The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification. NH2-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDS-PAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.

Sexual reproduction facilitates infection by the production of both a lineage advantage and infectious sexual spores in the ubiquitous human fungal pathogen Cryptococcus deneoformans. However, the regulatory determinants specific for initiating mating remain poorly understood. Here, we identified a velvet family regulator, Cva1, that strongly promotes sexual reproduction in C. deneoformans. This regulation was determined to be specific, based on a comprehensive phenotypic analysis of cva1Δ under 26 distinct in vitro and in vivo growth conditions. We further revealed that Cva1 plays a critical role in the initiation of early mating events, including sexual cell-cell fusion, but is not important for the late sexual development stages or meiosis. Thus, Cva1 specifically contributes to mating activation. Importantly, a novel mating-responsive protein, Cfs1, serves as the key target of Cva1 during mating, since its absence nearly blocks cell-cell fusion in C. deneoformans and its sister species C. neoformans. Together, our findings provide insight into how C. deneoformans ensures the regulatory specificity of mating. IMPORTANCE The human fungal pathogen C. deneoformans is a model organism for studying fungal sexual reproduction, which is considered to be important to infection. However, the specific regulatory determinants for activation of sexual reproduction remain poorly understood. In this study, by combining transcriptomic and comprehensive phenotypic analysis, we identified a velvet family regulator Cva1 that specifically and critically elicits early mating events, including sexual cell-cell fusion. Significantly, Cva1 induces mating through the novel mating-responsive protein Cfs1, which is essential for cell-cell fusion in C. deneoformans and its sister species C. neoformans. Considering that Cva1 and Cfs1 are highly conserved in species belonging to Cryptococcaeceae, they may play conserved and specific roles in the initiation of sexual reproduction in this important fungal clade, which includes multiple human fungal pathogens.

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion.

In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.

The Leotiomycetes is a hugely diverse group of fungi, accommodating a wide variety of important plant and animal pathogens, ericoid mycorrhizal fungi, as well as producers of antibiotics. Despite their importance, the genetics of these fungi remain relatively understudied, particularly as they don't include model taxa. For example, sexual reproduction and the genetic mechanisms that underly this process are poorly understood in the Leotiomycetes. We exploited publicly available genomic and transcriptomic resources to identify genes of the mating-type locus and pheromone response pathway in an effort to characterize the mating strategies and behaviors of 124 Leotiomycete species. Our analyses identified a putative a-factor mating pheromone in these species. This significant finding represents the first identification of this gene in Pezizomycotina species outside of the Sordariomycetes. A unique mating strategy was also discovered in Lachnellula species that appear to have lost the need for the primary MAT1-1-1 protein. Ancestral state reconstruction enabled the identification of numerous transitions between homothallism and heterothallism in the Leotiomycetes and suggests a heterothallic ancestor for this group. This comprehensive catalog of mating-related genes from such a large group of fungi provides a rich resource from which in-depth, functional studies can be conducted in these economically and ecologically important species.

Transcriptional repression of the silent mating-type loci HMLalpha and HMRa in Saccharomyces cerevisiae is regulated by chromatin structure. Sas2p is a catalytic subunit of the SAS histone acetyltransferase (HAT) complex. Although many HATs seem to relieve chromosomal repression to facilitate transcriptional activation, sas mutant phenotypes include loss of SIR1-dependent silencing of HMLalpha. To gain insight into the mechanism of the SAS complex mediated silencing at HMLalpha, we investigated the expression and chromatin structure of the alpha2 gene in the HMLalpha locus. We found that deletion of SAS2 in combination with a null allele of SIR1 changed the chromatin structure of the precisely positioned nucleosome, which includes the mRNA start site of the alpha2 gene and derepressed alpha2 transcription. The Sas2p HAT domain was required for this silencing. Furthermore, chromatin immunoprecipitation analysis revealed that the SAS complex was associated with the HMLalpha locus, and ASF1 (which encodes chromatin assembly factor Asf1p), but not SIR1 and SIR2, was necessary for this localization. These data suggest that the HAT activity and ASF1-dependent localization of the SAS complex are required for SIR1-dependent HMLalpha silencing.

Saccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native α-factor preproleader (αnat) and a top-down design over the fittest evolved signal peptide obtained in our lab (α9H2 leader). The goal was to analyse the effect of mutations accumulated in the signal sequence throughout iterations of directed evolution, or of other reported mutations, and their possible epistatic interactions. Both approaches agreed in the positive synergism of four mutations (Aα9D, Aα20T, Lα42S, Dα83E) contained in the final optimised leader (αOPT), which notably enhanced the secretion of several fungal oxidoreductases and hydrolases. Additionally, we suggest a guideline to further drive the heterologous production of a particular enzyme based on combinatorial saturation mutagenesis of positions 86th and 87th of the αOPT leader fused to the target protein.

In the fission yeast Schizosaccharomyces pombe, the TOR (target of rapamycin) and PKA (protein kinase A) signaling transduction pathways regulate the expression of genes required for cell growth and sexual differentiation in response to the nutritional environment. Inhibition of Tor2 signaling results in the induction of genes involved in sexual differentiation, and the cells undergo mating and meiosis, even under good nutritional conditions. The same phenotype is observed in mutants in which the PKA pathway is inactive. By contrast, Tor2 overexpression or mutations that hyperactivate PKA signaling impair sexual differentiation, even under poor nutritional conditions. Accordingly, a very important question is to understand the molecular mechanism by which these two pathways coordinately regulate gene expression in response to nutrients.

Anisogamy, the size difference between small male and large female gametes, is known to enable selection for sexual dimorphism and behavioral differences between sexes. Nevertheless, even isogamous species exhibit molecular asymmetries between mating types, which are known to ensure their self-incompatibility. Here, we show that different properties of the pheromones secreted by the MATa and MATα mating types of budding yeast lead to asymmetry in their behavioral responses during mating in mixed haploid populations, which resemble behavioral asymmetries between gametes in anisogamous organisms. MATa behaves as a random searcher that is stimulated in proportion to the fraction of MATα partner cells within the population, whereas MATα behaves as a short-range directional distance sensor. Mathematical modeling suggests that the observed asymmetric responses can enhance efficiency of mating and might thus provide a selective advantage. Our results demonstrate that the emergence of asymmetric mating behavior did not require anisogamy-based sexual selection.

There are around 400 internationally recognized dog breeds in the world today, with a remarkable diversity in size, shape, color and behavior. Breeds are considered to be uniform groups with similar physical characteristics, shaped by selection rooted in human preferences. This has led to a large genetic difference between breeds and a large extent of linkage disequilibrium within breeds. These characteristics are important for association mapping of candidate genes for diseases and therefore make dogs ideal models for gene mapping of human disorders. However, genetic uniformity within breeds may not always be the case. We studied patterns of genetic diversity within 164 poodles and compared it to 133 dogs from eight other breeds.

The breeding time of many species has changed over the past 2-3 decades in response to climate change. Yet it is a key reproductive trait that affects individual's parturition time and reproductive success, and thereby population dynamics. In order to predict how climate change will affect species' viability, it is crucial to understand how species base their reproductive efforts on environmental cues.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4-8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation-perhaps as a first step toward prezygotic isolation in S. pombe.

The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production.

