Mobility of the gradient tracking machine in mating yeast depends on Bud1 inactivation and actin-independent vesicle delivery

The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through the secretion of mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Because strong positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking after its assembly is complete. What prevents the GTM from triggering polarized growth at the DS? Here we describe two mechanisms that enable tracking. First, the Ras GTPase Bud1 must be inactivated to release the GTM. Second, actin-independent – but not actin-dependent – vesicle delivery must be targeted upgradient to effect GTM redistribution.

Genetic Dissection of T-DNA Insertional Mutants Reveals Uncoupling of Dikaryotic Filamentation and Virulence in Sugarcane Smut Fungus

The biotrophic basidiomycetous fungus Sporisorium scitamineum causing smut disease in sugarcane is characterized by a life-cycle composed of a yeast-like nonpathogenic haploid basidiosporial stage outside the plant and filamentous pathogenic dikaryotic hyphae within the plant. Under field conditions, dikaryotic hyphae are formed after mating of two opposite mating-type strains. However, the mechanisms underlying genetic regulation of filamentation and its association with pathogenicity and development of teliospores are currently unclear. This study has focused on the characterization and genetic dissection of haploid filamentous mutants derived from T-DNA insertional mutagenesis. Our results support the existence of at least three genotypes among the six haploid filamentous mutants that differentially contribute to virulence and development of the whip and teliospore, providing a novel foundation for further investigation of the regulatory networks associated with pathogenicity and teliospore development in S. scitamineum.

Phosphorylated Gβ is a directional cue during yeast gradient tracking

Budding yeast cells interpret shallow pheromone gradients from cells of the opposite mating type, polarize their growth toward the pheromone source, and fuse at the chemotropic growth site. We previously proposed a deterministic, gradient-sensing model that explains how yeast cells switch from the intrinsically positioned default polarity site (DS) to the gradient-aligned chemotropic site (CS) at the plasma membrane. Because phosphorylation of the mating-specific Gβ subunit is thought to be important for this process, we developed a biosensor that bound to phosphorylated but not unphosphorylated Gβ and monitored its spatiotemporal dynamics to test key predictions of our gradient-sensing model. In mating cells, the biosensor colocalized with both Gβ and receptor reporters at the DS and then tracked with them to the CS. The biosensor concentrated on the leading side of the tracking Gβ and receptor peaks and was the first to arrive and stop tracking at the CS. Our data showed that the concentrated localization of phosphorylated Gβ correlated with the tracking direction and final position of the G protein and receptor, consistent with the idea that gradient-regulated phosphorylation and dephosphorylation of Gβ contributes to gradient sensing. Cells expressing a nonphosphorylatable mutant form of Gβ exhibited defects in gradient tracking, orientation toward mating partners, and mating efficiency.

The Destructive Tree Pathogen Phytophthora ramorum Originates from the Laurosilva Forests of East Asia

As global plant trade expands, tree disease epidemics caused by pathogen introductions are increasing. Since ca 2000, the introduced oomycete Phytophthora ramorum has caused devastating epidemics in Europe and North America, spreading as four ancient clonal lineages, each of a single mating type, suggesting different geographical origins. We surveyed laurosilva forests for P. ramorum around Fansipan mountain on the Vietnam-China border and on Shikoku and Kyushu islands, southwest Japan. The surveys yielded 71 P. ramorum isolates which we assigned to eight new lineages, IC1 to IC5 from Vietnam and NP1 to NP3 from Japan, based on differences in colony characteristics, gene x environment responses and multigene phylogeny. Molecular phylogenetic trees and networks revealed the eight Asian lineages were dispersed across the topology of the introduced European and North American lineages. The deepest node within P. ramorum, the divergence of lineages NP1 and NP2, was estimated at 0.5 to 1.6 Myr. The Asian lineages were each of a single mating type, and at some locations, lineages of “opposite” mating type were present, suggesting opportunities for inter-lineage recombination. Based on the high level of phenotypic and phylogenetic diversity in the sample populations, the coalescence results and the absence of overt host symptoms, we conclude that P. ramorum comprises many anciently divergent lineages native to the laurosilva forests between eastern Indochina and Japan.

