Next Generation Sequencing for Studying Viruses and RNA Silencing-Based Antiviral Defense in Crop Plants
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Next Generation Sequencing for studying viruses and RNA silencing-based antiviral defense in crop plants Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Jonathan Seguin von Frankreich Basel, 2016 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Thomas Boller, PD Dr. Mikhail M. Pooggin und Prof. Mihaela Zavolan. Basel, 9 dezember 2014 Prof. Dr. Jörg Schibler General Preface Financial support of this PhD work was provided by the COST action 'Food and Agriculture' (FA) 0806, which has for final objective to create a RNA-based vaccine to immunize crop plants against viral infection. This work was done within a collaboration between the Fasteris SA company, directed by Dr. Laurent Farinelli, and the team of Dr. Mikhail Pooggin from the Plant Physiology research group in Botany at the University of Basel. The expertise of Dr. Laurent Farinelli's company was requested in the objective to use Illumina-Solexa technology to sequence small RNA and perform bioinformatic analysis. The expertise of Dr. Mikhail Pooggin was requested in order to study the defense mechanisms based on sRNA within plant infected by Geminiviruses, Pararetroviruses and Tobamoviruses. Consequently, this work as part of the action FA0806 involved several different other collaborations with COST member european scientists. Acknowledgments I would like to thanks and prove my gratitude to all people who help me during my whole doctoral study and who make this thesis possible. First, I would like to thank my supervisors, PD Dr. Mikhail Pooggin and Dr Laurent Farinelli for giving me the chance to do my thesis in collaboration with Fasteris and the University of Basel. I want to thank Dr. Pooggin for making me share his knowledge and guidance in data analysis and writing of the thesis. I want to thank Dr. Farinelli to have always welcomed me in his company, and have provided me all the technical and IT resources of Fasteris that I needed for the good progress of my thesis. I thank my lab members : Rajeswaran Rajendran, Nachelli Malpica, Anna Zvereva, Victor Golyaev, Silvia Turco and Katya Ivanova for their warm hospitality and their invaluable assistance in understanding their respective project. I would especially like to thank Prof. Thomas Boller for accepting to be my representative in the faculty of botany and Prof. Mihaela Zavolan for being a member of my PhD committee. I also thank Prof. Thomas Hohn and all the other members of the Botanical Institute for their kindness. I want to thank the Bioinformatic team of Fasteris SA: Patricia Otten especially who supervised me at Fasteris and teached me in the analysis of sequenced sRNA; Loic Baerlocher, William Baroni, Nicolas Gonzalez, and Julien Prados for their valuable advice. I would also like to thank Cécile Deluen and Magne Osteras for their explanation of the Solexa-Illumina NGS technologies, and to finish thanks to all Fasteris employees for their hospitality. Special acknowledgements go to our collaborators: Matthew Chabannes, Pierre-Olivier Duroy and Marie-line Caruana from CIRAD for their collaboration on the analysis of Banana samples infected with BSV; Valerian Dolja and his team for the analysis of infected vines samples in Oregon; Madurai Kamaraj University for the analysis of cassava infected with ICMV and SLCMV, and Dr Basanta Borah and Dr. Basavaprabhu L. Patil; and finally a big thank to Alejandro Fuentes for providing the tomatoes samples infected with TYLCV. I would also like to thank Prof. Andreas Voloudakis the coordinator of COST Action FA0806 for supporting the Farinelli/Pooggin project application to the Swiss COST fund for financial support of my thesis work . Finally, I would also like to thank my family for supporting me all these years in my personal and professional choices. I also want to thank my wife Hélène Méreau for her help and support, and my daughter Louise for the happiness and joy that inspires me daily. Abstract The main objectives of this work have been to use next generation sequencing (NGS) and develop bioinformatics tools for plant virus diagnostics and genome reconstruction as well as for investigation of RNA silencing-based antiviral defense. In virus-infected plants, the host Dicer-like (DCL) enzymes process viral double-stranded RNAs into 21-24 nucleotide (nt) short interfering RNAs (siRNAs) which can potentially associate with Argonaute (AGO) proteins and guide the resulting RNA-induce silencing complexes (RISCs) to target complementary viral RNA for post- transcriptional silencing and, in the case of DNA viruses, complementary viral DNA for transcriptional silencing. In the pioneering work, Kreuze et