Understanding endomembrane trafficking in plant cells using chemical genetics approach

Like other eukaryotic cells, plant cells contain an endomembrane system composed of compartmentalized organelles with specialized functions. Vesicle trafficking mediates the transport of materials between different organelles and between cells and the environment. The vesicle trafficking process is highly dynamic and plays essential roles in maintaining cellular homeostasis and environmental adaptation. Because of the essential roles of vesicle trafficking in plant growth and development, genes that are involved in vesicle trafficking often have redundant function when they exist as a large family or cause embryonic lethality when they exist as a signal gene or small gene family. Chemical genetics uses small molecule inhibitors to affect protein function without interfering with plant’s genome. Bioactive small molecules can generate a temporary perturbation of a biological system in a reversible and dose-dependent fashion, which allow us to observe dynamic cellular processes and discover new components in trafficking machineries. We recently discovered two small molecules named Endosidin2 (ES2) and Endosidin20 (ES20) that disrupt vesicle trafficking in plants. ES2 inhibits exocytosis by targeting the EXO70A1 subunit of the exocyst complex in plant cells. ES20 targets cellulose synthase (CESA) at the catalytic site and inhibits the delivery of Cellulose Synthase Complex (CSC) to the plasma membrane. This research thesis aims to characterize the specificity of ES2 on EXO70 homologs and identify new genes that mediate CSC trafficking. Drug Affinity Responsive Target Stability (DARTS) assay was used to test the specificity of ES2 in targeting different EXO70s in Arabidopsis. Chemical genetic screen for mutants that have increased sensitivity was conducted to identify novel genes related to CSC trafficking. This project provides new insights in the specificity of ES2 in targeting different EXO70s in plants and the regulatory mechanisms of CSC trafficking that control plant cellulose synthesis.