Characterization of the TOR Complex 1 in the model diatom Phaeodactylum tricornutum in response to nutrient availability.
Supervisors: Monia Teresa Russo, Alessandra Rogato (SZ Anton Dohrn, Naples)
Diatoms, one of the most important phytoplankton groups in the ocean, are known for their ubiquitous abundance and distribution in a wide range of aquatic environments. However, fundamental questions about their biology, in order to understand their ecological success, such as the ability to perceive and respond to environmental changes, remain largely unexplored. The TOR signaling pathway, which is highly conserved, is essential for integrating environmental cues coordinating cell growth with the cell cycle in eukaryotes. TOR (Target of Rapamycin), plays a fundamental role in signaling and regulating numerous essential functions for cell life, such as proliferation, protein synthesis, the regulation of metabolism and the process of autophagy and forms two functionally distinct complexes, TORC1 (TOR Complex 1) and TORC2 (TOR Complex 2). Therefore, the purpose of my thesis was to deepen the function of the TOR gene in the model diatom Phaeodactylum tricornutum with particular attention to the relationship with the nutritional state of the cell, through growth analysis, gene expression and methods of reverse genetics. Diatoms present only TORC1 composed of TOR, LST8 and Raptor genes. The expression profiles of TORC1 genes in wild-type cells grown in different N sources (NO3-, NH4+ and urea) indicated a significant downregulation of TOR for NH4+ and urea in comparison to the control (NO3-), with reductions of approximately 4 and 7 times, respectively. However, there was only a slight downregulation of LST8 and Raptor. In the case N deficiency condition (50 μM NO3-), there were no acceptable changes in gene expression observed compared to the control. In addition, a reverse genetics technique, CRISPR/Cas9 system, was adapted and carried out for generating knockout lines for these genes. Analysis of potential knockout mutants is ongoing through colony PCR screening. Now we are going through sequencing and analyzing the colonies. Next step will be functional and phenotypic characterization through physiological analysis under different parameters. Finally, the results allowed me to refine the information on the TORC1 complex genes in diatoms and their expression under different nutrient states.