Phenotypic Mating Research Articles

Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi

In ascomycetes, mating compatibility is regulated by the mating-type locus, MAT1. The objectives of this study were to identify and sequence genes at the MAT1 locus in the grape powdery mildew fungus, Erysiphe necator, to develop a PCR-based marker for determining mating type in E. necator, and to develop degenerate primers for amplification by PCR of conserved regions of mating-type idiomorphs in other powdery mildew fungi. We identified MAT1- 2- 1 of the MAT1- 2 idiomorph in E. necator based on the homologous sequence in the genome of Blumeria graminis f. sp. hordei and we found MAT1- 1- 1 and MAT1- 1- 3 of the MAT1- 1 idiomorph from transcriptome sequences of E. necator. We developed and applied a reliable PCR-based multiplex marker to confirm that genotype correlated with mating phenotype, which was determined by pairing with mating-type tester isolates. Additionally, we used the marker to genotype populations of E. necator from different Vitis spp. from throughout the USA. We found both mating types were present in all populations and mating-type ratios did not deviate from 1:1. The mating-type genes in E. necator are similar to those of other Leotiomycetes; however, the structure of the MAT1 locus in E. necator, like the MAT1- 2 idiomorph of B. graminis, is markedly different from other ascomycetes in that it is greatly expanded and may contain a large amount of repetitive DNA. As a result, we were unable to amplify and sequence either idiomorph in its entirety. We designed degenerate primers that amplify conserved regions of MAT1- 1 and MAT1- 2 in E. necator, Podosphaera xanthii, Microsphaera syringae, and B. graminis, representing the major clades of the Erysiphales. These degenerate primers or sequences obtained in this study from these species can be used to identify and sequence MAT1 genes or design mating-type markers in other powdery mildew fungi as well.

Cellular responses to extracellular guidance cues

Extracellular guidance cues have a key role in orchestrating cell behaviour. They can take many forms, including soluble and cell-bound ligands (proteins, lipids, peptides or small molecules) and insoluble matrix substrates, but to act as guidance cues, they must be presented to the cell in a spatially restricted manner. Cells that recognize such cues respond by activating intracellular signal transduction pathways in a spatially restricted manner and convert the extracellular information into intracellular polarity. Although extracellular cues influence a broad range of cell polarity decisions, such as mitotic spindle orientation during asymmetric cell division, or the establishment of apical-basal polarity in epithelia, this review will focus specifically on guidance cues that promote cell migration (chemotaxis), or localized cell shape changes (chemotropism).

Female asynchrony may drive disruptive sexual selection on male mating phenotypes in a Heliconius butterfly

Alternative male phenotypes may be a source of novel adaptive traits and may evolve under strong sexual selection. We studied interpopulation differences in male mating behavior related to receptive female synchrony in the monandrous pupal-mating butterfly Heliconius charitonia. In the population in which female-receptive pupae were more synchronous, larger males were unable to monopolize mates; variance in male mating success was lower; strength of sexual selection was weak; and all males competed for access to female pupae using the same strategy (pupal mating). In the population where no more than one female was receptive at a time (extreme asynchrony), only large males competed for pupae, and among these, only the largest individuals successfully mated. Thus, variance in mating success was higher, and sexual selection within pupal maters was stronger. In this population, smaller males patrolled large areas as an alternative mating behavior. When unmated females were experimentally released, small male size was associated with higher mating success. We suggest that alternative patrolling behavior may have evolved under strong sexual selection as a consequence of high asynchrony in receptive female availability in some populations.

The DEAD-box RNA helicase, Dhh1, functions in mating by regulating Ste12 translation in Saccharomyces cerevisiae

The DEAD-box RNA helicase, Dhh1, is a member of a highly conserved subfamily designated RCK/p54 helicases. Dhh1 functions as mRNA decapping activator, and is localized to discrete cytoplasmic foci known as processing bodies (P-bodies). Here, we describe the essential roles of Dhh1 in the yeast mating pathway. A dhh1 deletion mutation caused a significant decrease in the protein level of Ste12, a mating-specific transcription factor, resulting in severe mating defects. We examined the accumulation of Dhh1-GFP in P-bodies during mating. Following pheromone treatment, the P-body intensity and number increased in wild-type cells, while dhh1 mutant cells failed to show P-body formation. Both the mating and P-body phenotypes of dhh1 were suppressed by overexpression of STE12 or CAF20 encoding an eIF4E inhibitor. In wild-type cells, CAF20 overexpression led to an increased level of Ste12 protein as well as highly developed P-bodies. We propose that Dhh1 and Caf20 regulate the Ste12 protein expression and the Ste12 protein level is associated with P-body formation during mating.

