Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy John W. Mishler-Elmore May 2020 © 2020 John W. Mishler-Elmore. All Rights Reserved. 2 This dissertation titled Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase by JOHN W. MISHLER-ELMORE has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Michael A. Held II Associate Professor of Chemistry and Biochemistry Florenz Plassmann Dean, College of Arts and Sciences 3 ABSTRACT MISHLER-ELMORE, JOHN W., Ph.D., May 2020, Chemistry Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase Director of Dissertation: Michael A. Held II Extensin peroxidases play a critical role in plant cell growth and are believed to play equally important roles in defense from pathogenesis and mechanical stress. By catalyzing the covalent polymerization of extensin proteins, they participate in the formation of the cell plate for cell division and help to reinforce the wall—preventing pathogen infection. Due to it’s anionic character and catalytic processivity, TomEP is a particularly unique extensin peroxidase that requires much less time and enzyme than other extensin peroxidases to crosslink extensin substrate. Previous work identified the TomEP gene, and established methods to produce functional enzyme through heterologous expression in E. coli. This work aimed to expand upon these previous efforts by characterizing TomEP expression, TomEP function in vivo, and design a purification scheme to produce milligram-level quantities of pure enzyme for crystallization. An expression profile of TomEP was compiled using both qPCR analysis and promoter-GUS fusion experiments to provide data describing normal expression and response to wounding. Basal TomEP expression was demonstrated to be significantly higher in roots than in flowers, stems, or leaves. Through the same methods, wounding 4 treatments were shown to increase TomEP expression in tomato roots from one to four hours, followed by attenuation for the following sixteen hours. The foundations of gain and loss-of-function experiments were pursued in an attempt to discern TomEP’s influence on di-isodityrosine and pulcherosine content in tomato cell walls, using overexpression and CRISPR knock-out strategies. Overexpression lines of tomato and Arabidopsis were generated using Agrobacterium mediated methods, though these efforts failed to produce verifiable protein product, despite expression being observed on the RNA level. Transient expression in tobacco epidermal cells was successful however, allowing for in vivo analysis of TomEP activity, though no clear link between TomEP and di-isodityrosine or pulcherosine formation could be made. CRISPR knock-out lines of tomato were successfully generated, with two lines likely possessing homozygous or chimeric mutant alleles and fifteen others appearing to have heterozygous mutant alleles. No cell wall analysis of mutants could be performed without further breeding and characterization, but the path has been blazed for future research. Utilizing an established oxido-shuffling strategy to fold protein extracted from E. coli inclusion bodies, milligram quantities of recombinant TomEP were produced and purified. The previous purification scheme was unable to accommodate larger reaction volumes and additional protein product and required alterations to scale up production. Techniques utilized include ammonium sulfate precipitation, immobilized metal affinity purification, anion exchange chromatography, and size exclusion chromatography. Using 5 these methods, the milligram threshold was exceeded using the new purification scheme, enabling future x-ray crystallography. 6 DEDICATION To my wife Beth, whose faith was unwavering, my parents Cindy and Bill who supported us through this journey, and my grandparents Verda, Bill, and Liz who have always believed in me. A special mention goes to Shelby, Paddy, and most especially Louise for their companionship and unending love. 7 ACKNOWLEDGMENTS I would like to thank my advisor Dr. Michael Held for his endless support and advice that helped so much over the years, as well as during the production of this document. Overwhelming thanks goes to Dr. Marcia Kieliszewski and Dr. Allan Showalter who provided feedback throughout the process to help elevate the quality of this work. Additionally, I would like to thank Dr. Lauren McMills for agreeing to aid in the final steps of this journey. Thanks also goes to my peers and colleagues who provided both support and camaraderie. Tasleem Javaid for our continuing discussions on topics where our research overlapped and helping with plant care. Ali Aldhumani for technical assistance and friendship from beginning to end. And of course, my lab mates, past and present: Wen Dong, Daniel Nething, Yadi Zhou, Dare Ogunsekan, and Abhijit Sukul. 8 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 6 Acknowledgments............................................................................................................... 7 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 List of Abbreviations ........................................................................................................ 13 Chapter 1: Introduction ..................................................................................................... 14 1.1 The Plant Cell Wall ............................................................................................... 14 1.1.1 Function and Significance to Society .......................................................... 14 1.1.2 Components and Organization ..................................................................... 16 1.1.3 Class III Plant Peroxidases........................................................................... 32 1.1.4 TomEP ......................................................................................................... 38 Chapter 2: Materials and Methods .................................................................................... 42 2.1. Plant Material and Growth Conditions ................................................................ 42 2.1.1 Sterilization, Aseptic Culture, and Care of Tomato Seedlings .................... 42 2.1.2 Explant Collection and Plating for Tomato Transformations ...................... 42 2.1.3 Sterilization, Aseptic Culture, and Care of Arabidopsis plants ................... 44 2.1.4 Tobacco Sowing and Care ........................................................................... 45 2.2 Cloning and Binary Vector Assembly .................................................................. 45 2.2.1 Overexpression Construct Assembly ........................................................... 45 2.2.2 Promoter-GUS Construct Assembly ............................................................ 46 2.2.3 TomEP CRISPR Knock-out Construct Assembly ....................................... 48 2.2.4 Agrobacterium Transformation ................................................................... 49 2.3 Transformations and Selection ............................................................................. 50 2.3.1 Transient Expression of TomEP-mGFP and Extensin Analogs in Tobacco Epidermal Cells ..................................................................................................... 50 2.3.2 Generation of Transgenic Tomato Lines ..................................................... 51 2.3.3 Arabidopsis Transformation Using the Floral Dip Method ......................... 53 2.3.4 PCR Genotyping of Transgenic Plant Lines ................................................ 54 2.4 RNA Preparation, cDNA Synthesis, RT-PCR, and qPCR.................................... 55 2.4.1 RNA Extraction ........................................................................................... 55 2.4.2 Reverse Transcription of RNA Samples ...................................................... 56 2.4.3 Semi-Quantitative RT-PCR and qPCR ........................................................ 56 2.5 Analysis of Transgenic Plants and Tissues ........................................................... 58 2.5.1 Live Cell Imaging by Confocal Laser Scanning Microscopy ...................... 58 2.5.2 Fluorometric Detection of GUS Expression ................................................ 58 2.5.3 Preparation of Alcohol Insoluble Residue (AIR)......................................... 59 2.5.4 HPLC Analysis of Tyrosine Derivatives from AIR ..................................... 60 2.5.5 Western Blot Analysis of Overexpression Lines ......................................... 61 2.6 Heterologous Expression, Folding, and Purification of recombinant TomEP...... 62 9 2.6.1 ABTS Assay of POX Activity ..................................................................... 62 2.6.2 Inoculation of