LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia

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Science  18 Feb 2011:
Vol. 331, Issue 6019, pp. 909-912
DOI: 10.1126/science.1198181


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.

The rhizobial nodule symbiosis is widespread in the legume family (Fabaceae). Although this nitrogen-fixing symbiosis provides the plant with a major advantage, it is in principle restricted to a single family, and it is a major challenge for future agriculture to transfer this symbiosis to nonlegumes (1). The genus Parasponia could provide a key to this, because it encompasses the only nonlegume species that acquired also the rhizobium symbiosis (2, 3), where “rhizobium” refers to all species and genera that form nodules on legumes. Parasponia comprises several tropical tree species and belongs to Celtidaceae (4). Celtidaceae (order Rosales) and Fabaceae (order Fabales) are only remotely related. Further, not a single species phylogenetically positioned between Parasponia and Fabaceae is able to establish such rhizobium symbiosis. Hence, in all probability the common ancestor of present Parasponia species gained the rhizobium-nodule symbiosis independent from legumes. Therefore, a legume-Parasponia comparison provides a key to identifying genetic constraints underlying this symbiosis. In this study, we focused on parallel evolution of the recognition of the rhizobial signal that starts the symbiotic interaction, the Nod factor.

Parasponia makes lateral rootlike nodules that are associated with cell divisions in the root cortex (5). Rhizobium enters the Parasponia root intercellularly and becomes imbedded in a dense matrix. Rhizobium obtains an intracellular lifestyle when it reaches a nodule primordium. There, cortical cells are infected via threadlike structures that remain connected to the plasma membrane. These so-called fixation threads branch, fill up the cells, and provide a niche to rhizobia to fix nitrogen (5). This is illustrated by the expression, in these threads, of the rhizobium nifH gene that encodes one of the subunits of nitrogenase (fig. S1). In contrast, rhizobia enter most legume roots via root hair–based intracellular infection threads, and the bacteria are released in nodule cells as membrane-surrounded nitrogen-fixing organelle-like structures (symbiosomes) that harbor a single or only a few bacteria. Legume nodules are considered to be genuine organs with a unique ontogeny (6). The fact that the Rhizobium symbiosis is very common in 65-million-year-old Fabaceae led to the conclusion that the symbiotic interaction emerged as early as 60 million years ago (7). In contrast, the lateral rootlike nodule structure and more primitive rhizobium infections in Parasponia (5), together with the very close relation with the nonnodulating genus Trema (24), strongly suggest that Parasponia gained the Rhizobium symbiosis more recently than legumes.

A key step in rhizobium symbiosis is the recognition by the host of bacterial Nod factors, which are specific lipochito-oligosaccharides. This holds for (almost) all nodulated legumes but also for Parasponia (8). This implies that a nonlegume species evolved independently from legumes, a Nod factor perception mechanism. In legumes, Nod factors are perceived by specific LysM receptor kinases that coevolved with the Nod factor structure of their host-specific rhizobium species (912). Legume Nod factor receptors activate a common signaling cascade that is shared with and recruited from the more common and far more ancient arbuscular mycorrhizal symbiosis (13, 14). This common signaling pathway comprises an additional plasma membrane receptor kinase, several components in the nuclear envelope including a cation ion channel and subunits of nuclear pores, and a nuclear-localized calcium/calmodulin-dependent kinase (CCaMK) (13, 14). Rhizobium- and mycorrhizae-induced signaling diverge downstream of CCaMK, possibly because of a different nature of the induced calcium spiking (14, 15). Because legume Nod factor receptors are not essential for mycorrhization, it is generally assumed that mycorrhizal symbiosis is controlled by other receptors specific for mycorrhizal signals (i.e., Myc factor). Such Myc factor receptors, like Nod factor receptors, are presumed to activate the common symbiotic signaling pathway (1317). Two scenarios can be envisioned for how Nod factor receptors could have evolved. The complete mycorrhizal signaling pathway, including the Myc receptor, has been recruited by legumes, resulting in a common signaling pathway. In such a case, present Nod factor receptors have emerged upon gene duplication events and subsequently neofunctionalized during coevolution with specific rhizobium species. In this scenario, Myc receptors would be close homologs of known Nod factor receptors, as was argued previously (16, 17). However, such a scenario also implies that early in rhizobium symbiosis evolution a single receptor fulfilled a dual function, namely in mycorrhization as well as in Rhizobium symbiosis. A second scenario is that only the common signaling pathway devoid of a fungal-specific Myc receptor was recruited, and a novel receptor obtained the ability to activate this common signaling pathway upon Nod factor recognition. We favor the first hypothesis, because it is more simple and finds support in the fact that the chito-oligosaccharide backbone of Nod factors is a “fungal” characteristic; chitin is a major component in fungal cell walls. The occurrence of Nod factor signaling in Parasponia provides a possibility to investigate this hypothesis.