Variability of GnRH secretion in two goby species with socially controlled alternative male mating tactics

Male reproductive phenotypic plasticity related to environmental–social conditions is common among teleost fish. In several species, males adopt different mating tactics depending on their size, monopolizing mates when larger, while parasitizing dominant male spawns when smaller. Males performing alternative mating tactics are often characterized by a strong dimorphism in both primary and secondary reproductive traits. According to studies on sex-changing species and on species where only one male morph is reproductively active, male alternative phenotypes are expected to vary also in gonadotropin-releasing hormone (GnRH) neurons in forebrain preoptic area (POA). Here, we compared the intra- and inter-sexual variations in number and size of GnRH neurons, along with gonads and male accessory structure investment, in two goby species, the grass goby, Zosterisessor ophiocephalus, and the black goby, Gobius niger, characterized by male alternative mating phenotypes. In both species, older and larger males defend nests, court and perform parental care, while younger and smaller ones try to sneak territorial male spawning. We found that grass goby and black goby have different patterns of GnRH expression. Grass goby presents a clear intra-sexual dimorphism in GnRH expression, related to the occurrence of alternative mating tactics, while in the black goby, only inter-sexual differences are observed. The inter- and intra-specific variability in the GnRH neurons in these two goby species is discussed in light of the differences in migratory behavior, nest type, and mating system.

Cloning and characterization of the mating type ( MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny

Ubiquitin-dependent degradation of the yeast Mat(alpha)2 repressor enables a switch in developmental state.

Developmental transitions in eukaryotic cell lineages revolve around two general processes: the dismantling of the regulatory program specifying an initial differentiated state and its replacement by a new system of regulators. However, relatively little is known about the mechanisms by which a previous regulatory state is inactivated. Protein degradation is implicated in a few examples, but the molecular reasons that a formerly used regulator must be removed are not understood. Many yeast strains undergo a developmental transition in which cells of one mating type differentiate into a distinct cell type by a programmed genetic rearrangement at the MAT locus. We find that Mat(alpha)2, a MAT-encoded transcriptional repressor that is key to creating several cell types, must be rapidly degraded for cells to switch their mating phenotype properly. Strikingly, ubiquitin-dependent proteolysis of alpha2 is required for two mechanistically distinct purposes: It allows the timely inactivation of one transcriptional repressor complex, and it prevents the de novo assembly of a different, inappropriate regulatory complex. Analogous epigenetic mechanisms for reprogramming transcription are likely to operate in many developmental pathways.

An experimental approach to altering mating tactics in male horseshoe crabs (Limulus polyphemus)

Alternative reproductive tactics are often correlated with phenotype, density, environment, or social context. Male horseshoe crabs (Limulus polyphemus) have two mating tactics that are associated with phenotype. Males in good condition arrive at the nesting beach and spawn while attached to females, whereas those in poorer condition come ashore unattached and crowd around the nesting couples as satellites, fertilizing eggs through sperm competition. The correlation between mating tactic and phenotype may be due to males choosing tactics based on condition, or it may be that males that have not found a female choose to come ashore as satellites. To distinguish between these two possibilities, I conducted an experiment on male horseshoe crabs in the field at Seahorse Key on the northern Gulf coast of Florida. I prevented males from attaching to females by placing small plastic bags over the claws they use to attach. The results showed that males in poor condition came ashore as satellites, whereas males in good condition remained at sea. This means that mating tactics are cued by information about the male's condition and not about whether he found a female. The evolution of phenotype-correlated mating tactics can be represented by a model in which the fitness of each tactic changes with condition and fitness curves cross. I hypothesize that male horseshoe crabs in good condition have higher fitness when attached and that males in poorer condition to better when unattached.

Sequence variation in the rDNA ITS of Australian Armillaria species and intra-specific variation in A. luteobubalina

In contrast to northern hemisphere species, Australia’s Armillaria species have high variation in the rDNA Internal Transcribed Spacer (ITS) region. Isolates of the five different Australian species, five species from south-eastern Australia and one, A. luteobubalina, from Western Australia had distinct ITS sequences and PCR-RFLP profiles. Phylogenetic analyses grouped A. hinnulea away from the other Australian Armillaria species and with the seven northern hemisphere species. The 28 ITS sequences from A. luteobubalina isolates, 27 from Western Australia, gave rise to four distinct polymorphic groups. This ITS variation was not related to geographical locality, host species or mating phenotype.