Uniparental sexual reproduction following cell-cell fusion of opposite mating-type partners

Some animal species require an opposite-sex partner for their sexual development but discard the partner’s genome before gamete formation, generating hemi-clonal progeny in a process called hybridogenesis. In this study, we discovered hybridogenesis-like reproduction in a basidiomycete fungus, Cryptococcus neoformans. C. neoformans has two mating types, MATa and MATα, which fuse to produce a dikaryotic zygote that completes a sexual cycle producing recombinant meiotic progeny. Here, we discovered exclusive uniparental inheritance of nuclear genetic material in a fraction of the F1 progeny produced during bisexual reproduction of two opposite mating-type partners. By analyzing strains expressing fluorescent reporter proteins, we observed that dikaryotic hyphae were produced, but only one parental nuclei was found in the terminal basidium where sporulation occurs. Whole-genome sequencing revealed the nuclear genome of the progeny was identical with one or the other parental genome, whereas the mitochondrial genome was always inherited from the MATa parent. Uniparental sporulation was also observed in natural isolate crosses occurring in concert with biparental sporulation. The meiotic recombinase Dmc1 was found to be critical for uniparental reproduction. These findings reveal an unusual mode of eukaryotic microbial unisexual reproduction that shares features with hybridogenesis in animals.

Distinct specificity of two pheromone G-protein coupled receptors, Map3 and Mam2, in fission yeast species

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycetes including yeasts, potential mating between cells of opposite mating-type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focus on the differences in pheromone/receptor system between mating types. The fission yeast Schizosaccharomyces pombe has two mating types (sexes), Plus (P) and Minus (M). Here we investigated the specificity of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the molecular recognition for pheromone binding. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are essential for the specificity of M-factor recognition. Thus, the differences in these two GPCRs are likely to reflect the significantly distinct specificities of their respective pheromone/receptor systems; that is, the specificity of Map3 is more stringent than that of Mam2. We speculate that this sexual asymmetry might allow ascomycete fungi to generate novel prezygotic barriers within a population, while maintaining strong mate choice. Our genetic analyses also contribute to our understanding of the receptors that comprise the Class D GPCRs belonging to the fungal pheromone receptor family.Author summaryCourtship signals play a key role in the fertilization processes of living beings from animals to microorganisms and, in particular, sex pheromones are involved in differentiating among species. On the other hand, changes in a pheromone/receptor system might also alter species-specificity during the selection of a mating partner, which is likely to facilitate prezygotic isolation. Here we demonstrate a distinct difference in the specificity of GPCRs for pheromones between cells of opposite mating-type in the fission yeast Schizosaccharomyces pombe, by conducting a comprehensive genetic analysis and comparison of these proteins between closely related fission yeast species. Our finding of sexual asymmetry due to the relative strictness of receptors in S. pombe suggests that it might be one of the driving forces behind the formation of new species. Such differences in pheromone/receptor interactions between the sexes may occur in a variety of life-forms such as insects and amphibians; hence, the findings of this study might be extended to other organisms. In addition, this study of S. pombe pheromone receptors sheds light on the functions of the unique Class D GPCRs.

A simple, cheap, and robust protocol for the identification of mating type in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward as long as the mating type and ploidy of the strains are known. Haploid S. cerevisiae cells are either MATa or MATα mating type. Several techniques can be used to determine mating type (or ploidy), but are typically time-consuming, require specialized components, and/or the results are inconsistent and transient. Here we validated a simple, cheap and robust method to enable rapid identification of S. cerevisiae mating types. When cells of opposite mating type are mixed in liquid media, they creep up culture vessel sides, a phenotype that can easily be detected visually. In contrast, mixtures of cells of the same mating type or with a diploid strain(s) simply settle out. The method does not require specialized equipment, and is robust to different media, cell densities, temperatures and strains. It can be performed in 96-well plates, and the phenotype is observable for several days. The simplicity and robustness of this method makes it ideal for routine verification of S. cerevisiae mating type, and it could be used to screen for genes underlying the creeping phenotype.

