Rhizobium-root nodule symbiosis is generally considered to be unique for legumes. However, there is one exception, and that is Parasponia. In this nonlegume, the rhizobial nodule symbiosis evolved independently and is, as in legumes, induced by rhizobium Nod factors. We used Parasponia andersonii to identify genetic constraints underlying evolution of Nod factor signaling. Part of the signaling cascade, downstream of Nod factor perception, has been recruited from the more-ancient arbuscular endomycorrhizal symbiosis. However, legume Nod factor receptors that activate this common signaling pathway are not essential for arbuscular endomycorrhizae. Here, we show that in Parasponia a single Nod factor-like receptor is indispensable for both symbiotic interactions. Therefore, we conclude that the Nod factor perception mechanism also is recruited from the widespread endomycorrhizal symbiosis.

SummaryThe symbiosis between legumes and nitrogen-fixing rhizobia co-opted pre-existing endomycorrhizal features. In particular, both symbionts release lipo-chitooligosaccharides (LCOs) that are recognized by LysM-type receptor kinases. We investigated the evolutionary history of rhizobial LCO receptor genes MtLYK3-LjNFR1 to gain insight into the evolutionary origin of the rhizobial symbiosis.We performed a phylogenetic analysis integrating gene copies from nonlegumes and legumes, including the non-nodulating, phylogenetically basal legume Cercis chinensis. Signatures of differentiation between copies were investigated through patterns of molecular evolution.We show that two rounds of duplication preceded the evolution of the rhizobial symbiosis in legumes. Molecular evolution patterns indicate that the resulting three paralogous gene copies experienced different selective constraints. In particular, one copy maintained the ancestral function, and another specialized into perception of rhizobial LCOs. It has been suggested that legume LCO receptors evolved from a putative ancestral defense-related chitin receptor through the acquisition of two kinase motifs. However, the phylogenetic analysis shows that these domains are actually ancestral, suggesting that this scenario is unlikely.Our study underlines the evolutionary significance of gene duplication and subsequent neofunctionalization in MtLYK3-LjNFR1 genes. We hypothesize that their ancestor was more likely a mycorrhizal LCO receptor, than a defense-related receptor kinase.

Elucidation of the Nod factor structure was a major step in the molecular approach to unravel the signaling pathway in legumes that is essential for the establishment of rhizobium symbiosis. Rhizobial Nod factor molecules are lipochito-oligosaccharides (LCOs) consisting of three to five N-acetyl-glucosamines that on the amino group of the non-reducing glucosamine are acylated with a fatty acid of 16-20 C-atoms in length (C16 to C20). Furthermore, species specific substitutions can be present on the terminal glucosamines, thereby determining specific recognition of Nod factor structure by the legume host plants. 6 Examples of such modifications are glycosylation, sulfation, acetylation and methylation, for which the particular rhizobium species harbours specific nodulation (nod) genes.7,8 Therefore, it is generally assumed that the perception of Nod factors by legume host plants has co-evolved with their corresponding rhizobial symbionts. Some rhizobium species however, produce a diverse mixture Key words: parasponia, legumes, rhizobium, mycorrhizae, Nod factor of Nod factors often resulting in a large range of host plants. 9 Of such broad host range rhizobium species, Sinorhizobium sp. NGR234 is iconic, as it is well studied at a molecular and genetic level.10 This species not only nodulates hundreds of legume species, but also Parasponia; the only non-legume genus able to establish a similar symbiosis with rhizobium.