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: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:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used sucrose gradient centrifugation and Blue-Native PAGE to separate mitoribosomes of Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates.
Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.