Tracing the Evolution of the Arbuscular Mycorrhizal Symbiosis in the Plant Lineage
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Tracing the evolution of the arbuscular mycorrhizal symbiosis in the plant lineage Guru Vighnesh Radhakrishnan Thesis submitted for the degree of Doctor of Philosophy to the University of East Anglia Research conducted at the John Innes Centre September 2017 © This copy of thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract The Arbuscular Mycorrhizal (AM) symbiosis is formed by ~80% of land plants with a specific group of soil fungi, the AM fungi. Through this symbiosis, plants obtain nutrients that they otherwise would not be able to access. Based on data from fossils and extant plants, it has been predicted that the AM symbiosis evolved in early land plants. Research in the past two decades utilising angiosperm model plant species has identified several plant genes that regulate the AM symbiosis. These studies have also revealed that these symbiosis genes are highly conserved in the angiosperms but whether this conservation extends to the non-flowering plants has not been explored. In the present study, a comprehensive phylogenetic analysis of the symbiosis genes was conducted, using genomic and transcriptomic data from the non-flowering plant lineages, to gain insights into the evolution of these genes in plants. The results of this analysis indicate that these genes evolved in a stepwise fashion. While some genes appeared in the algal ancestors of the charophytes, others appeared in the early land plant ancestors of liverworts. To further study the AM symbiosis in the non-flowering plants, key methods and genomic resources were established for the liverwort Marchantia paleacea to enable its use as a model plant for the study of the evolution of the AM symbiosis. Using the resources established, phylogenomic comparisons were conducted between M. paleacea and a related liverwort species, M. polymorpha, that is predicted to have lost the ability to engage in the AM symbiosis. These analyses revealed that the homologs of angiosperm symbiosis genes are required for functional symbioses in the liverworts. The findings presented here provide insights into the processes that contributed to the evolution and maintenance of this ancestral symbiosis in the plant lineage. 2 List of contents ABSTRACT 2 LIST OF CONTENTS 3 LIST OF TABLES 7 LIST OF FIGURES 8 LIST OF ACCOMPANYING MATERIALS 11 LIST OF ABBREVIATIONS 12 PUBLICATIONS 13 ACKNOWLEDGEMENTS 14 CHAPTER 1: GENERAL INTRODUCTION 16 1.1. Evolution of the plant lineage 16 1.2. The Arbuscular mycorrhizal symbiosis 19 1.2.1. The AM symbiosis in angiosperms 20 1.2.2. Fossil evidence for the AM symbiosis 22 1.2.3. The AM symbiosis in extant lineages of non-flowering plants 24 1.2.4. The AM symbiosis in the context of land plant evolution 26 1.3. The genetic pathway controlling the AM symbiosis 27 1.3.1. The symbiosis signalling pathway 28 1.3.1.1. Symbiotic signal perception at the plant plasma membrane 30 1.3.1.2. Generation of oscillatory nuclear calcium signatures 32 1.3.1.3. Calcium signature decoding and activation of downstream symbiosis genes 33 1.3.2. Downstream symbiosis genes 34 1.4. Evolution of symbiosis genes 38 1.5. The aims of the current study 41 CHAPTER 2: COMPREHENSIVE PHYLOGENETIC ANALYSES PROVIDE INSIGHTS INTO THE STEPWISE ACQUISITION OF THE SYMBIOTIC TOOLKIT IN PLANTS 44 2.1. Introduction 44 2.2. Aim and experimental design 44 2.3. Results 47 2.3.1. Components of the symbiotic toolkit predated the evolution of land plants 47 3 2.3.2. The evolutionary mechanisms that shaped the origin of the symbiotic toolkit 48 2.3.2.1. Several genes in the symbiotic toolkit arose as a result of gene duplications 49 2.3.2.2. Duplication-independent evolutionary mechanisms also contributed to the evolution of the symbiotic toolkit 58 2.3.3. Conservation of structural and topological features of genes in the symbiotic toolkit in the plant lineage 59 2.3.4. Revisiting previously proposed phylogenetic hypotheses on the evolution of the symbiotic toolkit using new data 61 2.3.4.1. CCaMKs and CDPKs are monophyletic groups of calcium kinases predating green plant evolution 62 2.3.4.2. Domain loss explains the evolution of SYMRK architecture and rejects the predisposition of SYMRK for nodulation 64 2.4. Discussion 69 2.4.1. Stepwise evolution of the symbiotic toolkit in the plant lineage 69 2.4.2. Phylotranscriptomics provides novel insights into the evolution of symbiosis 71 2.4.3. Improved phylogenetic sampling challenges previously favoured hypotheses about the evolution of symbiosis genes 72 CHAPTER 3: ESTABLISHMENT OF A LIVERWORT