Male dimorphism, sperm traits and immunology in the corkwing wrasse ( Symphodus melops L.)

The evolution of alternative male mating phenotypes inevitably involves variation in risk or intensity of sperm competition. Males that typically mate in disfavoured roles (i.e. sneakers/parasitic males) are predicted to experience higher magnitudes of sperm competition than males mating in favoured roles (preferred by females/exhibiting mate monopolisation). Here, we investigated whether two distinct male phenotypes in corkwing wrasse (Symphodus melops) differ with respect to adaptations for sperm competition in terms of sperm quantity, sperm motility and fertilising capacity. We found that males exhibiting female mimicry (disfavoured role) had both absolutely and relatively larger gonads than males with typical secondary sexual characters (favoured role). Moreover, a higher proportion of sperm from female mimics was motile 5 min after activation compared to sperm of territorial males. There were, however, no differences in sperm concentration or in in vitro fertilisation ability between the two male morphs. We also examined whether the male phenotypes differed in some immune parameters and if any such variation is associated with sperm traits. Current theories predict that ejaculate quality should be negatively correlated with the level of immunocompetence. Territorial males had relatively heavier spleens than female mimics, but whether this reflects differing immunocompetence is uncertain, since the spleen is also an important organ for erythrocyte storage. Furthermore, there were no differences in the densities of circulating lymphocytes or granulocytes, and the immune parameters did not covary with the sperm traits. Differences in immunocompetence are therefore unlikely to provide a major proximate explanation for variations in sperm traits in corkwing wrasse.

Sperm storage by females of the polyandrous noctuid moth Heliothis virescens

Female tobacco budworm moths, Heliothis virescens, generally mate with more than one male, receiving from each mate both fertilizing sperm (eupyrene) and nonfertilizing anucleate sperm (apyrene), which is thought to play a role in sperm competition. One male typically gains sperm precedence, but it is not consistently the last or the first male to mate. I investigated the mechanism of this variable pattern of paternity by examining the patterns of storage of both types of sperm in the female's spermatheca as a function of multiple mating and male phenotype. The number of stored apyrene sperm varied with mating history, being greatest in twice-mated females and least in females mated to one nonvirgin male. In contrast, only one ejaculate's worth of eupyrene sperm was stored regardless of female mating history (once or twice mated). Thus, while they store two complements of apyrene sperm, twice-mated females apparently store only one ejaculate's worth of eupyrene sperm. This biased pattern of sperm storage may contribute to the variable pattern of paternity observed in this species. Eupyrene sperm storage also correlated positively with female size, male age and spermatophore size. Finally, a new sperm storage site was identified and described. It is a bulged region in the seminal duct.

A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function.

SUCCESSFUL FATHERS SIRE SUCCESSFUL SONS.

The theory of sexual selection holds a central role in evolutionary biology. Its key assumption is the heritability of traits associated with reproductive success. Strong indirect evidence supporting this assumption comes from the numerous studies that have identified heritable traits associated with mating success. However, there remain only a handful of studies that have attempted to demonstrate directly that successful fathers have successful sons. We present the results of an experimental study of the mating success and phenotype of male field crickets Gryllus bimaculatus (Orthoptera: Gryllidae) and their offspring. These reveal that sons of successful males obtain significantly more copulations than sons of unsuccessful males. There was no difference in body size of sons of either group, but sons of successful males had significantly longer development times. This may represent a naturally selected cost to traits associated with success that could balance their sexually selected advantages.

Switch-domain mutations in the Saccharomyces cerevisiae G protein alpha-subunit Gpa1p identify a receptor subtype-biased mating defect.

The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (alpha subunit), Ste4p (beta) and Ste18p (gamma). The switch II region of G alpha subunits is involved in several protein-protein interactions and an intrinsic GTPase activity. To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations. The Q323 analog in G alpha subunits and Ras is implicated in GTP hydrolysis. Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity. Mutation of residue A59 of Ras and the analogous G alphas residue has had quite different effects. The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in alpha cells than in a cells. In addition the G321T mutation resulted in an unusual pheromone response phenotype. We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and alpha-factor receptors.

