Parasponia: a missing piece of the evolutionary puzzle of nitrogen-fixing nodule symbiosis

Abstract

Nitrogen-fixing root nodule symbiosis occurs in ten taxonomic lineages from four related orders -Fagales, Fabales, Rosales and Cucurbitales- that together are called the nitrogen-fixing clade (NFC). Nodulating plants within the NFC are scattered by non-nodulating species, as well as can interact either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. Besides, it is generally assumed that nodulation evolved independently multiple times, though molecular genetic support for this hypothesis is lacking. Parasponia is the only non-legume plant which can establish nitrogen-fixing endosymbiosis with rhizobium, and it is the only nodulating plant within the Cannabaceae. The Parasponia lineage represents five species and phylogenetic analysis shows that this lineage is embedded within the non-nodulating Trema clade. As Parasponia and Trema are closely related, F1 hybrids could be created by crossing of the diploid Parasponia andersonii (2n=20) and the allotetraploid Trema tomentosa (2n=4X=40). Conceptually, P. andersonii x T. tomentosa F1 hybrid plants reflects a diploid T. tomentosa with a haploid genome of P. andersonii introduced. The F1 hybrid between diploid Parasponia andersonii and tetraploid Trema tomentosa can form nodules, whereas it is devoid of intracellular infection when inoculated with either Mesorhizobium plurifarium BOR2 or Bradyrhizobium elkanii WUR3. Based on its genetic composition and symbiotic phenotype, we argue that the F1 hybrid may mimic future engineer results. Therefore, we aimed to obtain a better understanding of the deviation in nodulation phenotype of wild type P. andersonii and F1 hybrid plants. To do so, we compared nodulation efficiencies and intracellular infection within nodule cells upon inoculation with a range of rhizobium strains, as Parasponia can interact with a wide range of rhizobia. This revealed that the host range of hybrid plants is narrower when compared to P. andersonii. We also show that the block in intracellular infection within hybrid nodules is consistent for all nodulating strains identified, cannot be overcome by increased LCO biosynthesis nor by mutating the type III or IV secretion systems of nodulating strains. The hybrid plants can establish arbuscular mycorrhization effectively, suggesting that the block of intracellular infection is rhizobium specific. Taken together, this indicates the occurrence of a yet unknown mechanism leading to an impaired host range and block of intracellular infection of hybrid plants. To answer evolution and genetic basis of nodulation, comparative genomic and transcriptomic analysis has been conducted using Parasponia species (Cannabaceae), the only non-legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that in legumes are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related non-nodulating plant species show evidence of parallel loss in non-nodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these non-nodulating lineages lost the potential to nodulate. By making use of the highly efficient Parasponia transformation platform, we conducted promoter:GUS expression analysis as well as CRISPR-Cas9 mutagenesis. Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are co-expressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF-YA1 commit conserved symbiotic functions in non-elgume plant species. As Rosales, Fabales and Fagales diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis. Taken together, these results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ~100 million years ago in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.