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Investigation of the Anti-Virc2 Transcript in Agrobacterium Tumefaciens Abbagail Lauren Johnson Iowa State University

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Investigation of the Anti-Virc2 Transcript in Agrobacterium Tumefaciens Abbagail Lauren Johnson Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2016 Investigation of the anti-virC2 transcript in Agrobacterium tumefaciens Abbagail Lauren Johnson Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agricultural Science Commons, Agriculture Commons, Agronomy and Crop Sciences Commons, Biology Commons, and the Botany Commons Recommended Citation Johnson, Abbagail Lauren, "Investigation of the anti-virC2 transcript in Agrobacterium tumefaciens" (2016). Graduate Theses and Dissertations. 15943. https://lib.dr.iastate.edu/etd/15943 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigation of the anti-virC2 transcript in Agrobacterium tumefaciens by Abbagail Johnson A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Plant Biology Program of Study Committee: Kan Wang, Major Professor Gwyn Beattie W. Allen Miller Iowa State University Ames, Iowa 2016 Copyright © Abbagail Johnson, 2016. All rights reserved. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ......................................................................................... vii ABSTRACT………………………………. .............................................................. ix CHAPTER 1 INTRODUCTION .......................................................................... 1 General Introduction ............................................................................................ 1 Importance of Agrobacterium .............................................................................. 2 The Process of Plant Transformation ................................................................... 5 Importance of the virC Operon ............................................................................ 9 Non-coding Regulatory RNA .............................................................................. 14 Research Objectives and Thesis Organization ..................................................... 24 References ......................................................................................................... 27 CHAPTER II MATERIALS AND METHODS .................................................... 45 Bacterial Strains and Plasmids ............................................................................. 45 Transcript Quantification ..................................................................................... 51 Rapid Amplification of cDNA Ends .................................................................... 54 Western Blot Protocol ......................................................................................... 55 AGROBEST Experiments ................................................................................... 57 Tumorigenesis Assays Using Kalanchoe daigremontiana .................................. 62 iii Statistical Analysis ............................................................................................... 64 References ......................................................................................................... 68 CHAPTER III RESULTS .......................................................................................... 73 Selection of a Regulatory RNA in A. tumefaciens for Further Investigation ...... 73 Examination of anti-vir2 Transcription Start and Stop Sites ............................... 75 Generation of Deletion Mutants C58 ∆Pavc2 and C58 ∆virC2 ........................... 77 Analysis of Transcript Expression Level Changes in anti-virC2 Promoter Deletion Mutant C58 ∆Pavc2 .............................................................................. 78 Analysis of VirC2 Protein Expression in anti-virC2 Promoter Deletion Mutant ∆Pavc2 ..................................................................................................... 82 Examination of Virulence in A. tumefaciens Mutant Strains C58 ∆Pavc2 and C58 ∆virC2 .............................................................................. 84 References ......................................................................................................... 92 CHAPTER IV DISCUSSION AND CONSLUSIONS ........................................... 108 References ......................................................................................................... 115 APPENDIX A PRIMERS, FRAGMENTS, PLASMIDS AND STRAINS ............ 117 APPENDIX B SUPPLEMENTAL INFORMATION ............................................ 127 Introduction ......................................................................................................... 127 Overexpression of Individual anti-virC2 Regions and Intergenic Region .......... 128 pAJ025; Constitutive Expression of virC2 and Inducible Expression of anti-virC2......................................................................................................... 131 Additional FLAG-tagged Proteins ....................................................................... 132 References ......................................................................................................... 136 iv LIST OF FIGURES Page Figure 1 The T-DNA transfer process ...................................................................... 43 Figure 2 Illustration of virulence genes on Ti plasmid in A. tumefaciens ................ 44 Figure 3 General strategy for homologous recombination used to alter genomic sequences of A. tumefaciens. ..................................................................... 69 Figure 4 Cloning strategy used to generate pAJ007................................................. 70 Figure 5 Cloning strategy used to generate pAJ021................................................. 71 Figure 6 Cloning strategy used to generate pKL1007 .............................................. 72 Figure 7 Expression of virC and anti-virC2 ............................................................ 94 Figure 8 Illustration of 3’ and 5’ sites for anti-virC2 regions 1, 2 and 3 ................. 95 Figure 9 Illustration of deleted anti-vir2 promoter in A. tumefaciens strain C58 ∆Pavc2 ...................................................................................... 96 Figure 10 Strand specific qRT-PCR shows transcript abundance in C58 and C58 ∆Pavc2 ................................................................................................ 97 Figure 11 qRT-PCR shows anti-virC2 transcript abundance in C58 and C58 ∆Pavc2 ................................................................................................ 98 Figure 12 Examination of FLAG-tagged VirC2 protein levels ................................. 99 Figure 13 Scale for qualitative scoring of tumors from Kalanchoe daigremontiana tumorigenesis assays. ...................................................... 100 Figure 14 Data from all Kalanchoe daigremontiana tumorigenesis assays comparing wild type C58 to C58 ∆virC2 .................................................. 101 Figure 15 Data from all Kalanchoe daigremontiana tumoregensis assays comparing C58 wild type and C58 ∆Pavc2 ............................................... 102 Figure 16 Levels of GUS are shown in A. thaliana plants infected by various strains of A. tumefaciens ............................................................................ 103 v Figure S1 pKL1004 through pKL 1001 were used to overexpress each of the three regions of the anti-virC2 transcript as well as the intergenic region between the virC and virG operons. ............................................... 137 Figure S2 Vir Gene transcript expression is examined in C58 ∆Pavc2 strains overexpressing a portion of the anti-virC2 transcript. ............................... 138 Figure S3 Vector pAJ025........................................................................................... 139 Figure S4 Relative abundance of transcript from the virC2/anti-virC2 region is shown for C58(pTF505) (empty vector) and C58(pAJ025). ............................................................................................. 140 Figure S5 Construction of constructs used to generate A. tumefaciens FLAG-tag mutants. .................................................................................... 141 vi LIST OF TABLES Page Table 1 Summary of 3’ and 5’ ends for anti-virC2 regions 1, 2 and 3 as observed using RACE. ............................................................................... 104 Table 2 Strand Specific qRT-PCR was performed to examine transcript levels in promoter deletion mutant C58 ∆Pavc2 compared to C58 wild type. .... 105 Table 3 Non-strand specific qRT-PCR was performed on cDNA from wild type c58, c58 ∆Pavc2, and c58 ∆virC2 in order to compare transcript levels .......................................................................................... 106 Table 4 Data from all Kalanchoe daigremontiana tumorigenicity assays.............. 107 Table 5 IntaRNA was used to predict possible mRNA targets of the entire anti-virC2 transcript ......................................................................... 116 Table 6 Primer list ..................................................................................................
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