Project description:Through transcriptome profiling using RNA-seq, we investigated the mechanisms behind bacterial endosymbiont (Burkholderia rhizoxinica) control over host (Rhizopus microsporus) reproductive biology. By analyzing differential expression across six different conditions, including fungal opposite mates growing independently with or without endosymbionts, as well as opposite mates growing together with endosymbionts (mating) or without endosymbionts (no mating), we were able to identify that endosymbionts control expression of a Ras signaling protein critical for sexual reproduction in many fungi (Ras2). As little is known regarding sexual reproduction in Mucoromycotina, we also used these data to investigate conservation of sex-related genes across all fungi, as well as predict potential genes involved in sensing of trisporic acid, the mating pheromone used by these fungi. 6 different conditions were analyzed, each consisting of two biological replicates. These included Rhizopus microsporus ATCC52813 (sex +) growing alone with endosymbionts, R. microsporus ATCC52814 (sex -) growing alone with endosymbionts, ATCC 52813 growing alone without endosymbionts, ATCC52814 growing alone without endosymbionts, ATCC52813 and ATCC52814 growing together with endosymbionts (successfully mating), and ATCC52813 and ATCC52814 growing together without endosymbionts (failure to mate). In each condition, fungi were cultivated on half-strength PDA and plugs of mycelium were placed at the edge of the plate. After 6 days, approximately 2.5 cm of tissue were harvested from the center of the plate. Each biological replicate consists of 5 plates which were pooled prior to RNA extraction to ensure sufficient tissue was collected.
Project description:Through transcriptome profiling using RNA-seq, we investigated the mechanisms behind bacterial endosymbiont (Burkholderia rhizoxinica) control over host (Rhizopus microsporus) reproductive biology. By analyzing differential expression across six different conditions, including fungal opposite mates growing independently with or without endosymbionts, as well as opposite mates growing together with endosymbionts (mating) or without endosymbionts (no mating), we were able to identify that endosymbionts control expression of a Ras signaling protein critical for sexual reproduction in many fungi (Ras2). As little is known regarding sexual reproduction in Mucoromycotina, we also used these data to investigate conservation of sex-related genes across all fungi, as well as predict potential genes involved in sensing of trisporic acid, the mating pheromone used by these fungi.
Project description:Wolbachia endosymbionts are widespread intracellular, maternally inherited bacteria which manipulate the host to favour their spread through a population. Cytoplasmic incompatibility (CI) and viral suppression are two such manipulations driven by uncharacterised mechanisms. To gain insight into potential molecular mechanisms responsible for these effects, we performed the first in-depth proteomic characterisation of the host response to Wolbachia infection. We find that the presence of the Wolbachia wMelPop in Aedes aegypti mosquito cells alters levels of proteins involved in cell cycle control, DNA replication, autophagy, vesicular trafficking, iron homeostasis, amino acid degradation, purine metabolism, lipid metabolism and immunity. The majority of the cell cycle/DNA replication proteins were downregulated in the presence of Wolbachia, in particular, a member of the anoctamin family was strongly down regulated which may be related to the chromosomal segregation defects observed in CI crosses. We found clear evidence for perturbed lipid metabolism, Wolbachia infected cells expressed higher levels of Apolipoprotein D and the cholesterol efflux transporter ABCA1, while the LDL receptor and fatty acid synthase were downregulated. ABCA1 was also upregulated at the mRNA level in adult Wolbachia infected Aedes aegypti mosquitoes. Wolbachia therefore perturbs cholesterol homeostasis causing the host cell to respond to an apparent cholesterol excess which may contribute to viral inhibition.