MODEL SYSTEM FOR THE STUDY OF PLANT-MICROBE INTERACTIONS 75 3.1. Introduction 75 3.2. Results 77 3.2.1. Establishment of an in vitro AM symbiosis in a Marchantia species 77 3.2.2. Developing methods to establish M. paleacea as a laboratory model to study the AM symbiosis 80 3.2.2.1. In vitro culture of M. paleacea 80 3.2.2.2. Induction of the M. paleacea sexual cycle 84 3.2.2.3. Agrobacterium tumefaciens mediated genetic transformation of M. paleacea 85 3.2.2.4. Fungal pathogen of M. paleacea 90 3.2.3. Developing genomic resources for M. paleacea 92 3.2.4. CRISPR/Cas9-based mutagenesis for reverse genetics in M. paleacea 97 3.3. Discussion 102 4 CHAPTER 4: DECIPHERING THE EVOLUTIONARY TRAJECTORIES OF SYMBIOSIS GENES IN LAND PLANTS 108 4.1. Introduction 108 4.2. Results 113 4.2.1. Reconstruction of the evolutionary history of genes committed to symbiosis in the angiosperms 113 4.2.2. Homologs of symbiosis-committed angiosperm genes are present in the liverworts 115 4.2.3. Homologs of genes committed to symbiosis in the angiosperms are transcriptionally induced during symbiosis in the liverworts 116 4.2.4. Signatures of co-elimination reveal symbiosis-committed genes in the liverworts 119 4.2.5. Loss of symbiosis was accompanied by a change in the selective constraints on homologs of symbiosis genes in the liverworts 124 4.2.6. A conserved set of genes is committed to symbiosis in the land plants 131 4.3. Discussion 133 CHAPTER 5: GENERAL DISCUSSION 137 5.1. Conclusions and outlook 150 CHAPTER 6: MATERIALS AND METHODS 153 6.1. Bioinformatics 153 6.1.1. A brief introduction to the methods used for the study of gene evolution 153 6.1.1.1. Phylogenetic trees 153 6.1.1.2. The concept of homology 154 6.1.1.3. Phylogenetic tree construction 155 6.1.2. Workflow for phylogenetic analysis 158 6.1.2.1. Datasets used 158 6.1.2.2. Sequence search 158 6.1.2.3. Sequence alignment 161 6.1.2.4. Tree construction 161 6.1.2.5. Tree viewing and editing 164 6.1.3. Generation of sequence resources 164 6.1.3.1. Nucleic acid extraction from M. paleacea 164 6.1.3.2. Genome sequencing and assembly 165 5 6.1.3.3. Transcriptome sequencing and assembly 166 6.1.3.4. Annotation of the M. paleacea genome 166 6.1.4. Intron-exon structure analysis 166 6.1.5. Orthogroup construction 167 6.1.6. Differential expression analysis of L. cruciata 167 6.1.7. Pseudogene identification and analysis 168 6.1.8. Estimation of selective constraints using PAML and RELAX 168 6.2. Experimental methods 168 6.2.1. Plant growth and media 168 6.2.2. Media and antibiotics used 169 6.2.3. In vitro mycorrhization assays 171 6.2.4. Sectioning and imaging of M. paleacea thalli 171 6.2.4.1. Ink staining 171 6.2.4.2. Wheat Germ Agglutinin (WGA) staining 172 6.2.5. Comparison of M. paleacea growth on different media 172 6.2.6. Sexual cycle induction of M. paleacea 173 6.2.7. Inoculation of M. paleacea with Trichoderma virens 173 6.2.8. Transformation of M. paleacea thalli 173 6.2.8.1. Golden Gate cloning 173 6.2.8.2. Plasmid amplification using Escherichia coli 174 6.2.8.3. Transformation of Agrobacterium tumefaciens to facilitate plant transformation 175 6.2.8.4. Transformation of M. paleacea through blending of thalli 175 6.2.8.5. Agartrap transformation of M. paleacea gemmalings 176 6.2.9. CRISPR/Cas9-mediated mutagenesis of M. paleacea 177 REFERENCES 179 APPENDIX 214 A1. List of genomes and transcriptomes used for the phylogenetic analyses conducted in the current study 214 A2. UNIX scripts used for phylogenetic analysis 226 Script for BLAST, alignment and initial tree building 226 Script for hmmsearch, alignment and initial tree building 231 Script for hmmscan and extracting sequences containing specific domains 235 Script for building final trees using RAxML 235 6 List of tables Table 2.1. Description of the genes of the symbiotic toolkit, the families they belong to, and the occurrence of Pfam domains in the proteins they encode. ..... 45 Table 2.2. The occurrence of proteins from the symbiotic toolkit with well- characterised domains and their constituent domains in the charophytes and chlorophytes. ........................................................................................................ 59 Table 3.1. In vitro mycorrhization experiments in Marchantia species using the AM fungus Rhizophagus irregularis. ................................................................... 78 Table 3.2. Sexual cycle induction of Marchantia species in vitro. ...................... 85 Table 4.1. Results of the PAML analysis to determine whether purifying selection on homologs of angiosperm symbiosis-committed genes was relaxed following the loss of symbiosis in M.