First, we confirmed and extended the idea that Parasponia-rhizobium symbiosis is induced by Nod factors. To this end, we used Parasponia andersonii, a species that can be nodulated by the broad host strain Sinorhizobium sp. NGR234 (18). A mutant of Sinorhizobium sp. NGR234 (NGR234ΔnodABC) that does not produce Nod factors was unable to trigger nodule formation or to infect roots of P. andersonii plantlets (0 of 30), whereas wild-type NGR234 does form nodules on ~40% of the plantlets [12 of 30; 8 weeks post inoculation (wpi)], similar as reported previously (5). Furthermore, root cortical cell divisions could be induced by local application of Nod factors (16 of 19; fig. S2). Next, we obtained evidence that, also in P. andersonii, the common symbiotic pathway is recruited to facilitate rhizobium symbiosis. A dominant active form of Medicago truncatula MtCCaMK was introduced in P. andersonii roots (19). In legumes, CCaMK is a key element in the common symbiotic pathway, and dominant active forms of this kinase result in spontaneous nodulation in absence of rhizobia (20, 21). In P. andersonii, we also observed spontaneous formation of nodule-like structures (6 of 30, Fig. 1), indicating that activation of the common signaling pathway is sufficient to induce nodule organogenesis. These data suggest that in P. andersonii the common signaling pathway is activated upon Nod factor perception.

Fig. 1

P. andersonii spontaneous nodule-like structure triggered by dominant active MtCCaMK. Scale bars indicate 50 μm. (A) Nodule-like structure on a transgenic P. andersonii root (selected on the basis of red fluorescence resulting from DsRED1 expression). Scale bar indicates 0.5 mm. (B) Longitudinal section of spontaneous nodule-like structure. Nodule-like structure originates from cortical and pericycle cell layers and has a rudimentary stele (s), reflecting the lateral rootlike origin of P. andersonii nodules.

