The Yeast Genomics* lab @ NOVA

(*evolutionary, functional, comparative and more)

Sexual reproduction has a fundamental role in the biology of many organisms and likely represents a defining point in the evolution the eukaryotes.

In fungi, the first description of sexual development goes back nearly two centuries. Ever since, a myriad of fungal species have had their sexual stages established, but the sexual system itself remains unknown or uncertain for many of these cases.

Within the Basidiomycota, emphasis has been given to sexual reproduction of dimorphic plant or animal fungal pathogens classified in two major lineages within the Basidiomycota (Ustilaginomycotina and Agaricomycotina) where sexual development and pathogenicity are usually interconnected. In contrast, considerably less is known for fungal species belonging to the third and earliest-branching lineage of Basidiomycota (Pucciniomycotina). This lineage also comprises many important plant parasites like the rust fungi (e.g. the wheat rust Puccinia graminis), in addition to saprobic yeast taxa such as the red yeasts of the genera Rhodosporidium and Sporidiobolus and the psychrotolerant yeasts of the genus Leucosporidium.

Using whole-genome sequencing, population and comparative genomics and a broad phylogenetic sampling, one topic we are currently exploring is the molecular characterization of the mating system of several Pucciniomycotina yeast species.

Transitions in modes of sexual reproduction in fungi seem to have continuously occurred over time, with most studies highlighting transitions from outcrossing tetrapolar multiallelic systems, to bipolar biallelic systems that promote selfing, and then to homothallic (self-fertile), where sexual development is initiated without the presence of two compatible mating partners. Although homothallism is thought to produce minimal genetic variability, this form of sex employs meiosis and may thus be maintained in fungi as an adaptation for surviving in specific ecological niches.

Although many basidiomycetes are homothallic, the genetic makeup underlying this mating behavior was been much less investigated. We are therefore exploring the molecular underpinings of the homothallic sexual behavior using the yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous) as a model.

Overall, we reason that such endeavors will provide a much better understanding on how the different modes of reproduction (selfing vs. outcrossing), mating systems (bipolar vs. tetrapolar) and lifestyles (pathogenic vs. saprobic) interplay in fungal evolution. Some of our ongoing studies are highlighted below:

Evolution of Sexual Reproduction in Basidiomycetes

Paula Gonçalves

Our initial studies in the red yeast Sporidiobolus salmonicolor uncovered a mating system that appeared to deviate from the classical bipolar and tetrapolar mating paradigms. This was based on the observation that only two alleles existed at the MAT pheromone/ receptor locus (P/R), but each allele was always associated to multiple, yet specific, alleles at the MAT homeodomain locus (HD). In other words, we did not detect random assortment of P/R and HD alleles in natural populations. However, the progeny of a laboratory cross between compatible strains indicated that recombination/assortment of P/R and HD loci could occur, albeit at a low frequency. We also confirmed that heterozygosity at both MAT regions is required for mating. To denote the distinction between the strictly bipolar mating behavior of S. salmonicolor natural isolates and the apparent tetrapolar pattern of inheritance of MAT genes in laboratory crosses we named this system pseudo-bipolar. We consider that this system may represent the initial steps of transition towards bipolarity.

(read the full story here)

First insight into the evolution of mating systems in the Pucciniomycotina

(A) Life cycle of the smut fungus Ustilago maydis (adapter from Morrow and Fraser 2009). The yeast form of U. maydis is non-pathogenic and invasion of the plant host requires switching to a filamentous form that occur upon mating of compatible yeast cells. (B) The red yeast Rhodosporidium toruloides has a very similar, but saprophytic, life cycle that can be completed in culture media. (C) The homothallic life cycle of Phaffia rhodozyma may involve conjugation between mother-cell and bud (pedogamy), conjugation of independent cells, or a single yeast cell may possibly undergo autopolyploidization (i.e. endoreplication).

A phylogeographic study of Phaffia rhodozyma (Xanthophyllomyces dendrorhous), an orange-coloured yeast that produces the biotechnologically relevant carotenoid astaxanthin, revealed that the genetic structure of the various populations results from the adaptation to different niches (host trees). This study also uncovered Australasia as a diversity hotspot for Phaffia suggesting that the genus evolved originally in this region in association with Nothofagus.

(read the full story here)

The prospective use of P. rhodozyma for astaxanthin production spurred the development of genetic tools, like targeted gene deletion, so that it is currently accessible to genetic modification. However, the mechanisms of sexual reproduction remain unknown. We are currently taking advantage of the biological diversity now uncovered to elucidate important features of Phaffia biology, with an emphasis on the molecular and functional characterization of the genes involved in sexual reproduction and how they interplay to originate the homothallic sexual behavior.

Molecular characterization of the homothallic sexual behavior of Phaffia rhodozyma

Geographic Distribution, phylogeny, host tree association of Phaffia.

(A) Diversity and phylogeny of HD alleles in natural isolates of S. Salmonicolor. (B) Comparison of the number and distribution of MAT alleles in emblematic species representing the different mating systems (HD, homeodomain locus; PR, pheromone/receptor locus). (C) Sexual proficiency of the T7.1 meiotic progeny resulting from assortment/recombination between PR and HD parental alleles. Heterozygosity at both MAT regions  is required for sexual reproduction.

(Top panel) Phaffia rhodozyma sexual stage (a slender holobasidium with two basidiospores arising from a single cell can be observed). (Bottom panel) Yeast cells (left); colony surface showing developing holobasidia (middle); holobasidium with two basidiospores (right).

The tetrapolar mating system of the yeast Leucosporidium scottii

((Left) Phylogeny of L. scottii species complex and MAT allele diversity. (Middle) Gene content and organization of the P/R MAT locus in L. scottii strains CBS 5931 (A1) and CBS 5930 (A2). (Rigth) Micrographs of crosses between the parental strains and selected progeny exhibiting nonparental combination of MAT alleles.

The tetrapolar system, in which mating is dictated by two alternate sets of MAT genes at genetically unlinked regions, has been proposed to be the ancestral state in basidiomycete fungi. Support for this model has however been gathered only from representatives of two more recently derived lineages. In this paper, published in Genetics we used whole-genome sequencing, chromoblot analysis and mating tests to show that Leucosporidium scottii, a psychrotolerant  basidiomycete yeast belonging to the earliest-branching lineage (Pucciniomycotina), has a tetrapolar system governed by two physically unlinked gene clusters: a multiallelic homeodomain (HD) locus and a biallelic pheromone/receptor (P/R) locus. Analysis of the distribution of MAT genes in natural populations showed that the two loci were in strong linkage disequilibrium, but independent assortment of MAT alleles was observed upon sexual reproduction. Fertile progeny with similar proportions of the four mating types was observed, but approximately 2/3 of the progeny was found to be nonhaploid and relatively unstable. This study adds to others in reinforcing tetrapolarity as the ancestral state of all basidiomycetes.

Highlighted Article

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