Using CRISPR Induced Mutations to Investigate Proteins Contributing to Polarized Growth in Plants
Abstract
Recently, a novel member of the Vacuolar Protein Sorting 26 gene family (VPS26C) has been characterized in Arabidopsis thaliana and human cell culture. Classically known as a member of the heterotrimeric large retromer subunit, VPS26C has been shown to be a member of a retriever complex required for the recycling of plasma membrane proteins in human cells and polarized growth and cell wall organization in Arabidopsis thaliana roots (McNally et al., 2017; Jha et al., 2018). vps26c lines of Arabidopsis expressing a GFP fusion to the Arabidopsis or human ortholog of VPS26C resulted in the complementation of the short root hair phenotype of vps26c plants, demonstrating the deep conservation of VPS26C function between plant and animal cells. While a repertoire of proteins associated with the human VPS26C ortholog are known, little research has been done to elucidate proteins that interact with VPS26C in plant cells. Here, we begin to characterize two Arabidopsis homologs known to interact with VPS26C in humans, CCDC22 and CCDC93. Arabidopsis lines containing T-DNA insertions in CCDC22 exhibit root hairs that are significantly shorter than those of wild type seedlings when grown on standard growth media. When grown on media supplemented with mannitol, a condition that creates osmotic stress, CCDC93 T-DNA mutants also exhibit a short root hair phenotype. These results suggest that CCDC22 and CCDC93 play a role in the polarized growth of plant cells. To investigate the possible intersection of the VPS26C/retriever pathway and the functions of CCDC22 and CCDC93 in plants, co-localization studies are underway and double mutants are being generated using CRISPR.
Primary Faculty Mentor Name
Mary Tierney
Status
Graduate
Student College
College of Agriculture and Life Sciences
Program/Major
Plant Biology
Primary Research Category
Biological Sciences
Using CRISPR Induced Mutations to Investigate Proteins Contributing to Polarized Growth in Plants
Recently, a novel member of the Vacuolar Protein Sorting 26 gene family (VPS26C) has been characterized in Arabidopsis thaliana and human cell culture. Classically known as a member of the heterotrimeric large retromer subunit, VPS26C has been shown to be a member of a retriever complex required for the recycling of plasma membrane proteins in human cells and polarized growth and cell wall organization in Arabidopsis thaliana roots (McNally et al., 2017; Jha et al., 2018). vps26c lines of Arabidopsis expressing a GFP fusion to the Arabidopsis or human ortholog of VPS26C resulted in the complementation of the short root hair phenotype of vps26c plants, demonstrating the deep conservation of VPS26C function between plant and animal cells. While a repertoire of proteins associated with the human VPS26C ortholog are known, little research has been done to elucidate proteins that interact with VPS26C in plant cells. Here, we begin to characterize two Arabidopsis homologs known to interact with VPS26C in humans, CCDC22 and CCDC93. Arabidopsis lines containing T-DNA insertions in CCDC22 exhibit root hairs that are significantly shorter than those of wild type seedlings when grown on standard growth media. When grown on media supplemented with mannitol, a condition that creates osmotic stress, CCDC93 T-DNA mutants also exhibit a short root hair phenotype. These results suggest that CCDC22 and CCDC93 play a role in the polarized growth of plant cells. To investigate the possible intersection of the VPS26C/retriever pathway and the functions of CCDC22 and CCDC93 in plants, co-localization studies are underway and double mutants are being generated using CRISPR.