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Development and Application of Tobacco Rattle Virus Induced Gene Silencing in Gerbera Hybrida

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Development and Application of Tobacco Rattle Virus Induced Gene Silencing in Gerbera Hybrida View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Helsingin yliopiston digitaalinen arkisto Department of Agricultural Sciences Faculty of Agriculture and Forestry University of Helsinki Finnish Doctoral Program in Plant Science Development and Application of Tobacco Rattle Virus Induced Gene Silencing in Gerbera hybrida Xianbao Deng ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry, University of Helsinki, for public examination in lecture room B2, B-building (Latokartanonkaari 7-9), on 22 November 2013, at 12 o’clock noon. Supervisors: Professor Teemu Teeri Department of Agricultural Sciences University of Helsinki Professor Paula Elomaa Department of Agricultural Sciences University of Helsinki Professor Jari Valkonen Department of Agricultural Sciences University of Helsinki Member of the follow-up group: Dr. Heiko Rischer VTT Technical Research Centre of Finland Reviewers: Professor Steve Whitham Department of Plant Pathology Iowa State University Docent Laura Jaakola Department of Arctic and Marine Biology University of Tromsø Opponent: Docent Kirsi Lehto University of Turku Custos: Professor Paula Elomaa Department of Agricultural Sciences University of Helsinki ISBN 978-952-10-9451-4 (paperback) ISBN 978-952-10-9452-1 (PDF) Helsinki University Printing House Helsinki 2013 Cover figure: Viruse-induced gene silencing (VIGS) phenotypes in Gerbera hybrida. Left image: Silencing of the gerbera phytoene desaturase (GPDS) gene in the gerbera cultivar Terraregina. Middle and right images: Silencing of the gerbera chalcone synthase 1 (GCHS1) gene in the gerbera cultivar President at the petal developmental stage 5 (middle) and stage 8 (right). Table of Contents List of original publications ........................................................................... 1 Abbreviations ................................................................................................. 2 Abstract .......................................................................................................... 3 1. INTRODUCTION ...................................................................................... 5 1.1 Gerbera hybrida ũ a model for Asteraceae ................................... 5 1.2 RNA silencing .................................................................................... 7 1.2.1 The history of RNA silencing ...................................................... 7 1.2.2 RNA silencing pathways ............................................................. 9 1.3 Virus-induced gene silencing (VIGS) ............................................ 13 1.3.1 Viral suppressors of RNA silencing .......................................... 14 1.3.2 VIGS as a functional genomics approach ............................... 16 1.4 Tobacco Rattle Virus ....................................................................... 18 1.4.1 The role of the 16K protein ....................................................... 20 1.4.2 TRV as VIGS vector .................................................................. 21 1.5 Flavonoid biosynthesis ................................................................... 22 1.5.1 Chalcone synthase .................................................................... 25 1.5.2 Flavonoid biosynthesis and chalcone synthase in Gerbera hybrida ....................................................................................................... 26 2. AIMS OF THE STUDY ........................................................................... 28 3. MATERIALS AND METHODS ............................................................... 29 4. RESULTS AND DISCUSSION .............................................................. 30 4.1 TRV 16K helps in balancing the induction and suppression of the host anti-viral RNA silencing (I) ................................................................. 30 4.1.1 Disruption of 16K enhanced viral symptoms ........................... 30 4.1.2 16K enhances PDS silencing ................................................... 31 4.1.3 16K stabilizes recombinant RNA2 genome ............................ 32 4.2 TRV 29K movement protein is an RNA silencing suppressor (I) 33 4.3 Application of TRV VIGS in gerbera (I, II) .................................... 34 4.3.1 Factors affecting VIGS in gerbera ........................................... 34 4.3.2 TRV VIGS in gerbera ................................................................ 36 4.4 Functional characterization for gerbera CHSs (III) ...................... 36 4.4.1 Gerbera CHS is represented by a three-gene family............. 