Lisa Wasserstrom:
Molecular mechanisms regulating sporulation in the filamentous fungus Ashbya gossypii

Date: 06-08-2014    Supervisor: Steen Holmberg

Sporulation is a camplex developmemal program that fungi enter to ensure survival in unfavorable environmental conditions. Many fungal species are able to produce spores sexually through meiosis, which is beneficial since it imroduces genetic variability into a population. The sexually reproducing ascomycete Saccharomyces cerevisiae has a well-defined sexual cycle, in which diploid cells can undergo meiosis and produce haploid spores in response to nutriem starvation. The diploid state is a requirement for meiosis and results from fusion of two haploid cells of the opposite mating type, which is regulated by the pheromone response pathway. Most ascomycetes have been reported to produce meiotic spores, however, a sexual cycle has not yet been identified in the filamenrous fungus Ashbya gossypii. The main focus of my doetoral thesis has therefore been to understand the mechanisms behind sporulation in this fungus.

The lifecycle of A. gossypii starting with a haploid spore that matures imo spore-comaining mycelia can be completed without the need for a mating partner. Spores in A. gossypii are thought to be derived sexually like all other Saccharomycetaceae species, but the sexual cycle remains unidentified. In this thesis I provide a comprehensive functional analysis of genes importam for sporulation in A. gossypii. Previous results, together with findings presemed in this work show that the role of the pheromone response pathway in A. gossypii has been rewired to regulate sporulation negatively instead of comrolling mating. In line with this, a mating partner might not be required since the multinudeate compartments could still enable nuclear fusion (karyogamy) and meiosis. The presence of karyogamy is supported by our results that deledon of the A. gossypii homolags Kar3 and Kar4, involved in karyogamy in S. cerevisiae, results in severely crippled sporulation.

This work also identifies the main regulators of sporulation in A. gossypii, namely IME1, IME2, IME4, KAR4 and NTD80. Using large scale RNA sequencing data of these non-sporulating deledon strains we were able to identifY 67 down-regulated genes, most of which were up-regulated in an oversporulating mutant, namely stei2. Imerestingly, transcription of the main regulator of meiosis in yeast, IME1, is regulared by a significandy smaller promoter in A. gossypii and is independem of the transcription factors Msn2 and Sok2. Furthermore, the role of Sok2 in A. gossypii has been rewired from a transcriptional repressar to an activator of sporulation. This is in comrast to S. cerevisiae, where Sok2 is a repressar of IME1 transcription while Msn2 and Msn4 function as activators.

A prerequisite for meiosis is meiotic recombination rhat allows eross-over between homologous chromosomes. In S. cerevisiae, this requires double strand break (DSB) formation and subsequem repair via the components Spol l and Dmcl. The work in rhis thesis show that the A. gossypii Spol l and Dmcl homolags are not required for sporulation, thus suggesting that other proteins generate DSBs in this fungus.

In summary, this work has led to better understanding of the componems regulating sporulation in A. gossypii and rheir hierarchical organization.