Elucidating a Conserved Myosin XI-RabE GTPase Interaction that is Required for Polarized Cell Growth in Plants A Dissertation Submitted to the Faculty of WORCESTER POLYTECHNIC INSTITUTE in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biology and Biotechnology July 31, 2020 by Robert George Orr Approved by: Dr. Luis Vidali Dr. Mary Munson Associate Professor Professor Dept. Biology & Biotechnology Dept. Biochemistry and Molecular Worcester Polytechnic Institute Pharmacology University of Massachusetts Medical School Dr. Elizabeth F. Ryder Dr. Amity L. Manning Associate Professor Assistant Professor Dept. Biology & Biotechnology Dept. Biology & Biotechnology Worcester Polytechnic Institute Worcester Polytechnic Institute Table of Contents Page List of Figures .................................................................................................................. iii Acknowledgements ......................................................................................................... v Preface ............................................................................................................................vi Abstract ........................................................................................................................... 1 Chapter 1 : Polarized Cell Growth in Plants and the Loss-of-Function Tools that Facilitate Our Understanding ........................................................................................... 3 1.1 Introduction ............................................................................................................ 4 1.2 Physcomitrella/Physcomitrium patens: A Versatile Plant Model for Cellular Polarized Growth ......................................................................................................... 7 1.3 Cytoskeletal Coordination ...................................................................................... 9 1.4 Exocytosis ............................................................................................................ 14 1.4.1 Rab GTPases ................................................................................................ 16 1.5 The Apical Plasma Membrane ............................................................................. 17 1.6 Endocytosis .......................................................................................................... 18 1.7 Outlook and Insights from Other Tip Growing Systems ....................................... 20 1.8 Directed Loss-of-Function Approaches in Plant Functional Genomics ................ 22 1.8.1 Homology Directed Repair ............................................................................. 23 1.8.2 CRISPR/Cas .................................................................................................. 24 1.8.3 RNA Interference ........................................................................................... 29 1.9 Concluding Remarks ............................................................................................ 33 Chapter 2 : Robust survival-based RNAi of gene families using in tandem silencing of adenine phosphoribosyltransferase .............................................................................. 35 2.1 Abstract ................................................................................................................ 36 2.2 Introduction .......................................................................................................... 36 2.3 Results ................................................................................................................. 41 2.3.1 Development of A Novel Positive Selection RNAi Methodology Based on Endogenous APT Activity ....................................................................................... 41 2.3.2 APTi Experimental Design for High-throughput Phenotyping ........................ 45 i 2.3.3 The APTi System Silences the Myosin XI Family with High Efficacy ............. 49 2.3.4 APTi-based Silencing of the Lyk5 Family Disrupts P. patens’ Response to Chitin Oligosaccharides .......................................................................................... 56 2.4 Discussion ............................................................................................................ 62 2.5 Conclusions ......................................................................................................... 67 2.6 Materials and Methods ......................................................................................... 67 2.6.1 Plant Materials and Culture Conditions .......................................................... 67 2.6.2 APT-based RNAi Construct Design ............................................................... 68 2.6.3 APTi Phenotyping Assay ............................................................................... 70 2.6.4 Analysis of Myosin XI Protein Abundance ..................................................... 71 2.6.5 Lyk5-RNAi Construct Design and Functional Assay ...................................... 72 Chapter 3 : RabE and its Interaction with Myosin XI are Essential for Cell Polarization and Growth .................................................................................................................... 75 3.1 Abstract ................................................................................................................ 76 3.2 Introduction .......................................................................................................... 77 3.3 Results ................................................................................................................. 80 3.3.1 RabE Localizes to Sites of Polarized Secretion and Colocalizes with Myosin XI ............................................................................................................................ 80 3.3.2 RabE is Required for Normal Growth in P. patens, and A. thaliana RabE1c Rescues the rabE mutant ....................................................................................... 85 3.3.3 Myosin XI is a Conserved Effector of RabE, and A. thaliana Myosin Isoforms XI-K and XI-E Rescue Loss of Endogenous Myosin XI in P. patens. ...................... 94 3.3.4 Myosin XI:RabE Interface Prediction ............................................................. 97 3.4 Discussion .......................................................................................................... 107 3.5 Materials and Methods ....................................................................................... 111 3.5.1 Yeast Two-Hybrid Assay.............................................................................. 111 3.5.2 Construction of Fluorescently Tagged RabE Moss Lines ............................ 112 3.5.3 Live-cell Confocal Imaging and Kymograph Analysis .................................. 112 3.5.4 Homology Modeling and Interface Prediction .............................................. 113 3.5.5 CRISPR Knockout of RabE14 and RabE15................................................. 114 3.5.6 RNAi Growth Assays ................................................................................... 115 3.5.7 Phylogenetic Analysis .................................................................................. 116 3.5.8 Western Blot Analysis .................................................................................. 117 3.5.9 Myosin XI Protoplast Expression ................................................................. 117 ii 3.5.10 Myosin XI-CCT Purification ........................................................................ 118 3.5.11 Statistical Analyses .................................................................................... 119 Chapter 4 : Future Directions and Conclusions ........................................................... 125 4.1 Extension of the APTi System for Long-Term Imaging and Conditional Activation ................................................................................................................................. 126 4.2 Further Characterization of the Myosin XI:RabE Interaction .............................. 127 4.3 Conclusions ....................................................................................................... 130 Chapter 5 : References ............................................................................................... 132 List of Figures Figure 1.1 Drawings depicting tip-growing cell types in mosses, liverworts, and flowering plants. .............................................................................................................................. 5 Figure 1.2 A stylized perspective of three tip growing plant systems and a working model of exocytosis ....................................................................................................... 12 Figure 1.3 Model depicting the apex of a generic tip-growing plant cell as a cycling center that coordinates exo- and endocytic activity. ...................................................... 21 Figure 1.4 CRISPR\Cas9 mediated loss-of-function approaches. ................................. 26 Figure 1.5 RNAi-mediated loss-of-function. ................................................................... 31 Figure 2.1 Proof-of-principle and construction of APT-based RNAi (APTi) vectors to enable positive selection of actively silencing plants. .................................................... 44 Figure 2.2