After mating, the physiology of Drosophila females undergo several important changes, some of which are reflected in their rest-activity cycles. To explore the hypothesis that mating modifies the temporal organization of locomotor activity patterns, we recorded fly activity by a video tracking method. Monitoring rest-activity patterns under light/dark (LD) cycles indicated that mated females lose their ability to anticipate the night-day transition, in stark contrast to males and virgins. This postmating response is mediated by the activation of the sex peptide receptor (SPR) mainly on pickpocket (ppk) expressing neurons, since reducing expression of this receptor in these neurons restores the ability to anticipate the LD transition in mated females. Furthermore, we provide evidence of connectivity between ppk+ neurons and the pigment-dispersing factor (PDF)-positive ventral lateral neurons (sLNv), which play a central role in the temporal organization of daily activity. Since PDF has been associated to the generation of the morning activity peak, we hypothesized that the mating signal could modulate PDF levels. Indeed, we confirm that mated females have reduced PDF levels at the dorsal protocerebrum; moreover, SPR downregulation in ppk+ neurons mimics PDF levels observed in males. In sum, our results are consistent with a model whereby mating-triggered signals reach clock neurons in the fly central nervous system to modulate the temporal organization of circadian behavior according to the needs of the new status.

Sex offers advantages even in primarily asexual species. Some ciliates appear to utilize such reproductive strategy with many mating types. However, the factors determining the composition of mating types in the unicellular ciliate Tetrahymena thermophila are poorly understood, and this is further complicated by non-Mendelian determination of mating type in the offspring. We therefore developed a novel population genetics model to predict how various factors influence the dynamics of mating type composition, including natural selection. The model predicted either the coexistence of all seven mating types or fixation of a single mating type in a population, depending on parameter combinations, irrespective of natural selection. To understand what factor(s) may be more influential and to test the validity of theoretical prediction, five replicate populations were maintained in laboratory such that several factors could be controlled or measured. Whole-genome sequencing was used to identify newly arising mutations and determine mating type composition. Strikingly, all populations were found to be driven by strong selection on newly arising beneficial mutations to fixation of their carrying mating types, and the trajectories of speed to fixation agreed well with our theoretical predictions. This study illustrates the evolutionary strategies that T. thermophila can utilize to optimize population fitness.

Pathogens generate ubiquitous selective pressures and host-pathogen interactions alter social behaviours in many animals1-4. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal5-13. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for beta-galactosidase (beta-gal) expression in the presence and absence of alpha factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: beta-gal and Fig2::beta-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

Mating systems are an important factor influencing the variance in reproductive success among individuals within natural populations and thus have important ecological and evolutionary implications. We used molecular pedigree reconstruction techniques with microsatellite DNA data to characterize the genetic mating system and mate selection in adult smallmouth bass spawning in Lake Opeongo. The genetic mating system of smallmouth bass in this system can be characterized as predominantly monogamous with a low rate of polygynandry particularly among larger individuals. Iteroparous individuals showed a complete absence of interannual mate fidelity, presumably due to the low annual return rate of spawning adults. Within a season, individuals from both sexes pursued additional mating opportunities with males showing greater variance in mate number than females. Female mate selection appeared to be largely random with little evidence for elevated levels of inbreeding in this population. Multiple mating females pursued additional males to whom they were less related than the first male with which they spawned within a given season, however, this pattern varied among years. The mating pattern observed in this population would likely limit the strength of sexual selection and thus could account for the lack of sexual dimorphism and the absence of alternative reproductive tactics in this species.

The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is a haploid yeast that is able to form diploid cells by mating once nitrogen becomes limiting. Activation of the mating response requires the secretion of a- and α-factor pheromones, which bind to G-protein coupled receptors on cells of opposite mating type. In K. phaffii, the genes coding for the α-factor (MFα), the pheromone surface receptors and the conserved a-factor biogenesis pathway have been annotated previously. Initial homology-based search failed to identify potential a-factor genes (MFA). By using transcriptome data of heterothallic strains under mating conditions, we found two K. phaffiia-factor genes. Deletion of both MFA genes prevented mating of a-type cells. MFA single mutants were still able to mate and activate the mating response pathway in α-type cells. A reporter assay was used to confirm the biological activity of synthetic a- and α-factor peptides. The identification of the a-factor genes enabled the first characterization of the role and regulation of the mating pheromone genes and the response of K. phaffii to synthetic pheromones and will help to gain a better understanding of the mating behavior of K. phaffii.