Characterization Of A Novel Vps26c-Retromer Complex And Its Interaction With An Endosomal Trafficking Pathway Regulated By The Snare Vti13 In Controlling Polarized Growth And Cell Wall Organization In Arabidopsis Thaliana

The endosomal trafficking system is a network of highly coordinated cellular pathways that control the growth and function of cells. The coordination of secretion and endocytosis in cells is one of the primary drivers of polarized growth, where new plasma membrane and cell wall components are deposited at the growing apex. In plants, one of the cell types exhibiting polarized growth are the root hairs. Root hairs are regulated extensions of epidermal cells called trichoblasts and are essential for anchorage, absorption of water and nutrients, and plant-microbe interactions. In this thesis, I characterize a previously undescribed protein involved in retromer function and endosomal trafficking pathways that regulate tip growth in root hairs of Arabidopsis thaliana. The large retromer complex functions in recycling receptors in endosomal trafficking pathways essential for diverse developmental programs including cell polarity, programmed cell death, and shoot gravitropism in the model plant, Arabidopsis thaliana. I have characterized VPS26C, a novel member of the large retromer complex, that is essential in maintaining root hair growth in Arabidopsis. We used Bimolecular Fluorescence Complementation (BiFC) analysis to demonstrate thatVPS26C interacts with previously characterized core retromer subunits VPS35A and VPS29. Genetic analysis also indicates that vps26c suppresses the root hair growth and cell wall organization phenotypes of a null mutant of the SNARE VTI13 that localizes to early endosomes and the vacuole membrane, indicating a crosstalk between the VPS26C-retromer and VTI13-dependent vesicular trafficking pathways. Phylogenetic analysis was used to show that VPS26C genes are present in most angiosperms but appear to be absent in monocot genomes. Moreover, using a genetic complementation assay, we have demonstrated that VPS26C shares deep conservation of biochemical function with its human ortholog (DSCR3/VPS26C). We also used an affinity purification-based proteomic analysis to identify proteins associated with VTI13 in young seedlings. Preliminary results suggest that a number of proteins linked to cell plate organization in plants are associated with the VTI13 proteome, emphasizing the potential role of this pathway in new cell wall biosynthesis/organization. Additionally, we have identified endoplasmic reticulum (ER)-body proteins, involved in plant defense response pathways, suggesting that either the VTI13 endosomal trafficking pathway is functioning in plant defense responses, or the ER-body proteins have additional independent function(s) in Arabidopsis roots that depend on VTI13. In summary, I have described a novel retromer complex essential for polarized growth in Arabidopsis. VPS26C is an ancient gene and shares sequence and functional homology between human and Arabidopsis. vps26c is a genetic suppressor of the vti13dependent root hair growth and cell wall organization pathways. Proteomic analysis of VTI13 endosomes in young seedlings suggests that a number of proteins associated with cell plate formation are associated with VTI13 compartments, supporting the genetic analysis described here and serves as a starting point to further describe the role of this pathway in controlling polarized growth in plants.