Abstract

Some of the most unique and compelling survival strategies in the natural world evolved long ago, and are fixed in now-isolated species. Molecular insight into these adaptations has been limited, as classic experimental genetics has focused on the interfertile individuals of a population. Here we dissect a complex thermotolerance difference between yeast species that diverged millions of years ago. Using a new mapping approach that screens mutants in a sterile interspecific hybrid, we identified eight genes that underlie the growth advantage of Saccharomyces cerevisiae over its sister species S. paradoxus at high temperature. All eight encode housekeeping factors with no known direct function in heat-shock or stress response. Pro-thermotolerance alleles at these mapped loci were required for the adaptive trait in S. cerevisiae and sufficient for its partial reconstruction in S. paradoxus. Together, our data reveal the genetic mechanism by which S. cerevisiae acquired its high-temperature growth advantage in the distant past. And our study lays the groundwork for the mapping of genotype to phenotype in clades of sister species across Eukarya.

Abstract

The evolution of obligate plant pathogens like smut fungi is often characterized by lineage tracking resulting in host specificity and one-to-one relationships. Therefore, adaptation and specialization to the host plant seems to be crucial and should involve genes or regulatory pathways governing host specificity. To identify genes relevant for host specificity of Microbotryum species, we produced artificial hybrids between the two host-specific species M. lychnidis-dioicae and M. silenes-acaulis and applied strong experimental selection on different host plants to identify genes necessary for successful infections. Genome comparison of the two species revealed that most gene families are shared and the majority of genes are conserved, indicating very similar biological features of both species, including host adaptation and infection processes. Lower nucleotide identity of genes encoding for secreted proteins might indicate their importance for host specific interaction, as it is known from other plant pathogens. Moreover, we identified 211 candidate genes that occur in each hybrid and backcross genome that were posed under host-driven selection and might therefore play a crucial role in host specialization. The analysis of hybrid genomes also demonstrates the effect of genetic homogeneity on the fitness of hybrid individuals including the occurrence of species-specific mating type chromosomes. We analyze the evolution of candidate genes in the context of the whole genus and will discuss their potential contribution to host specificity. These studies should contribute to our functional understanding of the evolution of host specificity and its relevance for systematics of the genus Microbotryum.

Abstract

Cellulose represents the most abundant carbohydrate in terrestrial ecosystems and a major carbon and energy source for saprotrophic fungi. Plant cell-wall cellulose is found in an amorphous form, where cellulose chains are loosely interwoven and in a crystalline form, where the chains interact with hydrogen bonds and Van der Waals forces to form highly crystalline resistant to decomposition fibers. White-rot wood decayers employ a diverse set of enzymes during decomposition that cause a combination of hydrolysis (cellobiohydrolases, endoglucanases) and oxidation (lytic polysaccharide monooxygenases) of cellulose. While decomposition progresses, large amounts of crystalline cellulose are left behind in the decomposed wood. In contrast, brown-rot fungi secrete mostly endoglucanase during cellulose decomposition, while their genomes mostly lack cellobiohydrolase and lytic polysaccharide monooxygenase genes. In place of the costly enzymes brown-rot fungi are thought to employ a non-enzymatic mechanism that involves the generation of the Fenton reaction and the subsequent generation of hydroxyl radicals. The hydroxyl radicals in return are thought to affect the crystallinity of cellulose disrupting its structure rendering the fibers susceptible to degradation from endoglucanases. Brown-rot wood decay is efficient and results in the complete decomposition of all carbohydrates in wood. To what degree enzymatic versus non-enzymatic decomposition affects the crystallinity of cellulose is not well established. To examine this, we grew a strain of a Gloeophyllum species (brown rot) and a strain of Phanerochaete laevis (whit rot) on high-quality filter paper as a carbon source for 40 days and we examined the effect of the two fungi on the crystallinity of cellulose using Raman spectroscopy. Our results show that Gloeophyllum had a pronounced impact on the structure of cellulose, while P. laevis had a very small effect. This suggests that brown-rot fungi might target the higher structure of cellulose in order to make it more accessible to the action of endoglucanases, while white-rot fungi degrade cellulose without altering its higher structure. To further test this method we performed the same experiment for eight litter decomposers across Agaricales. The distinction between white-rot and brown-rot fungi cannot be applied for litter decomposers and therefore, a way to explore further the decomposition of cellulose by such species is needed. Six of the species investigated had a very small effect on the structure of cellulose, similarly to P. laevis. However, two species modified the structure of cellulose to an intermediate degree between P. laevis and Gloeophyllum. Our results suggest that using Raman spectroscopy is a promising method to distinguish cellulose degradation between white-rot and brown-rot fungi. Furthermore, the results for litter decomposers indicate that using this method we will be able to explore the mechanisms of cellulose degradation in fungi with less studied decomposition strategies.

