The fission yeast clade, which has a distinct life history from other yeasts, can provide important clues about evolutionary changes. To reveal these changes the large S. cryophilus supercontigs were assembled into chromosomes using synteny relationships and the conserved pericentromeric, subtelomeric genes. Togetherness of the supercontigs was confirmed by PCR. Investigation of the gene order revealed localisation of the rDNA arrays, more than 300 new conserved orthologues and proved that S. cryophilus supercontigs were mosaics of collinear blocks. PFGE analysis showed that size of the S. cryophilus chromosomes differ from the S. pombe chromosomes. Comparative genomic analyses of the newly assembled chromosomes confirmed that the closest relative of S. cryophilus was S. octosporus not just in sequence similarity but also in a structural way, and revealed that preservation of the conserved regions did not arise from the lower number of chromosomal rearrangements. Translocations were more typical in the closely related species, while the number of inversions increased with the phylogenetic distances. Our data suggested that sites of the chromosomal rearrangements were not random and often associated with repetitive sequences, structural- and nucleotide evolution might correlate. Chromosomal rearrangements of the fission yeasts compared to other lineages were also discussed.

The fission yeasts (Schizosaccharomyces) representing a highly divergent phylogenetic branch of Fungi evolved from filamentous ancestors by gradual transition from mycelial growth to yeast morphology. For the transition, a mechanism had been developed that separates the sister cells after the completion of cytokinesis. Numerous components of the separation mechanism have been characterised in Schizosaccharomycespombe, including the zinc-finger transcription factor Ace2p and the fork-head transcription factor Sep1p. Here we show that both regulators have regions conserved within the genus. The most conserved parts contain the DNA-binding domains whose amino-acid sequences perfectly reflect the phylogenetic positions of the species. The less conserved parts of the proteins contain sequence blocks specific for the whole genus or only for the species propagating predominantly or exclusively as yeasts. Inactivation of either gene in the dimorphic species Schizosaccharomycesjaponicus abolished cell separation in the yeast phase conferring hypha-like morphology but did not change the growth pattern to unipolar and did not cause extensive polar vacuolation characteristic of the true mycelium. Neither mutation affected the mycelial phase, but both mutations hampered the hyphal fragmentation at the mycelium-to-yeast transition. Ace2p(Sj) acts downstream of Sep1p(Sj) and regulates the orthologues of the Ace2p-dependent S.pombe genes agn1(+) (1,3-alpha-glucanase) and eng1(+) (1,3-beta-glucanase) but does not regulate the orthologue of cfh4(+) (chitin synthase regulatory factor). These results and the complementation of the cell separation defects of the ace2(-) and sep1(-) mutations of S.pombe by heterologously expressed ace2(Sj) and sep1(Sj) indicate that the cell separation mechanism is conserved in the Schizosaccharomyces genus.

The capability of RNA isolation with good efficiency and high quality is essential for a downstream application such as RNA sequencing. It requires successful cell culturing and an effective RNA isolation method. Although effective methods are available, production of the homogenous mycelia and extraction of good-quality mycelial RNA from true invasive hyphae, which penetrated into the agar plates, are difficult. To overcome these problems, the aim of this study was to develop technical modifications which allow production of homogenous mycelial biomass without extra stimuli agents and improve quality of the RNA extracted from the fungal hyphae. Our alternative culture medium was suitable for production both yeast-phase cells and hyphae of the Schizosaccharomyces japonicus and other dimorphic species, such as the Candida albicans, Saccharomyces cerevisiae, and Jaminaea angkorensis. To improve quality of the mycelial RNA, we developed an isolation procedure of the hyphal tip, which eliminated the unnecessary vacuoles-containing parts of the hyphae. To increase RNA quantity, we used glass beads in the RNA extraction protocol to achieve stronger breaking of the mycelial walls. All these modifications can also be useful for researchers working with other dimorphic fungi and can contribute to the higher comparability of the transcriptional data coming from yeast-phase cells and hyphae or even from different species.

The plant microbiome is an increasingly intensive research area, with significance in agriculture, general plant health, and production of bioactive natural products. Correlations between the fungal endophytic communities and plant chemistry can provide insight into these interactions, and suggest key contributors on both the chemical and fungal side. In this study, roots of various horseradish (Armoracia rusticana) accessions grown under the same conditions were sampled in two consecutive years and chemically characterized using a quality controlled, untargeted metabolomics approach by LC-ESI-MS/MS. Sinigrin, gluconasturtiin, glucoiberin, and glucobrassicin were also quantified. Thereafter, a subset of roots from eight accessions (n = 64) with considerable chemical variability was assessed for their endophytic fungal community, using an ITS2 amplicon-based metagenomic approach using a custom primer with high coverage on fungi, but no amplification of host internal transcribed spacer (ITS). A set of 335 chemical features, including putatively identified flavonoids, phospholipids, peptides, amino acid derivatives, indolic phytoalexins, a glucosinolate, and a glucosinolate downstream product was detected. Major taxa in horseradish roots belonged to Cantharellales, Glomerellales, Hypocreales, Pleosporales, Saccharomycetales, and Sordariales. Most abundant genera included typical endophytes such as Plectosphaerella, Thanatephorus, Podospora, Monosporascus, Exophiala, and Setophoma. A surprising dominance of single taxa was observed for many samples. In summary, 35.23% of reads of the plant endophytic fungal microbiome correlated with changes in the plant metabolome. While the concentration of flavonoid kaempferol glycosides positively correlated with the abundance of many fungal strains, many compounds showed negative correlations with fungi including indolic phytoalexins, a putative glucosinolate but not major glucosinolates and a glutathione isothiocyanate adduct. The latter is likely an in vivo glucosinolate decomposition product important in fungal arrest. Our results show the potency of the untargeted metabolomics approach in deciphering plant–microbe interactions and depicts a complex array of various metabolite classes in shaping the endophytic fungal community.

Fungal pathogens, from phytopathogenic fungus to human pathogens, are able to alternate between the yeast-like form and filamentous forms. This morphological transition (dimorphism) is in close connection with their pathogenic lifestyles and with their responses to changing environmental conditions. The mechanisms governing these morphogenetic conversions are still not fully understood. Therefore, we studied the filamentous growth of the less-known, non-pathogenic dimorphic fission yeast, S. japonicus, which belongs to an ancient and early evolved branch of the Ascomycota. Its RNA sequencing revealed that several hundred genes were up- or down-regulated in the hyphae compared to the yeast-phase cells. These genes belonged to different GO categories, confirming that mycelial growth is a rather complex process. The genes of transport- and metabolic processes appeared especially in high numbers among them. High expression of genes involved in glycolysis and ethanol production was found in the hyphae, while other results pointed to the regulatory role of the protein kinase A (PKA) pathway. The homologues of 49 S. japonicus filament-associated genes were found by sequence alignments also in seven distantly related dimorphic and filamentous species. The comparative genomic analyses between S. japonicus and the closely related but non-dimorphic S. pombe shed some light on the differences in their genomes. All these data can contribute to a better understanding of hyphal growth and those genomic rearrangements that underlie it.