Comparative and functional genomics of nitrogen-fixing rhizobium symbiosis in plants
Given that nodulating plants do not require exogenous nitrogen fertilizer, engineering nodulation in non-nodulating crops has been a longstanding ambition. From an evolutionary perspective, identifying the genetic changes that led to nodulation can provide key engineering targets. The occurrence of nitrogen-fixing root nodule symbiosis with rhizobium or Frankia bacteria is limited to ten plant lineages in four orders: Fagales, Fabales, Rosales, and Cucurbitales. These four orders together form a clade, referred to as the nitrogen-fixing clade. The scattered phylogenetic distribution of nodulating lineages previously led to the hypothesis that nodulation evolved independently multiple times, possibly preceded by a predisposition event at the root of the nitrogen-fixing clade. This thesis presents comparative genomic and transcriptomic analyses to identify genetic changes leading to the evolution of nodulation, as well as innovations in the computational tools required for such analyses.Chapter 2 consists of a review of known molecular mechanisms in two plant-bacteria symbioses (with rhizobia and Frankia) and in two plant-fungus symbioses (arbuscular mycorrhizae and ectomycorrhizae). Specifically, I explore to what extent molecular mechanisms are shared between these four symbioses. The two main commonalities in symbiotic signalling are (1) rhizobium and Frankia symbioses are known to co-opt various elements of arbuscular mycorrhizal symbiotic signalling, and (2) plant-secreted flavonoids and strigolactones act as attractants to the symbiont in all four symbioses. Placing the known symbiotic molecular mechanisms in a comparative context will provide a targeted approach at studying the molecular evolution of nodulation.In chapter 3 I studied the molecular evolution of nodulation in the only lineage outside of the legumes that engages in rhizobium symbiosis – Parasponia – and its non-nodulating sister lineage Trema, both from the Cannabaceae family in the order Rosales. I started with assembling and annotating reference genomes for Parasponia andersonii and Trema orientalis using newly generated data, taking care to avoid lineage specific annotation errors. Using targeted and untargeted approaches, I performed a comparative genomic analysis to identify genetic changes that correlate with the nodulation trait. Following the multiple gain hypothesis, I expected to find evidence for a gain of nodulation in Parasponia. However, such evidence could not be found. Instead, I found pseudogenes in Trema genomes of genes known to be essential for nodulation, which indicated a loss of the nodulation trait in Trema species. In an extended evolutionary perspective, I analyzed public data for several non-nodulating lineages in the Rosales, revealing consistent gene loss of NOD FACTOR PERCEPTION 2 (NFP2), NODULE INCEPTION (NIN), and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Combined with the identification of 290 conserved genes that are transcriptionally upregulated in nodules of Parasponia andersonii and the legume Medicago truncatula (order Fabales), I conclude that the evolutionary origin of nodulation lies at least at the root of the Rosales, and that the trait was subsequently lost multiple times in non-nodulating lineages within the nitrogen-fixing clade.As genes do not function in isolation, Chapter 4 extends the perspective of chapter 3 to the evolution of transcriptional networks in nodulation. In model legumes, multiple transcriptional regulators are known to be crucial for nodulation, including NIN. However, it is not known if the transcriptional networks controlled by these transcriptional regulators are conserved among nodulating species. Furthermore, since NIN is lost in most non-nodulating lineages in the nitrogen-fixing clade, it is likely thatthe transcriptional networks involving NIN are different between nodulating and non-nodulating lineages. I develop a bioinformatic stategy to compare predicted transcriptional networks from RNA sequencing dataacross multiple species. Unfortunately, however, a critical inspection of the accuracy of the predicted networks revealed a false-positive rate of 90%-99%, rendering a comparative analysis infeasible. As is, this chapter provides a complete framework to study the evolution of transcriptional networks, once it is technically feasible to identify such networks on a genome-wide scale.In chapter 5 I present GeneNoteBook, a web-based genome-browser for comparative genomics studies in model and non-model organisms. The GeneNoteBook user interface is optimized to facilitate browsing genes and to query genes based on a variety of metadata, including protein domains, orthogroups, and gene ontology terms. Furthermore, GeneNoteBook enables users to edit and add custom notes and attributes to individual genes. This editing feature was used extensively in the targeted analysis of chapter 3, where a team of several domain experts manually curated homologs of known symbiosis genes in the genomes of Parasponia and Trema. As such, the Parasponia GeneNoteBook represents an accessible single source of genomic and transcriptomic data for the Parasponia-Trema comparative study system. In general, the GeneNoteBook architecture is not confined to the Parasponia-Trema system but can be applied to a wide range of comparative genomics studies.In conclusion, this thesis represents a milestone in the study of the evolutionary origin of nodulation, and the accompanying molecular changes. As a result of the widespread loss of essential nodulation genes in the Rosales, a single evolutionary origin of nodulation has now become most likely. This new evolutionary hypothesis raises a variety of novel questions on the molecular mechanisms involved in nodulation, which can be experimentally verified. As such, this thesis is a key example of hypothesis generation through data-driven bioinformatics research.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
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Wageningen University
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| Subjects: | Life Science, |
| Online Access: | https://research.wur.nl/en/publications/comparative-and-functional-genomics-of-nitrogen-fixing-rhizobium- |
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