Abstract

Comparative genomic approaches, such as the comparison of gene content or gene expression levels, among related species with distinct niches offer powerful tools to understand the genomic basis of adaptation in non-model organisms. Combining both classic comparative and functional genomic approaches in an integrated fashion is essential to our ability to distinguish changes of functional importance for a particular phenotype from incidental ones, or to understand the relative contribution of different evolutionary processes. Here, we use an integrated approach, based on phylogenomic reconstruction of gene content in combination with gene expression profiling on key substrates to study specialisation of wood decay in the house invading fungus Serpula lacrymans (Basidiomycota, Boletales, Serpulaceae). This fungus is invasive to the built environment in Europe and distinguishes itself from its wild relatives through a particularly aggressive wood decay and ability to infect dry and patchy habitats. Our analysis includes four individuals of three species within the Serpulaceae: two individuals of the invasive house-living S. lacrymans var. lacrymans, the wild sister species S. lacrymans var. shastensis and the widely distributed wild relative S. himantioides as an outgroup. Using an integrated analysis strategy, we have investigated i) the relative importance of gene duplication and loss compared to changes in gene expression for the fine-tuning of wood decay, and ii) the timing of such changes with respect to invasion of the built-environment. Results show a shift from generalist to specialist decay strategy in the ancestor of the Serpula lacrymans varieties. This is reflected in strong, conserved differentiation of gene sets active on spruce or pine substrates in these species, while the generalist S. himantioides expresses largely the same genes on both types of wood. Functional analysis of differentially regulated genes indicate that gene sets expressed on spruce and pine may reflect different decay stages. Among all wood-induced genes in the var. lacrymans strain from Europe, 7.6% arose in the common ancestor of vars. lacrymans and shastensis, 2.1% arose in the common ancestor of var. lacrymans and 6.1% were strain-specific, suggesting a considerable contribution of gene duplication.

Abstract

Virtually all terrestrial plants depend on symbiotic interactions with fungi. Symbiotic Arbuscular mycorrhizal (AM) fungi evolved over 450 million years ago and were instrumental for the colonization of land. Mediating nutrient uptake and sequestering carbon in soil this symbiosis lies at the core of all terrestrial ecosystems. AM fungi are obligate biotrophs and cannot complete their life cycle without obtaining carbon from host roots. In return they provide their host with nutrients, such as phosphorus. In contrast to the fungi, plants are facultative mycotrophs, but under natural conditions all host roots are colonized as a result of multiple beneficial effects of AM fungi. The evolutionary stability of this symbiosis is exceptional given that both host plants and symbiotic fungi are promiscuous, forming interactions across individuals and species. In the absence of host - symbiont specificity and given their inability to discriminate among partners prior to interaction, evolutionary theory predicts that “free riders” would evolve and spread. Free riders are cheaters that benefit from the interaction without providing significantly in return. However, under natural conditions this has not crippled the function of the AM symbiosis.

In our group we combine single nuclei sequencing methods with estimates of symbiotic efficiency experiments to study the evolutionary stability of AM fungi. Specifically, we try to evolve cheating strains by allowing AM fungi to selectively adapt to different host plants and comparing how symbiotic efficiency develops in response to host adaptation. If cheater strains evolve these are expected to trade less phosphorus per unit carbon obtained from the host, because phosphorus is limiting in our study system. The link between rates of trade, RNA expression in roots and strain genome structure will be explored.

Earlier hypothesis that AM fungi may be heterokaryotic, meaning that they harbor genetically distinct nuclei in their coenocytic mycelia has been challenged by recent genome sequencing of the AM fungi Rhizophagus irregularis. We are now able to explore the generality of these new findings thanks to our single nuclei sequencing method, originally developed to generate reference genomes for our experimental studies.

Abstract

Fungal endophytes are ubiquitous in plant leaves. While some endophytes are commensal symbionts, others can modify plant disease severity either by interacting directly with pathogens (e.g., mycoparisitism, antibiosis) or by altering the plant defense response. It is often assumed that species within common fungal endophyte genera, e.g. Cladosporium, Alternaria, Epicoccum, exhibit cosmopolitan distributions. However, few studies have investigated the phylogeographic structure of common endophytes, or tested for variation in ecological function within clades. We examined Cladosporium, a ubiquitous, wind-dispersed endophyte associated with the leaves of the model tree, Populus trichocarpa (black cottonwood). Cladosporium populations were sampled by amplicon metabarcoding leaf samples (ITS1) and multilocus sequence typing (5 genes: ITS and partial actin, β-tubulin, ef1α, rpb2) of 96 Cladosporium cultures collected from eight sites spanning the core of the tree’s geographic range and a strong climatic gradient from west (wet) to east (dry) of the Cascade Range in the Pacific Northwest of North America. Our ongoing experiments test the degree of rust pathogen antagonism (via mycoparisitism) across the 96 strains using both in agaro and in planta assays. Our multigene phylogeny supports ~15 previously described species within Cladosporium, and as many as five undescribed species. While the majority of these species are thought to have near global distributions, ITS1 metabarcodes and multigene data both indicate strong phylogeographic structure across the study area with particular species largely restricted to either west or east of the Cascade Range. We are working to link the phylogenetic structure of Cladosporium endophytes to variation in mycoparisitism. In addition, we are sequencing the genomes of a subset of isolates and will use comparative genomics to explore environmental adaptation and the genomic basis for mycoparasitism.