al. (2009) have demonstrated that an RNA virus genome can be reconstructed from multiple siRNA contigs generated by the short sequencing read assembler Velvet. In this PhD study, we developed a bioinformatics pipeline to analyze viral siRNA populations in various model and crop plants experimentally infected with known viruses and naturally infected with unknown viruses. First, we developed a bioinformatics tool (MISIS) to view and analyze maps of small RNAs derived from viruses and genomic loci that generate multiple small RNAs (Seguin et al. 2014b). Using MISIS, we discovered that viral siRNAs cover the entire genomes of RNA and DNA viruses as well as viroids in both sense and antisense orientation without gaps (Aregger et al. 2012; Seguin et al. 2014a; Rajeswaran et al. 2014a, 2014b), thus allowing for de novo reconstruction of any plant virus or viroid from siRNAs. Then, we developed a de novo assembly pipeline to reconstruct complete viral genomes as single contigs of viral siRNAs, in which Velvet was used in combination with other assemblers: Metavelvet or Oases to generate contigs from viral redundant or non-redundant siRNA reads and Seqman to merge the contigs. Furthermore, we employed the mapping tool BWA and the map viewing tool IGV to verify the reconstructed genomes and identify a consensus master genome and its variants present in the virus quasispecies. The approach combining deep siRNA sequencing with the bioinformatics tools and algorithms, which enabled us to reconstruct consensus master genomes of RNA and DNA viruses, was named siRNA omics (siRomics) (Seguin et al. 2014a). We utilized siRomics to reconstruct a DNA virus and two viroids associated with an emerging grapevine red leaf disease and generate an infectious wild type genome clone of oilseed rape mosaic virus (Seguin et al. 2014a). Furthermore, siRomics was used to investigate siRNA- based antiviral defense in banana plants persistently infected with six distinct banana streak pararetroviruses (Rajeswaran et al. 2014a) and rice plants infected with rice tungro bacilliform pararetrovirus (Rajeswaran et al. 2014b). Our results revealed that multiple host DCLs generate abundant and diverse populations of 21-, 22- and 24-nt viral siRNAs that can potentially associate with multiple AGO proteins to target viral genes for post-transcriptional and transcriptional silencing. However, pararetroviruses appear to have evolved silencing evasion mechanisms such as overexpression of decoy dsRNA from a short non-coding region of the virus genome to engage the silencing machinery in massive siRNA production and thereby protect other regions of the virus genome from repressive action of viral siRNAs (Rajeswaran et al. 2014b). Furthermore, despite massive production of 24-nt siRNAs, the circular viral DNA remains unmethylated and therefore transcriptionally active, while the host genome is extensively methylated (Rajeswaran et al. 2014b). These findings shed new light at the siRNA generating machinery of economically-important crop plants. Our analysis of plant small RNAs in banana and rice revealed a novel class of highly abundant 20-nt small RNAs with 5'-terminal guanidine (5'G), which has not been identified in dicot plants. Interestingly, the 20-nt 5'G-RNA-generating pathway does not target the pararetroviruses, which correlates with silencing evasion (Rajeswaran et al. 2014a, 2014b). This thesis work is a part of the European Cooperation in Science and Technology (COST) action that aims develop an RNA-based vaccine to immunize crop plants against viral infection. Our analysis of viral siRNA profiles in various virus-infected plants allowed to identify the regions in the viral genomes that generate low-abundance siRNAs, which are the candidate regions to be targeted by RNA interference (RNAi). Our analysis of RNAi transgenic tomato plants confirmed that targeting of the low-abundance siRNA region of Tomato yellow leaf curl virus (TYLCV) by transgene-derived siRNAs renders immunity to TYLCV disease, one of the major constraints for tomato cultivation worldwide. Table des matières List of abbreviations.............................................................................................................................3 1. Introduction......................................................................................................................................5 1.1 Descriptions of plant virus families...........................................................................................6 1.2 Viruses investigated in this study...............................................................................................9 1.2.1 Cauliflower mosaic virus...................................................................................................9