Defining the Molecular and Physiological Function of the Promiscuous Pontin/Reptin ATPase Complex in Plants and its Role in TOR Signaling

This work addresses the role of TARGET OF RAPAMYCIN (TOR) and the R2TP complex in plant development and defense, and also whether or not the R2TP complex is conserved in plants. TOR is the focus of many researchers interested in metabolic diseases and cancers since TOR dysregulation is a major cause of such diseases. The role of TOR in plants is relatively unexplored, although more work is being done on its role in key plant processes. TOR is a conserved Ser/Thr protein kinase that integrates many metabolic processes to coordinate growth and development with nutritional status in eukaryotes. TOR is often dysregulated in many human diseases including diabetes and cancer, and as such, has been relatively well studied in mammalian systems. The TOR protein and complex are well-conserved across the eukaryotic kingdom; however, their role in plant growth, development, and defense is not as well understood. Recent work in this emerging field has shown that TOR plays a key role in plants and may serve as a way for plants to fine-tune their balance between growth and defense. Chapter 1 is a summary of different plant defense processes that TOR controls in plants, and ways that it modulates the growth vs. defense tradeoffs. In chapter 2, I switch focus to the R2TP complex, which may play a role in TORC1 dimerization in plants. I use virus-induced gene silencing (VIGS) to silence two key components in the R2TP complex, Reptin and Pontin, which are two AAA+ ATPases. I also used VIGS to silence some of their putative interactors, including TOR, and look at the roles that Reptin, Pontin, and their interactors play in plant development and viral defense. Based off of phenotypic and transcriptomic analyses, it was shown that Reptin and Pontin knockdown plants show less viral spread and impaired growth. In chapter 3, we used a proximity labeling approach with TurboID, which is a promiscuous biotin ligase, to investigate the conservation of the R2TP complex in plants. The R2TP complex was first discovered in Saccharomyces cerevisiae as consisting of Reptin, Pontin, Spaghetti, and Pih1. When we did bioinformatic analyses, we saw that Pih1 does not exist in flowering plants, which leaves us to believe that the R2TP complex might exist with Pih1 in flowering plants, or the or the role of Pih1 is taken over by another candidate. We tagged Reptin, Pontin, and Spaghetti with TurboID and found that the R2TP complex is indeed conserved in A. thaliana, while also identifying some putative replacements for Pih1. In summary, TOR controls growth and defense both in plants, and it might be doing so through its relationship with Reptin and Pontin, which function in the R2TP complex. The work presented here also provides evidence to the conservation of the R2TP complex in A. thaliana. The R2TP complex and TOR both play important roles in growth, stress responses, and defense against pathogens, and so continued research directly focused on this topic in crop plants will aid agricultural efforts to breed resilient high-yield crops.

Advisor: Baker, Barbara