The asymmetric chemical structures of two mating pheromones reflect their differential roles in conjugation of Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the mating reaction is controlled by two mating pheromones, M-factor and P-factor, secreted by M- and P-type cells, respectively. M-factor is a C-terminally farnesylated lipid peptide, whereas P-factor is a simple peptide. To examine whether this chemical asymmetry in the two pheromones is essential for conjugation, we constructed a mating system in which either pheromone can stimulate both M- and P-cells, and examined whether the resulting autocrine strains can mate. Autocrine M-cells responding to M-factor successfully mated with P-factor-less P-cells, indicating that P-factor is not essential for conjugation; by contrast, autocrine P-cells responding to P-factor were unable to mate with M-factor-less M-cells. The sterility of the autocrine P-cells was completely recovered by expressing the M-factor receptor. These observations indicate that the different chemical characteristics of the two types of pheromone, a lipid and a simple peptide, are not essential; however, a lipid peptide is absolutely required for successful mating. Our findings allow us to propose a model of the differential roles of M-factor and P-factor in conjugation of S. pombe.Summary statementLipid pheromone peptides secreted locally from one cell may be concentrated at the fusion site with an opposite mating-type cell, which then polarizes to enable successful conjugation in S. pombe.

Unisexual reproduction promotes competition for mating partners in the global human fungal pathogenCryptococcus deneoformans

Courtship is pivotal for successful mating. However, courtship is challenging for theCryptococcus neoformansspecies complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type,C. deneoformanscan undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain’s ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor geneGPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggestC. deneoformansemploys hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition.Author SummarySexual reproduction plays a pivotal role in shaping fungal population structure and diversity in nature. The global human fungal pathogenCryptococcus neoformansspecies complex evolved distinct sexual cycles: bisexual reproduction between mating partners of the opposite mating types, and unisexual reproduction with only one mating type. During both sexual cycles, cells undergo a yeast-to-hyphal morphological transition and nuclei diploidize through either cell-cell fusion followed by nuclear fusion during bisexual reproduction or endoreplication during unisexual reproduction. Despite the complex sexual life cycle, the majority of Cryptococcal isolates are α mating type. Albeit the scarcity ofMATacells in the environment, meiotic recombination is prevalent. To decipher this conundrum, we ask whether there is an underlying mechanism in whichCryptococcusspecies increase their mating opportunities. In this study, we showed that the undirected hyphal growth during unisexual reproduction enablesMATα cells to forage for mating partners over a larger surface area, and whenMATα hyphae come into close proximity of rareMATacells, pheromone response pathway activation in bothMATα andMATacells can further enhance mating. This mating enhancement could promote outcrossing and facilitate genome reshuffling via meiotic recombination.

Temporal Genetic Dynamics of an Experimental, Biparental Field Population of Phytophthora capsici

Defining the contributions of dispersal, reproductive mode, and mating system to the population structure of a pathogenic organism is essential to estimating its evolutionary potential. After introduction of the devastating plant pathogen, Phytophthora capsici, into a grower’s field, a lack of aerial spore dispersal restricts migration. Once established, coexistence of both mating types results in formation of overwintering recombinant oospores, engendering persistent pathogen populations. To mimic these conditions, in 2008, we inoculated a field with two P. capsici isolates of opposite mating type. We analyzed pathogenic isolates collected in 2009-13 from this experimental population, using genome-wide single-nucleotide polymorphism markers. By tracking heterozygosity across years, we show that the population underwent a generational shift; transitioning from exclusively F1 in 2009-10; mixed generational in 2011; and ultimately all inbred in 2012-13. Survival of F1 oospores, characterized by heterozygosity excess, coupled with a low rate of selfing, delayed declines in heterozygosity due to inbreeding and attainment of equilibrium genotypic frequencies. Large allele and haplotype frequency changes in specific genomic regions accompanied the generational shift, representing putative signatures of selection. Finally, we identified an approximately 1.6 Mb region associated with mating type determination, constituting the first detailed genomic analysis of a mating type region (MTR) in Phytophthora. Segregation patterns in the MTR exhibited tropes of sex-linkage, where maintenance of allele frequency differences between isolates of opposite mating type was associated with elevated heterozygosity despite inbreeding. Characterizing the trajectory of this experimental system provides key insights into the processes driving persistent, sexual pathogen populations.