In legumes, two different Nod factor receptor types are involved. One of these, MtLYK3/LjNFR1 in M. truncatula/Lotus japonicus, has several paralogous genes that resulted from recent duplication events (9, 11, 16, 17, 22, 23). In contrast, the second Nod factor receptor (MtNFP/LjNFR5) has only one paralog in M. truncatula and L. japonicus (11, 23), and a putative orthologous gene is absent in Arabidopsis, a species that is unable to establish mycorrhizal symbiosis (11, 16, 17). Interestingly, in M. truncatula this paralog, MtLYR1, is transcriptionally up-regulated during mycorrhization (24). Therefore, we focused on the putative MtNFP/LjNFR5 orthologous gene in P. andersonii. To clone P. andersonii homologs a bacterial artificial chromosome (BAC) library was constructed and screened with MtNFP as probe. All eight positive BACs came from a single locus and shared the region containing one MtNFP/LjNFR5-like LysM receptor that we named PaNFP (Parasponia andersonii NOD FACTOR PERCEPTION). Southern blotting as well as sequencing of P. andersonii MtNFP/LjNFR5–like sequences generated by polymerase chain reaction (PCR) using degenerated primers and genomic DNA, as well as nodule and root cDNA, confirmed that P. andersonii has a single NFP-like gene. Next we searched for MtNFP/LjNFR5-like genes in available genome sequences of other Fabidae (Rosid I) species (19). Apple (Malus x domestica), a close relative of P. andersonii, also has only a single MtNFP/LjNFR5-like gene that we named MdLYR1 [Malus x domestica LYK-RELATED1 (11)]. Subsequent phylogenetic analysis revealed that PaNFP and MdLYR1 are close homologs of legume MtNFP/LjNFR5 and MtLYR1/LjLYS11 (Fig. 2). On the basis of this result, we conclude that, in contrast to legumes, P. andersonii contains only a single MtNFP/LjNFR5-like gene. The legume-specific nature of the gene duplications is supported by the presence of two conserved deletions in the legume genes (fig. S3). Also a substantial level of microsynteny in paralogous regions as well as a low level of nucleotide substitutions in paralogous gene pairs supports the recent nature of the duplication (17, 23). To determine whether this duplication predates the Fabaceae, we searched for MtNFP/LjNFR5-like sequences in a collection of cDNA clones from the basal legume Chamaecrista fasciculata (25). We identified a single clone (named CfNFP1) that is phylogenetically ancestral to the duplication observed in M. truncatula and L. japonicus (fig. S4). Therefore, we conclude that the duplication of MtNFP/LjNFR5 in the legume lineage was not essential to gain symbiosis with Rhizobium.

Fig. 2

Maximum-likelihood phylogeny of MtNFP/LjNFR5-like genes in the Rosid I (Fabidae) clade. P. andersonii (Pa), apple (Md), and castor bean (Rc) contain only a single gene, whereas in poplar (Pt), cucumber (Cs), and legumes (Gm/Lj/Mt) lineage-specific duplications have occurred. In legumes, LjNFR5, MtNFP, GmNFR5a, and GmNFR5b are Rhizobium Nod factor receptors. Branch lengths are proportional to the number of amino acid substitutions per site. Branch support was obtained from 1000 bootstrap repetitions. LjLYS16 and the closest MtNFP/LjNFR5 homolog in Oryza sativa (OsLYR1) were used as outgroups.

Reverse transcription PCR studies revealed that PaNFP is expressed in roots (fig. S5). To study whether PaNFP has a symbiotic function, we performed RNA interference (RNAi) knockdown experiments (19). P. andersonii roots transformed with the empty vector (control roots) could be nodulated effectively with Sinorhizobium sp. NGR234 (Fig. 3A; 11 out of 30 plants formed nodules, and in total 55 nodules were formed 8 wpi). Transgenic P. andersonii roots that express a PaNFP RNAi construct have markedly reduced PaNFP expression levels (often below detection level, and, in cases where it is detected, it is ≥50% reduced; fig. S5). Inoculation of such RNAi roots with Sinorhizobium sp. NGR234 resulted only in a few nodules (PaNFP RNAi roots had 13 nodules on 30 plants, 8 wpi), and these were much smaller than nodules on control roots (Fig. 3, A and D). Sectioning of NFP RNAi nodules showed that they harbored rhizobia intercellularly, but fixation thread formation was completely blocked in all nodules investigated (n = 10) (Fig. 3, B and E). This demonstrated that PaNFP is involved in nodule formation and is essential for the switch to an intracellular lifestyle of rhizobia. Also in legumes, MtNFP/LjNFR5 is essential for nodule formation as well as intracellular accommodation of rhizobia (11, 12). On the basis of these results, we conclude that P. andersonii has recruited a gene orthologous to the MtNFP/LjNFR5 Nod factor receptor in legumes to control rhizobium symbiosis. This points to constraints in evolution of Nod factor perception mechanisms. As hypothesized above, a Nod factor receptor could have been recruited from the mycorrhizal signaling pathway. Because P. andersonii has only a single MtNFP/LjNFR5-like gene, we determined whether PaNFP is also essential for endomycorrhization. PaNFP RNAi knockdown and control roots were inoculated with Glomus intraradices. This experiment showed that both are equally well infected by fungal hyphae. However, arbuscle formation is blocked in PaNFP RNAi roots, whereas in control roots arbuscules were effectively formed (Fig. 3, C and F, and fig. S6). PaNFP therefore is also essential for successful intracellular infection during arbuscle formation by mycorrhizal fungi. We conclude that in P. andersonii a single MtNFP/LjNFR5-like receptor, PaNFP, fulfills a dual symbiotic function and controls the intracellular life style of both arbuscular mycorrhizae fungi and rhizobium.