36 4.4.2 Spatial and temporal expression patterns of gerbera CHSs . 37 4.4.3 Silencing GCHS1 and GCHS4 separately by VIGS revealed that GCHS1 is the major CHS in gerbera petals for anthocyanin biosynthesis .............................................................................................. 38 4.4.4 GCHS4 is regulated post-transcriptionally in gerbera petals 39 5. CONCLUSIONS AND PROSPECTS ................................................... 40 ACKNOWLEDGEMENTS .......................................................................... 42 REFERENCES ........................................................................................... 44 REPRINT OF ORIGINAL PUBLICATIONS………………………………65 List of original publications This thesis is based on the following publications, which are referred to in the text by their roman numerals. The publications have been reprinted with the kind permission of the publishers. I Deng X, Kelloniemi J, Haikonen T, Vuorinen AL, Elomaa P, Teeri TH, Valkonen JPT. 2013. Modification of Tobacco rattle virus RNA1 to serve as a VIGS vector reveals that the 29K movement protein is an RNA silencing suppressor of the virus. Molecular Plant-Microbe Interactions 26: 503–514. II Deng X, Elomaa P, Nguyen CX, Hytönen T, Valkonen JPT, Teeri TH. 2012. Virus-induced gene silencing for Asteraceaeņa reverse genetics approach for functional genomics in Gerbera hybrida. Plant Biotechnology Journal 10: 970–978. III Deng X, Bashandy H, Ainasoja M, Kontturi J, Pietiäinen M, Laitinen RAE, Albert VA, Valkonen JPT, Elomaa P, Teeri T. Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida. New Phytologist, in press. My contribution to the above publications: I I was involved in the experimental design, constructed the trv30K16Kstop mutants, and carried out all the experiments. I was involved in the data interpretation, wrote the manuscript draft and revised the manuscript together with other co-authors. II I was involved in the experimental design, screened gerbera cultivars that are sensitive to TRV infection and carried out all VIGS experiments. I wrote the manuscript draft and revised the manuscript together with other co-authors. III I was involved in the experimental design, analyzed gerbera CHS sequences and carried out the experiments on CHS expression analysis, in situ hybridization, VIGS, Western blotting, and polysome RNA analysis. I wrote the manuscript draft and revised the manuscript together with other co-authors. 1 Abbreviations AGO ARGONAUTE protein CHL Mg-chelatase CHS Chalcone synthase CP Coat protein DCL Dicer-like protein DFR Dihydroflavonol-4-reductase dpi Days post infiltration dsRNA Double-stranded RNA EST Expressed sequence tag miRNA Micro RNA MP Movement protein PDS Phytoene desaturase PKS Polyketide synthase 2PS 2-pyrone synthase PTGS Post transcriptional-gene silencing RdDM RNA-directed DNA methylation RDR/RdRp RNA-directed RNA polymerase RISC RNA-induced silencing complex sgRNA Subgenomic RNA siRNA Small interfering RNA sRNA Small RNA ssRNA Single-stranded RNA tasiRNA Trans-acting small interfering RNA TF Transcription factor TRV Tobacco rattle virus VIGS Virus-induced gene silencing VSR Viral suppressors of RNA silencing 2 Abstract RNA silencing is a conserved mechanism that occurs in a broad range of eukaryotes, which is regulated by small RNAs (sRNAs). RNA silencing operates to control gene expression and maintain genome integrity. Virus- induced gene silencing (VIGS) in plants is a natural antivirus mechanism that has adapted from the general RNA silencing system. To counter the antivirus RNA silencing, plant viruses have evolved to encode viral suppressors of RNA silencing (VSRs). Nowadays VIGS is usually referred to as the technology that uses recombinant viruses to knock down the expression of plant endogenous genes. Gerbera hybrida (gerbera) is a model species in the family of Asteraceae. As a highly heterozygous species, gerbera lacks efficient functional genetic approaches other than gene transfer. The aim of the present study was to develop a Tobacco rattle virus (TRV, genus Tobravirus) induced gene silencing system for gerbera, and use TRV VIGS to characterize functions of chalcone synthase (CHS) encoding genes in the plant. Preliminary VIGS experiments on the cultivar Terraregina, by syringe infiltration and applying previously developed TRV vectors, did not result in visible VIGS phenotypes due to the absent of TRV RNA2 in the up non- infiltrated leaves. Consequently, I first aimed to study the mechanism of TRV VIGS, and tried to develop new VIGS vectors based on TRV RNA1. I investigated the role of two important TRV proteins of the 16K VSR and the 29K movement protein (MP) on TRV infection and TRV VIGS, and developed TRV RNA1 based VIGS vectors. For accomplishing this, a series of TRV RNA1 mutants have been
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