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.

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.

Ustilago maydis Mating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bolker, M., and Kahmann, R. 1996. Ustilago maydis mating hyphae orient their growth toward pheromone sources. Fungal Genetics and Biology 20, 299-312. When small drops of Ustilago maydis sporidia were placed 100-200 µm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example, a2b2 mating hyphae grew toward a1b1 and a1b2 mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with the a2 allele began elongating before sporidia with the a1 allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous at b but not at a formed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, the a2a2b1b2 strain formed filaments more quickly than the a1a1b1b2 strain. Taken together, these results suggest that the a2 pheromone diffuses less readily or is degraded more quickly than the a1 pheromone.

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.

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.

The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter.

Expression of the phage Mu mom gene is transcriptionally regulated by DNA methylation. Three GATC sites upstream of the mom promoter have to be methylated by the Escherichia coli deoxyadenosine methylase (Dam) to allow initiation of transcription. An E. coli dam strain was mutagenized with Tn5 in an attempt to isolate mutants which allow mom gene expression. Three independent Tn5 mutants were isolated, each mapped to a gene at 89.6 min which we designate momR. The wildtype gene was cloned and sequenced, it encodes a protein of 305 amino acids. The protein belongs to a group of related bacterial activators recently identified as the LysR family (Henikoff et al., 1988). MomR protein was overproduced and purified. Expression of momR is autoregulated; MomR binds to a 43 bp region upstream of its coding sequence. In the mom promoter MomR protects a 43 bp region containing the three GATC sites. Specific binding to these sequences was observed only with unmethylated DNA. Fortuitously, we learned that MomR is identical to OxyR, a regulatory protein responding to oxidative stress. We discuss the implications of this control for Mu development.

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.

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.

The a mating type locus of U. maydis specifies cell signaling components.

The a mating type locus of the phytopathogenic fungus U. maydis controls fusion of haploid cells and filamentous growth of the dikaryotic mycelium. The a locus exists in two alleles, termed a1 and a2, which are defined by nonhomologous DNA regions comprising 4.5 kb for a1 and 8 kb for a2, flanked by identical sequences. Based on functional assays, mutants, and sequencing, we demonstrate that the mating type in each allele is determined by a set of two genes. One encodes a precursor for a lipopeptide mating factor, and the other specifies the receptor for the pheromone secreted by cells of opposite mating type. Thus, U. maydis employs a novel strategy to determine its mating type by providing the primary determinants of cell-cell recognition directly from the mating type locus.

Translation of the bacteriophage Mu mom gene is positively regulated by the phage com gene product.

Expression of the bacteriophage Mu mom gene is subject to posttranscriptional regulation by the phage com gene product. We have used mom-lacZ translational fusion genes to define the sequence requirements for stimulation of mom expression by Com. We show that the mom translation initiation region (TIR) is inactive in the absence of Com. We suggest that this repressed state is due to mRNA secondary structure in the TIR, since a deletion that destabilizes a stem-loop structure in the TIR results in high levels of Com-independent translation. We identify sequences on the mRNA, adjacent to the stem and loop, that are required for stimulation by Com. We propose that Com acts to stimulate initiation of translation by relieving the structural repression of the mom TIR. Indirect evidence is presented suggesting that Com binds to a site in the TIR.

Role of bacteriophage Mu C protein in activation of the mom gene promoter.

The phage Mu C gene product is a specific activator of Mu late gene transcription, including activation of the mom operon. Fusion of the C gene to the efficient translation initiation region of the Escherichia coli atpE gene allowed significant overproduction of C protein, which was subsequently purified and assayed for DNA binding by gel retardation and nuclease footprinting techniques. C protein binds to a site immediately upstream of the -35 region both of the mom promoter and the related phage D108 mod promoter. The location of the mom promoter has been determined by primer extension. Upstream deletions extending more than 3 base pairs into the C-binding site abolished activation of the mom promoter in vivo. In vitro binding of C was not significantly affected by DNA methylation. A second, C-dependent promoter was identified just downstream of the C coding region; comparison with the mom promoter revealed common structural elements.

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.

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.

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.

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.

A two-component regulatory system for self/non-self recognition in Ustilago maydis.

In U. maydis the multiallelic b locus controls sexual and pathogenic development. In the b locus a gene coding for a regulatory protein had been identified, and it was suggested that the interaction of two b polypeptides specified by different alleles programs sexual development in this fungus. We now demonstrate the existence of a second regulatory gene in the b locus. We term this gene bW and refer to the former as the bE gene. Both genes exist in many alleles. Although unrelated in primary sequence, both genes are similar in their overall organization. The gene products display allele-specific variability in their N-terminal domains, show a high degree of sequence conservation in the C-terminal domains, and contain a homeodomain-related motif. Genetic evidence is provided to show that the pair of bE and bW polypeptides encoded by different b alleles is the key regulatory species.

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.

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.

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.