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.     

The Pheromone Cell Signaling Components of the Ustilago a Mating-Type Loci Determine Intercompatibility between Species

The MAT region of Ustilago hordei, a bipolar barley pathogen, harbors distinct mating functions (a and b loci). Here, we show that the b locus is essential for mating and pathogenicity, and can induce pathogenicity when introduced into a strain carrying a b locus of opposite specificity. Transformation experiments using components of the a1 locus and analysis of resulting dual mating phenotypes revealed that this locus harbors a pheromone receptor gene (Uhpra1) and a pheromone gene (Uhmfa1). These U. hordei a1 genes, when introduced by transformation, are necessary and sufficient to make U. maydis, a tetrapolar corn pathogen, intercompatible with U. hordei MAT-2, but not MAT-1, strains. U. hordei strains transformed with the U. maydis a1 locus also become intercompatible with U. maydis a2, but not a1, strains. The interspecies hybrids produced dikaryotic hyphae but were not fully virulent on either corn or barley. Partial, natural intercompatibility was shown to exist between the sugarcane smut U. scitaminea and both U. hordei and U. maydis. These results show that the signal transduction pathway for mating responses is conserved between different smut species. We conclude that, apart from intraspecies compatibility, the Ustilago a locus also dictates intercompatibility in this group of fungi.     

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.     

Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti

Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.     

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.     

The Gbeta-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis, fusion of haploid cells is a prerequisite for infection. This process is controlled by a pheromone-receptor system. The receptors belong to the seven-transmembrane class that are coupled to heterotrimeric G proteins. Of four Galpha subunits in U. maydis, only gpa3 has a function during mating and cyclic AMP (cAMP) signaling. Activation of the cAMP cascade induces pheromone gene expression; however, it does not lead to the induction of conjugation tubes seen after pheromone stimulation. To investigate the possibility that a Gbeta subunit participates in pheromone signaling, we isolated the single beta subunit gene, bpp1, from U. maydis. bpp1 deletion mutants grew filamentously and showed attenuated pheromone gene expression, phenotypes associated with deltagpa3 strains. In addition, a constitutively active allele of gpa3 suppressed the phenotype of the bpp1 deletion strains. We suggest that Bpp1 and Gpa3 are components of the same heterotrimeric G protein acting on adenylyl cyclase. Interestingly, while deltagpa3 strains are impaired in pathogenicity, deltabpp1 mutants are able to induce plant tumors. This could indicate that Gpa3 operates independently of Bpp1 during pathogenic development.     

Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis

In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G₂ phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.     

Methionineless Death in Escherichia coli

Methionine auxotrophs of strains derived from Escherichia coli 15 lose their colony-forming ability when deprived of this amino acid. Late addition of methionine to liquid cultures did not restore plating efficiency but permitted growth of surviving cells. This phenomenon, termed methionineless death (mld), was not observed with methionine auxotrophs of E. coli strains B, W, or K(12), nor was a similar amino acidless death observed with corresponding auxotrophs of E. coli 15 for arginine, tryptophan, proline, isoleucine, and leucine. Mld was not dependent upon the genetic site determining methionine auxotrophy, nor did it affect the decarboxylation of methionine or the stability of methionyl-transfer ribonucleic acid synthetase activity of starved cells. Death was not altered by the presence of spermine or spermidine but was abolished by the methionine analogue, alpha-methylmethionine. Simultaneous starvation of another amino acid in a multiple auxotroph also significantly reduced mld, suggesting a possible role of protein synthesis. The onset of mld is correlated with a lower net increase of deoxyribonucleic acid.     

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.     

Identification and Functional Analysis of Pheromone and Receptor Genes in the B3 Mating Locus of Pleurotus eryngii

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.     

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 α-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.     

Is it possible to isolate methionine auxotrophs in Chlamydomonas reinhardtii?

Summary In order to address the problem of amino acid auxotroph scarcities in photosynthetic organisms, an attempt was made to recover methionine auxotrophs in a unicellular green alga, Chlamydomonas reinhardtii. Evidence of methionine permeation into the algal cells was provided by the successful competition of the amino acid with its antimetabolite, methionine sulfoximine. No methionine auxotrophs were isolated despite the frequent recovery of both nicotinamide and arginine auxotrophs, selected as controls, and of methionine sensitive mutants. The use of either N-methyl-N-nitro-N-nitrosoguanidine or ultraviolet light as mutagens appeared not to alter the mutation spectrum. Medium containing methionine was shown to be inhibitory to the growth of cells in culture under fluorescent light and this toxicity was further stimulated in the presence of riboflavin. In view of our results and the observations of other studies, the non-recoverability of methionine auxotrophs is discussed as a function of the photodynamic action of methionine.     

Physical-chemical plant-derived signals induce differentiation in Ustilago maydis

Ustilago maydis is able to initiate pathogenic development after fusion of two haploid cells with different mating type. On the maize leaf surface, the resulting dikaryon switches to filamentous growth, differentiates appressoria and penetrates the host. Here, we report on the plant signals required for filament formation and appressorium development in U. maydis. In vitro , hydroxy-fatty acids stimulate filament formation via the induction of pheromone genes and this signal can be bypassed by genetically activating the downstream MAP kinase module. Hydrophobicity also induces filaments and these resemble the dikaryotic filaments formed on the plant surface. With the help of a marker gene that is specifically expressed in the tip cell of those hyphae that have formed an appressorium, hydrophobicity is shown to be essential for appressorium development in vitro . Hydroxy-fatty acids or a cutin monomer mixture isolated from maize leaves have a stimulatory role when a hydrophobic surface is provided. Our results suggest that the early phase of communication between U. maydis and its host plant is governed by two different stimuli.     

Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.