Nodule symbiosis with nitrogen-fixing bacteria is initiated when plants of the nitrogen-fixing clade (NFC) perceive bacterial lipo-chitooligosaccharide (LCO) signals through LysM-type cell-surface receptors. Whether the NFP/NFR5-type LCO receptor essential for nodulation was recruited once in the common ancestor of the clade, or convergently in separate lineages, is unresolved, as is the molecular basis of its specialization. Rutten et al. (2020) showed in the non-legume nodulator Parasponia andersonii that the NFP1/NFP2 receptor pair predates nitrogen-fixing nodule symbiosis: the NFP2 copy is essential for rhizobial LCO perception, the NFP1 paralog cannot substitute for it even when driven by the NFP2 promoter, and Parasponia receptors only partially restore signaling in Lotus LCO-receptor mutants. Finegan et al. (2025) have since challenged the single-origin model of the NFP1/NFP2 duplication with phylogenomic and synteny evidence for a tandem duplication in a Cucurbitales–Rosales ancestor and independent, convergent recruitment of the legume NFP. The open problem is to establish the duplication history and orthology of NFP-type LCO receptors across the NFC and to identify the amino-acid determinants, receptor-complex compatibility constraints, and residual arbuscular mycorrhizal functions that together explain how this receptor lineage was specialized for nodulation.

Background

Root nodule symbiosis (RNS), in which plants host nitrogen-fixing bacteria (rhizobia, or Frankia bacteria in the so-called actinorhizal plants) in specialized root organs called nodules, occurs exclusively within the "nitrogen-fixing clade" (NFC) of flowering plants, comprising the orders Fabales (including the legumes), Fagales, Cucurbitales, and Rosales. The symbiosis is initiated when the plant perceives lipo-chitooligosaccharides (LCOs) — chitin oligomer backbones decorated with a fatty acid, called Nod factors when produced by rhizobia — through LysM receptor-like kinases (LysM-RLKs), cell-surface receptors whose extracellular lysin motif (LysM) domains bind N-acetylglucosamine-containing ligands. In legumes, LCO perception requires a receptor pair at the plasma membrane: NFR5/NFP (an LYR-type LysM receptor whose intracellular kinase domain is catalytically inactive, functioning as a pseudokinase) and NFR1/LYK3 (a catalytically active kinase that transduces the signal). Downstream signaling proceeds through the "common symbiosis signaling pathway", a calcium-oscillation-based cascade shared with the far more ancient arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi. Parasponia (Cannabaceae, Rosales), the only non-legume genus that forms nodules with rhizobia, infects bacteria apoplastically via crack entry rather than through intracellular root-hair infection threads as most legumes do, making it the key comparative system for nodulation evolution. Comparative genomics of Parasponia (van Velzen et al., 2018) led to the model that the NFP-type genes (the LysM receptor subclade LYR-IA) duplicated in an ancestor of the NFC, producing NFP-I and NFP-II subclades, with all nodulation-functioning NFPs belonging to NFP-II. Rutten et al. (2020) tested this in P. andersonii by phylogeny reconstruction, trans-complementation in the Lotus japonicus LCO-receptor double mutant ljnfr1;ljnfr5, and CRISPR-Cas9 mutagenesis. They identified four LysM receptors functioning in LCO-driven nodulation (PanLYK1, PanLYK3, PanNFP1, PanNFP2), showed that PanNFP2 is the essential rhizobium LCO receptor and that a repaired Trema levigata nfp2 pseudogene (Trema being a non-nodulating close relative of Parasponia) can replace PanNFP2 whereas PanNFP1 driven by the same PanNFP2 promoter cannot, and found that pannfp1, pannfp2, and pannfp1;pannfp2 mutants are still effectively mycorrhized. The NFP-type receptors of Petunia and tomato (LYK10), from outside the NFC, can complement legume nfp/nfr5 mutants (Girardin et al., 2019), implying LCO perception is ancestral to the NFC and predates the NFP1/NFP2 duplication. Rutten et al. left open when the duplication occurred (legumes lack an NFP-I-type gene, so a duplication at the NFC root with subsequent Fabales loss could not be distinguished from a later duplication in a Fagales–Cucurbitales–Rosales ancestor), which amino-acid adaptations make NFP2-type proteins nodulation-competent, why Parasponia receptors only partially restore LCO signaling in Lotus, and whether PanNFP1/PanNFP2 retain weak functions in AM symbiosis. This framing was substantially revised in 2025: Finegan et al. showed that NFP1/NFP2 pairs are tandem duplicates restricted to Cucurbitales and Rosales, that the closest paralog (a gene related to another by duplication) of the legume MtNFP is MtLYR1, which lies on a different chromosome within a syntenic block — a stretch of conserved gene order between chromosomes, left here by a whole-genome duplication (an ancient genome-doubling event) in the papilionoid legumes, the largest legume subfamily — and that MtNFP is therefore not orthologous (the same gene separated only by speciation events) to the NFP2-like group: evidence for convergent, multiple origins of NFP-based LCO signaling that contradicts the clade-wide NFP-II model. The single-versus-multiple-origin question for nodulation itself remains actively debated (Doyle et al., 2025; Lee et al., 2026).

Problem Statement

Determine the evolutionary history and molecular basis of NFP-type lipo-chitooligosaccharide (LCO) receptor specialization for root nodule symbiosis in the nitrogen-fixing clade (NFC), comprising four linked elements that jointly answer how, when, and through which molecular changes an ancestral mycorrhiza-associated LysM receptor lineage was converted into the Nod factor receptor (Nod factors being the rhizobial LCO signals) of nitrogen-fixing nodulation: (1) resolve when and in which ancestral lineages the duplication(s) that produced the NFP1/NFP2 (NFP-I/NFP-II) gene lineages occurred, and whether the legume NFP/NFR5 genes (e.g., MtNFP, LjNFR5) are orthologous to the Parasponia and actinorhizal (Frankia-nodulated) NFP2 lineage — that is, discriminate the single-origin model of a clade-wide NFP-II group from the convergence model of a Cucurbitales–Rosales tandem duplication with independently recruited legume NFP — using phylogenomic and syntenic evidence across Fabales, Fagales, Cucurbitales, and Rosales; (2) identify the specific amino-acid differences that make NFP2-type receptor proteins competent to drive LCO-induced nodulation while their NFP1-type paralogs (e.g., PanNFP1 expressed under the native PanNFP2 promoter) are not, including whether legume NFP and Rosales/Cucurbitales NFP2-type proteins were co-opted through the same or different changes; (3) establish the mechanistic basis of the species barrier revealed by the only partial restoration of LCO signaling when P. andersonii receptors are expressed in L. japonicus LCO-receptor mutants — candidate mechanisms include receptor-intrinsic incompatibility with heterologous membrane partners of the receptor complex, different receptor-complex composition, and adaptations linked to the contrasting rhizobial infection modes (crack entry versus root-hair infection threads), among others; and (4) determine whether PanNFP1 and PanNFP2 retain quantitative (weak) functions in arbuscular mycorrhizal symbiosis that the existing CRISPR mutant analysis may have been unable to detect because of substantial biological variation in mycorrhization efficiency. All four elements are jointly required for completion: resolving any proper subset of them — however completely — does not by itself resolve the question.

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