UC Riverside

TANGLED1 (TAN1) is a microtubule binding, division-site-localized protein that plays a role in division plane orientation in plants. Although many potential TAN1 interacting proteins have been identified, how TAN1 interacts with other proteins to ensure correct division positioning is not well understood. Arabidopsis thaliana tan1 single mutants have very weak division plane orientation defects. However mutating a second gene that encodes the unrelated, division-site-localized protein AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9), results in strong synthetic phenotypes in the tan1 air9 double mutant including stunted growth, misoriented divisions, and increased cell file rotation. I designed a course-based undergraduate research experience as a high-throughput strategy to screen for TAN1 interactors. Students participated in a sensitized mutagenesis screen by learning how to use CRISPR-Cas9 technology to create gene knockouts of candidate TAN1 interactor genes in air9 single mutant Arabidopsis plants. I also investigated the functional role of TAN1 interaction with the division-site-localized kinesin PHRAGMOPLAST ORIENTING KINESIN1 (POK1). I found that POK1 was mislocalized in the tan1 air9 double mutant, which suggests that AIR9 and TAN1 are involved in POK1 localization to the division site. Using a yeast two-hybrid screen, I identified POK1 interaction sites within the first 132 highly conserved amino acids of TAN1. When mutagenized TAN1 that lost interaction with POK1 in the yeast two-hybrid system was introduced into the tan1 air9 double mutant, it failed to fully rescue phragmoplast positioning defects and disrupted TAN1 and POK1 recruitment to the division site during metaphase and early telophase. This demonstrated that TAN1 interaction with POK1 is important for stabilizing both proteins at the division site to ensure correct phragmoplast guidance during telophase, and that there are likely other unidentified proteins that further stabilize TAN1 and POK1 at the division site. Finally, I investigated the cause of defects in the nondividing cells of tan1 air9 double mutants, by driving TAN1 expression in the tan1 air9 double mutant using the well characterized G2/M-phase specific promoter of the KNOLLE gene. KNOLLE driven TAN1 fully rescued the tan1 air9 phenotypes, suggesting that defects that occur during mitosis can influence the organization of nondividing cells.