Using Chemical Genetics to Dissect Exocytosis in Arabidopsis

Exocytosis is crucial for delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces, playing a vital role in normal plant development as well as responses to biotic and abiotic stresses. One key molecular player, the exocyst, is an octameric protein complex that tethers secretory vesicles to the plasma membrane (PM). Chapter 1 is a literature survey that introduces the function of the exocyst, as well as the characterization of Endosidin2 (ES2), a synthetic molecule that targets the EXO70 subunit of exocyst. This chapter also defines existing knowledge gaps in the profiling of cargo proteins trafficked by the exocyst and the identification of novel modulators of exocytosis. Chapter 2 employs a comparative proteomics approach to examine the changes of PM proteome of root cells following ES2-treatment. Proteins with decreased abundance at the PM were considered candidate cargo proteins of ES2-targeted trafficking and several were validated with quantitative live-cell imaging. Chapter 3 describes the use of ES2 as a tunable and reversible chemical genetics tool as demonstrated by the development and deployment of a large-scale mutant screen in Arabidopsis that identified 70 ES2-hypersensitive mutants (es2s). Among these, candidate mutations for 14 non-allelic lines were mapped and reported. T-DNA insertion lines were subsequently screened as alternative alleles to identify causal mutations. In Chapter 4, the causal mutation of es2s-15-12 was confirmed as ArgJ with a second T-DNA insertion mutant allele as well as genetic and chemical complementation. ArgJ encodes an enzyme in the arginine biosynthesis pathway. It was demonstrated that arginine biosynthesis deficiency synergizes with ES2 to inhibit root growth in Arabidopsis. Root growth in argj mutants was not hypersensitive to other inhibitors with different modes of action, such as LatB, ES9-17, and BFA. Additionally, roots of argj-1 displayed a reduced abundance of PIN2 at the apical PM in epidermal cells; however, PIN2 polar distribution was not further reduced by ES2 treatment. Our findings point to a functional connection between arginine metabolism and exocytosis. Chapter 5 discusses potential future directions and experiments, including technological advances and the testing of new hypotheses. Overall, this study presents a detailed application of chemical genetics to dissect the exocytosis process in Arabidopsis and uncovers novel modulators of exocytosis in plants.