based system. After cell fusion, the other mating-type locus, which exists in multiple alleles, determines intracellular recognition. Each allele encodes a pair of homeodomain proteins that are active only in pairwise combinations in which the two partners originate from different alleles of the locus. Recent discoveries suggest that the underlying molecular recognition mechanism is the ability to form heterodimers. Whereas the proteins in all different allelic combinations interact, it is a specific feature of proteins from the same allele not to interact. This suggests the existence of a code for protein-protein recognition.

Alleles↗
PubMed1994

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.

Amino Acid Sequence↗
PubMed1994

Control of mating and development in Ustilago maydis.

In Ustilago maydis the a and b mating type loci control pathogenicity as well as sexual development. We review the function of these loci in controlling the cell fusion step, the switch from yeast-like to filamentous growth and subsequent pathogenic development. Our special emphasis will be the role of pheromones and pheromone signaling in these processes.

Alleles↗