Fig. 3

Rhizobium nodulation and mycorrhization on P. andersonii control (A to C) and PaNFP RNAi knockdown (D to F) roots. (A) Control nodule. Scale bar, 1.0 mm. (B) Rhizobium fixation threads in control nodule. Scale bar, 10 μm. (C) Arbuscle in inner root cortical cell of (slightly squashed) control roots. Scale bar, 50 μm. (D) PaNFP RNAi nodule. Scale bar, 1.0 mm. (E) Aborted fixation threads in PaNFP RNAi nodule. Scale bar, 10 μm. (F) Aborted intracellular infection of Glomus intraradices in PaNFP RNAi root. Scale bar, 50 μm.

Our findings in P. andersonii provide strong support for the hypothesis that during evolution a Myc factor receptor, as part of the common signaling cascade, was recruited to serve as Nod factor receptor in the rhizobial-plant symbiosis. Because in P. andersonii PaNFP fulfills a dual function, we suggest that only a few adaptions, if any at all, will have occurred to enable perception of a new ligand, rhizobium Nod factors. Also this result suggests that the Myc factor will have structural characteristics similar to those of Nod factors. In most legumes, MtNFP/LjNFR5 underwent at least one round of gene duplication (Fig. 2). However, our data suggest that this duplication occurred within the Papilionoideae subfamily of the Fabaceae (e.g., Medicago, Lotus, and Glycine), because CfNFP of Chamaecrista, as part of the basal Caesalpinioideae subfamily, is ancestral to the duplication events (fig. S4). Therefore it is likely that in Chamaecrista mycorrhization and Rhizobium symbiosis are controlled by just a single receptor, CfNFP. In more recent legumes like M. truncatula and L. japonicus, a duplication of this receptor has occurred, and only one of these has evolved as a Nod factor receptor. It seems very probable that the second copy functions as a Myc factor receptor.

The bacterial genera collectively named rhizobium that evolved the ability to establish a nodule symbiosis, in general, acquired this by horizontal transfer of nod genes (26). This event allowed them to produce fungal-like molecules, namely Nod factors, by which they could use the ancient mechanism by which endomycorrhizal fungi establish an intracellular life style and turned these rhizobia from free-living bacteria into nitrogen-fixing endosymbionts. However, although the endomycorrhizal symbiosis is widespread in the plant kingdom only very few plant lineages, namely legumes and Parasponia, have recruited this mechanism for the rhizobial nodule symbiosis. Studies on the constraints underlying this evolutionary event in Parasponia can provide insight into whether and how this nitrogen-fixing symbiosis can be transferred to other nonlegumes.

Supporting Online Material

Materials and Methods

Figs. S1 to S16


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank W. Broughton for NGR234; E. James, P. Hadobas, and T. J. Higgens for Parasponia seeds; P. van Dijk for flow cytometry; and J. Talag and W. Golser for BAC library construction. This research is funded by the Dutch Science Organization (Nederlandse Organisatie voor Wetenschappelijk Onderzoek) (VIDI 864.06.007).
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