A filamentous growth response mediated by the yeast mating pathway.

Haploid cells of the budding yeast Saccharomyces cerevisiae respond to mating pheromones by arresting their cell-division cycle in G1 and differentiating into a cell type capable of locating and fusing with mating partners. Yeast cells undergo chemotactic cell surface growth when pheromones are present above a threshold level for morphogenesis; however, the morphogenetic responses of cells to levels of pheromone below this threshold have not been systematically explored. Here we show that MATa haploid cells exposed to low levels of the alpha-factor mating pheromone undergo a novel cellular response: cells modulate their division patterns and cell shape, forming colonies composed of filamentous chains of cells. Time-lapse analysis of filament formation shows that its dynamics are distinct from that of pseudohyphal growth; during pheromone-induced filament formation, daughter cells are delayed relative to mother cells with respect to the timing of bud emergence. Filament formation requires the RSR1(BUD1), BUD8, SLK1/BCK1, and SPA2 genes and many elements of the STE11/STE7 MAP kinase pathway; this response is also independent of FAR1, a gene involved in orienting cell polarization during the mating response. We suggest that mating yeast cells undergo a complex response to low levels of pheromone that may enhance the ability of cells to search for mating partners through the modification of cell shape and alteration of cell-division patterns.     

The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.     

Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.     

Structure-function analysis of lipopeptide pheromones from the plant pathogen Ustilago maydis

Mating of two haploid cells is a prerequisite for the successful infection of corn by the pathogenic fungus Ustilago maydis. Cell-cell recognition is mediated by small lipopeptide pheromones. Genes encoding pheromone precursors as well as pheromone receptors are located in the a mating type locus. Two pheromones are known, the tridecapeptide a1 and the nonapeptide a2, both of which contain an S-prenylated cysteine methyl ester at the C-terminus. It has previously been shown that synthetic pheromones are active in a biological test system. Here, we used the same assay to perform a detailed analysis of synthetic a1 and a2 pheromones. Testing of truncated derivatives of a1 and a2 revealed that in both cases the pheromone function is less sensitive to N-terminal than to C-terminal truncations. Replacement of each amino acid in the a1 pheromone by either alanine or the corresponding D-amino acids revealed that four positions are important for function: the two central glycines (positions 5 and 9), proline at position 7 and tyrosine at position 10. By introducing different naturally occurring as well as synthetic amino acids at position 10, we demonstrate that the presence of an aromatic side chain at this position is necessary for function. We propose a model in which a cis peptide bond at proline 7 favours the formation of a type II' beta turn of the a1 pheromone backbone with glycine 9 in position i+1 (where i refers to the first position of the beta turn). As a result, tyrosine 10, at position i+2 of the turn, would be highly exposed and could be inserted into a structurally well-defined binding pocket of the receptor. The latter may represent an important facet of receptor specificity.     

Autocrine, mitogenic pheromone receptor loop of the ciliate Euplotes raikovi: pheromone-induced receptor internalization

The ciliate Euplotes raikovi produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone Er-1, it was first observed that the expression of the Er-1 receptor "p15," a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted Er-1 and gradually decreased in parallel with new Er-1 secretion. It was then shown that p15 is internalized through endocytic vesicles following Er-1 binding and that the internalization of p15/Er-1 complexes is specifically blocked by the paracrine p15 binding of Er-2, a pheromone structurally homologous to, and thus capable of fully antagonizing, Er-1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to Er-1 and that Er-1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/Er-1 complexes are internalized as a consequence of their unique property (not shared by p15/Er-2 complexes) of undergoing clustering.     

White Cells Facilitate Opposite- and Same-Sex Mating of Opaque Cells in Candida albicans

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTL a) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.     

Cell aging-induced methionine oxidation causes an autocrine to paracrine shift of the pheromone activity in the protozoan ciliate, Euplotes raikovi

Ciliates of the genus Euplotes rely on the autocrine (self) and paracrine (non-self) activities of their water-borne protein pheromones to control the two fundamental phenomena of their life cycle, i.e. vegetative (mitotic) growth and sex manifested as cell union in mating pairs. We observed that cell aging determines the synthesis of increasing concentrations of pheromones that are oxidized at the level of methionine residues which are more exposed on the molecular surface. The oxidized form of the E. raikovi pheromone Er-1 was purified and its interactions with its source cells were shown no longer to be of autocrine type directed to promote cell growth, but changed to interactions of the paracrine type directed to induce cell unions in mating pairs of the selfing type (i.e. involving genetically identical cells). These pairs generate viable offspring, like pairs formed between genetically different cells. It was therefore concluded that aging cells may paradoxically gain beneficial effects from the synthesis of oxidized forms of their pheromones. By undergoing mating in response to the interactions with these forms, they can re-initiate a new life cycle and, in fact, rejuvenate.     

Pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in Neurospora crassa

Neurospora crassa is a self-sterile filamentous fungus with two mating types, mat A and mat a. Its mating involves chemotropic polarized growth of female-specific hyphae (trichogynes) toward male cells of the opposite mating type in a process involving pheromones and receptors. mat A cells express the ccg-4 pheromone and the pre-1 receptor, while mat a strains produce mRNA for the pheromone mfa-1 and the pre-2 receptor; MFA-1 and CCG-4 are the predicted ligands for PRE-1 and PRE-2, respectively. In this study, we generated Deltaccg-4 and Deltamfa-1 mutants and engineered a mat a strain to coexpress ccg-4 and its receptor, pre-2. As males, Deltaccg-4 mat A and Deltamfa-1 mat a mutants were unable to attract mat a and mat A trichogynes, respectively, and consequently failed to initiate fruiting body (perithecial) development or produce meiotic spores (ascospores). In contrast, Deltaccg-4 mat a and Deltamfa-1 mat A mutants exhibited normal chemotropic attraction and male fertility. Deltaccg-4 Deltamfa-1 double mutants displayed defective chemotropism and male sterility in both mating types. Heterologous expression of ccg-4 enabled mat a males to attract mat a trichogynes, although subsequent perithecial differentiation did not occur. Expression of ccg-4 and pre-2 in the same strain triggered self-stimulation, resulting in formation of barren perithecia with no ascospores. Our results indicate that CCG-4 and MFA-1 are required for mating-type-specific male fertility and that pheromones (and receptors) are initial determinants for sexual identity during mate recognition. Furthermore, a self-attraction signal can be transmitted within a strain that expresses a pheromone and its cognate receptor.     

Structural requirements for activity of the pheromones of Ustilago hordei

Ustilago hordei, the cause of barley-covered smut, initiates mating with pheromones. Gene sequence analysis suggested that these pheromones, Uhmfa1 and Uhmfa2, would be farnesylated peptides. Although isolation of mating-type-specific activity was rarely possible, chromatographic separations of culture supernatants yielded fractions that stimulated or inhibited mating. Based on predicted amino acid sequences and mass spectra of stimulating fractions, a series of pheromone analogs were synthesized and their activities were determined. Underivatized Uhmfa1 (PGKSGSGLGYSTC) or Uhmfa2 (EGKGEPAPYC) peptides were inactive, while peptides that were farnesylated and/or methyl esterified specifically induced conjugation tubes by cells of the opposite mating type. Uhmfa1 truncated from the amino terminus beyond the lysine lost activity, while truncated Uhmfa2 remained partially active. In mating bioassays, a pheromone concentration-dependent default mating response was observed. In competition studies, shorter Uhmfa1 peptides lacking pheromone activity inhibited activity of full-length peptides most effectively when both had the same functional groups.     

Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

Signal transduction during mating and meiosis in S. pombe

When starved, the fission yeast Schizosaccharomyces pombe responds by producing mating factors or pheromones that signal to cells of the opposite sex to initiate mating. Like its distant relative Saccharomyces cerevisiae, cells of the two mating types of S. pombe each produce a distinct pheromone that binds to receptors on the opposite cell type to induce the morphological changes required for mating. While the pathways are basically very similar in the two yeasts, pheromone signalling in S. pombe differs in several important ways from that of the more familiar budding yeast. In this article, Olaf Nielsen describes the pheromones and their effects in S. pombe, and compares the signalling pathways of the two yeasts.     

Isolation of pheromone precursor genes of Magnaporthe grisea.

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.     

Pheromone communication in the fission yeast Schizosaccharomyces pombe

Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.     

Disentangling signaling gradients generated by equivalent sources

Yeast cells approach a mating partner by polarizing along a gradient of mating pheromones that are secreted by cells of the opposite mating type. The Bar1 protease is secreted by a-cells and, paradoxically, degrades the α-factor pheromones which are produced by cells of the opposite mating type and trigger mating in a-cells. This degradation may assist in the recovery from pheromone signaling but has also been shown to play a positive role in mating. Previous studies suggested that widely diffusing protease can bias the pheromone gradient towards the closest secreting cell. Here, we show that restricting the Bar1 protease to the secreting cell itself, preventing its wide diffusion, facilitates discrimination between equivalent mating partners. This may be mostly relevant during spore germination, where most mating events occur in nature.     

Remarkably simple sequence requirement of the M-factor pheromone of Schizosaccharomyces pombe

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMC(Far)-OCH(3)) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.     

Control of mating and development inUstilago maydis

InUstilago maydis thea andb 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.