The Chronicles of Nodulation : The LCOs, the Genes and the Evolutionary Trajectories

Plants take up nitrogen from the soil as ions or free amino acids. Some plant species form symbioses with nitrogen-fixing soil bacteria to carry out biological nitrogen fixation using the enzyme complex nitrogenase to convert atmospheric nitrogen to ammonia, which the plants can use. This involves the formation of specialized plant structures called root nodules and is known as root nodule symbiosis. Such bacteria include rhizobia and Frankia. The hosts of rhizobia are legumes (order Fabales) and one non-legume (Parasponia, order Rosales), while Frankia associates with actinorhizal plants from the orders Fagales, Cucurbitales, and Rosales. Phylogenetically, Frankia can be divided into four clusters. The earliest diverging cluster is Frankia cluster-2, which can be divided into the island and continental lineages, and, with one exception, contains species that have never been cultivated. It diverged from the non-symbiotic cluster-4. Subsequently, another symbiotic lineage branched off from cluster-4: the precursor of the two symbiotic clusters, cluster-1 and cluster-3. Cluster-1 contains two distinct groups: cluster-1a, whose species can infect Alnus, and cluster-1c, whose species can infect (Allo-)Casuarina. To date, some cluster-1a strains have not been cultivated. The as-yet-uncultivable strains of both cluster-1a and cluster-2 lineages show strong genome erosion (4.2 to 5.6 MB compared to 10 MB in cluster-4). This thesis aims to provide a broader understanding of the evolution and saprotrophic capabilities of the nitrogen-fixing Actinomycetota Frankia, and how these bacteria interact with their host plants. In Study I, we tried to determine why certain Frankia strains cannot be cultured. Frankia have an uptake hydrogenase enzyme (Hup), which recycles the hydrogen produced by nitrogen fixation to counter the loss of energy that occurs during the process. We analysed the different types of [NiFe] uptake hydrogenases found across Frankia clusters to understand whether they play a role in a strain’s ability to survive in culture. We found that the Frankia strains which could not be cultivated to date have lost the gene set required to produce type-1h [NiFe] uptake hydrogenase, which scavenges electrons from atmospheric hydrogen for respiration during carbon starvation. These strains contain either type-1f or type-2a hydrogenases, either of which can recycle the hydrogen produced during nitrogen fixation, allowing them to fix nitrogen during symbiosis in an energy-efficient manner. In Study II and Study III, we checked for the presence of undiscovered Frankia species exhibiting genome reduction in Casuarina or Coriaria hosts that escaped isolation via traditional culturing techniques. Nodules were collected from several countries and directly sequenced the nodule to obtain nodule metagenome assembled genomes (MAGs), bypassing the need to culture the microsymbionts. We identified five new Frankia species: one Casuarina-infective species from cluster-1c, and four from earlier studies: one novel Coriaria-infective species from the cluster-2 continental lineage, and three novel Coriaria-infective species from the cluster-2 island lineage. All these strains show evidence of erosion of the genes needed to produce type-1h [NiFe] uptake hydrogenase. In conclusion, Frankia strains follow one of two evolutionary trajectories – either towards obligate symbiosis (accompanied by strong genome erosion), or towards rhizosphere colonization (with limited genome erosion). In Study IV, we examined the lipochitooligosaccharide (LCO) compounds produced by some species from the continental lineage of cluster-2 Frankia. In rhizobia and arbuscular mycorrhizal (AM) fungi (i.e., in other plant microsymbionts), LCOs are used for microbe-host communication. In the evolutionarily older AM fungi, LCOs can also suppress host defences. Since Frankia LCOs are not produced by all Frankia species, a function in genus-wide microsymbiont-host communication seems unlikely. We therefore examined the effect of Frankia LCOs on plant defence. Our findings suggest that, like AM LCOs, Frankia LCOs suppress plant defence, enabling infection and nodulation under stressed conditions. Overall, these studies advance the understanding of how Frankia evolved during their association with actinorhizal plants and highlight some major differences between Frankia strains that can be grown in culture and those that cannot.