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Abstract Background Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. Results We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5’-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn’t contain the canonical dinucleotide ‘GT’ signature but the much less common motif ‘GC’. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5’-intron donor splice site ‘GA’. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. Conclusion SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5’-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

Abstract Background Nodule symbiosis with diazotrophic Frankia or rhizobium occurs in plant species belonging to ten taxonomic lineages within the related orders Fabales, Fagales, Cucurbitales, and Rosales. Phylogenomic studies indicate that this nitrogen-fixing nodulation trait has a single evolutionary origin. In legume model plants, the molecular interaction between plant and rhizobium microsymbiont is mapped to a significant degree. A specific LysM-type receptor kinase, LjEPR3 in Lotus japonicus and MtLYK10 in Medicago truncatula, was found to act in a secondary identity-based mechanism, controlling intracellular rhizobium infection. Furthermore, LjEPR3 showed to bind surface exopolysaccharides of Mesorhizobium loti, the diazotrophic microsymbiont of L. japonicus. EPR3 orthologous genes are not unique to legumes. Surprisingly, however, its ortholog EXOPOLYSACCHARIDE RECEPTOR (EPR) is pseudogenized in Parasponia, the only lineage of non-legume plants that nodulate also with rhizobium. Results Analysis of genome sequences showed that EPR3 orthologous genes are highly conserved in nodulating plants. We identified a conserved retrotransposon insertion in the EPR promoter region in three Parasponia species, which associates with defected transcriptional regulation of this gene. Subsequently, we studied the EPR gene of two Trema species as they represent the sister genus of Parasponia for which it is assumed it lost the nitrogen-fixing nodulation trait. Both Trema species possess apparently functional EPR genes that have a nodulation-specific expression profile when introduced into a Parasponia background. This indicates the EPR gene functioned in nodulation in the Parasponia-Trema ancestor. Conclusion We conclude that nodule-specific expression of EPR3 orthologous genes is shared between the legume and Parasponia-Trema lineage, suggesting an ancestral function in the nitrogen-fixing nodulation trait. Pseudogenization of EPR in Parasponia is an exceptional case in nodulating plants. We speculate that this may have been instrumental to the microsymbiont switch -from Frankia to rhizobium- that has occurred in the Parasponia lineage and the evolution of a novel crack entry infection mechanism.