Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis

Abstract

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysMtype receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Nuclear phylotranscriptomics and phylogenomics support numerous polyploidization events and hypotheses for the evolution of rhizobial nitrogen-fixing symbiosis in Fabaceae

“…The LjNFR1/MtLYK3 genes encode LysM receptor-like kinases, and previous phylogenetic analyses have indicated that eudicot LjNFR1/MtLYK3 homologs form two major clades (LYK-Ia and LYK-Ib) (Buendia et al, 2018;Rutten et al, 2020), although the duplication of LYK-1b genes in Fabaceae was not examined further. To investigate the evolution of LYK genes in Fabaceae, we performed a phylogenetic analysis with multiple homologs from many of the 19 legumes sampled here and recovered both LYK-Ia and LYK-Ib clades in eudicots (Supplemental Figure 30), consistent with previous results based on fewer legume genes (Rutten et al, 2020).…”

“…The LjNFR1/MtLYK3 genes encode LysM receptor-like kinases, and previous phylogenetic analyses have indicated that eudicot LjNFR1/MtLYK3 homologs form two major clades (LYK-Ia and LYK-Ib) (Buendia et al, 2018;Rutten et al, 2020), although the duplication of LYK-1b genes in Fabaceae was not examined further. To investigate the evolution of LYK genes in Fabaceae, we performed a phylogenetic analysis with multiple homologs from many of the 19 legumes sampled here and recovered both LYK-Ia and LYK-Ib clades in eudicots (Supplemental Figure 30), consistent with previous results based on fewer legume genes (Rutten et al, 2020). In addition, the legume genes in the LYK-Ib clade form three highly supported subclades, each with genes from multiple legumes (Supplemental Figure 30): (1) the NFR1 (SNF) clade, which includes the known SNF genes LjNRF1/ MtLYK3 and genes from other nodulating Papilionoideae and Caesalpinioidae species but lacks genes from Cercis canadensis or Nissolia schottii; (2) the NFRe1 clade with MtLYK1 and closely related homologs; and (3) the CERK clade with MtLYK9, LjCERK6 (LjLYS6), and closely related homologs.…”

“…The lack of NFR1 homologs in non-nodulating legumes suggests that these genes have undergone multiple losses. By contrast, the NFRe1 and CERK homologs in non-nodulating legumes probably have other functions, although some of them have been shown to contribute to nodulation, including those in Parasponia (Cannabaceae, Rosales), the only non-legume that can form rhizobial nodules (Rutten et al, 2020).…”

Comparative and functional genomics of nitrogen-fixing rhizobium symbiosis in plants

“…NFP2 is a lysin motif-containing (LysM) receptor involved in detecting rhizobial LCOs. Recent work showed that NFP2 arose from a duplication event at the base of the nitrogen-fixing clade, giving rise to NFP1 and NFP2 orthogroups [354]. The NFP2 orthogroup contains receptors that are essential for rhizobium LCO recognition in legumes (e.g.…”

“…In both rhizobium and Frankia, lineages that do and do not produce LCOs exist. Combined with the observation that only actinorhizal lineages interacting with (potential) LCO-producing Frankia have a functional NFP2 gene [354], the ancestral symbiosis likely predates the NFP1-NFP2 divergence and involved an LCO producing Frankia. To further substantiate this hypothesis, functional studies on NFP1 and NFP2 in actinorhizal lineages are indispensable.…”