12] Functional analysis of pfmdr1 gene of Plasmodium falciparum

This chapter discusses the functional analysis of pfmdr1 gene of Plasmodium falciparum . Multidrug resistance ( mdr ) gene homologs are implicated in mediating drug resistance in parasitic protozoans including Plasmodium and Leishmania. This chapter focuses on the functional characterization of these mdr genes and their role in drug resistance. Two experimental approaches, including heterologous expression of pfmdr1 in yeast and subcellular localization studies in P. falciparum address the functional nature of Pgh1 in a mechanism of drug resistance. The expression of pfmdr1 in yeast containing a null mutation in ste6 can complement STE6 function and restore mating to a level 1000-fold over background, demonstrating that Pgh1 functions as a transport molecule in this system. The expression of the pfmdr1 gene containing mutations associated with either chloroquine or mefloquine resistance in the wild abolishes this mating phenotype, suggesting that these mutations are important for the transport function of Pgh1.

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.

A ste12 allele having a differential effect on a versus alpha cells.

The transcriptional activator Ste12p is a key component of the yeast pheromone response pathway: phosphorylated as a consequence of signal transduction, it activates transcription of genes that promote mating and the subsequent fusion of the two cell types a and alpha. Activation by Ste12p requires three types of protein-protein interaction between DNA-binding activator proteins: (1) Ste12p by itself can induce non-cell-type-specific genes involved in mating; (2) cooperation of the transactivator Mcm1p with Ste12p induces a-specific genes; and (3) formation of a complex of the activator proteins Mcm1p and alpha 1 (a transcriptional activator of alpha-specific genes) with Ste12p is believed to induce alpha-specific genes. We isolated and characterized a partially functional ste12 allele (ste12-T50), that is defective only in the activation of alpha-specific genes. ste12-T50 was isolated as a second-site mutation conferring the a mating phenotype on mat alpha 2 mutant cells. In mat alpha 2 cells, where due to the lack of repressor, alpha 2, both sets of cell-type-specific genes are expressed, ste12-T50 apparently tips the balance in favor of a-specific gene expression. Thus, mat alpha 2 ste12-T50 cells mate like a cells. Additional ste12 mutants that confer the a mating phenotype on mat alpha 2 cells have also been isolated.

GENETIC VARIATION FOR FEMALE MATE DISCRIMINATION IN DROSOPHILA MELANOGASTER.

Comparisons between the Canton-S and Tai-Y strains of Drosophila melanogaster (both wild type) revealed variation in female mate discrimination based on chemical courtship signals present as hydrocarbons on the male cuticle. Mating tests indicated that 7-tricosene, which is the primary hydrocarbon on the Canton-S male cuticle but is nearly absent from Tai-Y, was a significant component of the signal. The discrimination was asymmetrical in that Canton-S females clearly distinguished between the two types of males in no-choice tests, but Tai-Y females did not. F1 females expressed an intermediate ability to discriminate, and female progeny of backcrosses expressed a mating phenotype very similar to that of the parental strain to which the backcross was made. Analysis of independent effects from the X and both major autosomes indicated that the discrimination is controlled by gene(s) on chromosome 3.

The effects of stress and parasites on the expression of dominance and secondary sexual characters

Male reproductive phenotypic plasticity related to environmental–social conditions is common among teleost fish. In several species, males adopt different mating tactics depending on their size, monopolizing mates when larger, while parasitizing dominant male spawns when smaller. Males performing alternative mating tactics are often characterized by a strong dimorphism in both primary and secondary reproductive traits. According to studies on sex-changing species and on species where only one male morph is reproductively active, male alternative phenotypes are expected to vary also in gonadotropin-releasing hormone (GnRH) neurons in forebrain preoptic area (POA). Here, we compared the intra- and inter-sexual variations in number and size of GnRH neurons, along with gonads and male accessory structure investment, in two goby species, the grass goby, Zosterisessor ophiocephalus, and the black goby, Gobius niger, characterized by male alternative mating phenotypes. In both species, older and larger males defend nests, court and perform parental care, while younger and smaller ones try to sneak territorial male spawning. We found that grass goby and black goby have different patterns of GnRH expression. Grass goby presents a clear intra-sexual dimorphism in GnRH expression, related to the occurrence of alternative mating tactics, while in the black goby, only inter-sexual differences are observed. The inter- and intra-specific variability in the GnRH neurons in these two goby species is discussed in light of the differences in migratory behavior, nest type, and mating system.

A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae.

The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.