Evolutionary duplication of lipo oligochitin-like receptor genes in soybean differentiates their function in cell division and cell invasion

Abstract

Gene duplication in evolution has long been viewed as a mechanism for functional divergence. We recently cloned two related lipo-oligochitin receptor genes (GmNFR1α and GmNFR1β) in Glycine max (soybean) that allowed the distinction of two nodulation factor (NF) responses during early legume nodule ontogeny, namely invasion of the root hair and concomitant cortical cell divisions. Root-controlled GmNFR1α mutants nod49 and rj1 failed to form curled root hairs, infection threads and nodules but develop subepidermal cortical cell divisions (CCD) and mycorrhizal associations. In contrast GmNFR1β mutant PI437.654 had full symbiotic abilities. However, GmNFR1α mutants formed normal nodules at reduced frequency when inoculated with high Bradyrhizobium titers. The mutation was complemented in Agrobacterium rhizogenes K599 transformed roots using both CaMV 35S and the native GmNFR1α promoters. GmNFR1α may encode a high affinity NF receptor responsible for the entire nodulation cascade while GmNFR1β with lower affinity to NF suffices to induce cell divisions but not early infection events.

Nodulation and symbiotic nitrogen fixation provide a major conduit for nitrogen into the earth's biosphere, capable of replacing synthetic fertilizer augmentation of high input food production. With ever-increasing fossil fuel costs needed for fertilizer production, storage, distribution and application, the understanding and concomitant optimization of the natural symbiotic process of plant-bacterium interaction is gaining emphasis.

General Concept of Nod Factor Perception in Legumes

The initiation of nodule ontogeny in legumes involves a complex set of signal exchanges as described in Figure 1. It requires the reception of a Rhizobium-derived ‘Nodulation Factor’ (NF, a lipooligosaccharide) by a presumptive receptor kinase complex (NFR1/LYK3 plus NFR5/NFP), both part of the LysM receptor kinase family.^1–5 These Nod factor receptors were not thought to be involved in Myc factor perception, but Maillet et al.⁶ suggest that NFP, not LYK3 is partly involved in the Myc-signal-elicited root branching stimulation response. In Parasponia andersonii, the only non-legume species that acquired rhizobia symbiosis, a single NFP/NFR5-like receptor fulfil a dual symbiotic function of both rhizobia and arbuscular mycorrhizae.⁷ Nod factor reception leads to induction of cortical cell divisions and, in parallel, the deformation, curling and eventual invasion of root hairs permitting the entry of soil-born Rhizobium bacteria and enrichment of NF signaling into the root interior. However, some β-rhizobia use an alternative pathway to initiate symbioses in some legumes, where a purine derivative may play a key role in triggering nodule formation.⁸ Rhizobia also produces exopolysaccharides (EPS)/lipopolysaccharides (LPS) that involved in invasion and nodule development, bacterial release from infection threads, and bacteroid development.⁹

Figure 1.

General concept of legume nodulation factor (NF) perception. Legumes secrete a flavone/isoflavone “cocktail” that is perceived by “Rhizobium/Bradyrhizobium” bacteria, activating as many as 50 genes to secrete a decorated, host-specific lipo-oligosaccharide (LCO) commonly called ‘Nod factor.’ Nod factors are perceived by LysM-type receptor kinase protein complexes consisting of NFR1 and NFR5. The activated NFR1/NFR5 heterodimer activates by unknown mechanism the NORK/SYMRK receptor kinase (required for both “Rhizobium” and mycorrhizal infection). Perception of the Nod-factor initiates two separate but interacting pathways,¹⁰ one leading to root hair deformation, bacterial entrapment and subsequent re-orientation of root hair growth to facilitate infection thread formation. The second leads to the activation of the cell cycle and mitosis in both cortex and pericycle opposite xylem poles.¹¹ Both pathways interact for nodule formation and endosymbiosis, leading to symbiotic nitrogen fixation. In parallel, some Rhizobium species secrete purine-derived factors that may act as mitogens in an NF-NFR1/5 independent fashion.⁷ Rhizobium also presents surface exopolysaccharide (EPS)/lipopolysaccharide (LPS) factors that influence effectiveness in nodulation.⁸

Isolation of GmNFR1α and GmNFR1β Genes

Following pioneering work in Lotus japonicus and Medicago truncatula,^1,3 the NF receptor 1 (NFR1) gene of the economically more significant soybean (Glycine max) was just recently cloned through map-based cloning and candidate gene approach and genetic complementation.⁵ This discovery parallels the cloning of the soybean NFR5 gene⁴ believed to encode the partner component of NFR1.

Two non-nodulation mutants [one EMS-induced (nod49), the other naturally occurring (rj1)] fail to nodulate.^12,13 Chemically induced mutant nod49 lacks root hair deformation, curling and infection thread growth but forms mycorrhizal associations.¹⁴ Histology of nodulation events in nod49, rj1 and wild type showed that despite the absence of infection-related events, nod49 and rj1 developed subepidermal cortical cell divisions; such “pseudo-infections” develop into “actual infections” if coupled with a successful root hair infection.^15,16 Significantly, both nod49 and rj1, when inoculated with high Bradyrhizobium japonicum titers (greater than 100 million cells per mL), sparingly form normal nodules through infection threads, illustrating that their signaling efficiency and not nodule ontogeny is impaired.

Soybean symbiotic genes have been mapped and isolated using map-based cloning.^17–19 We attempted a similar approach for nod49, which was crossed with wild-type (Nod⁺) Glycine soja; F2 segregation linked nod49 within 3 cM of SSR marker Satt459 on chromosome 2. Reflecting ancestral genome duplication in soybean, this region is directly duplicated on chromosome 14, maintaining the map order and distances of several RFLPs. The DNA sequence of two linked RFLPs, namely K411-1 and A343-2, showed high identity to the C and N terminal of LysM receptor kinases. Since Radutiou et al.¹ had shown that LysM receptor kinases LjNFR1 and LjNFR5 are likely NF receptors in Lotus japonicus, we shifted the map-based cloning to a candidate gene approach.

PCR primers were designed for K411-1 and A343-2 and genomic DNA of Bragg was amplified to yield a product of predicted size and DNA sequence. The PCR product was cloned and used to screen the Clemson BAC library made of soybean cultivar PI437.654. Several BAC clones were isolated and characterised by HindIII fingerprinting. Two distinct fingerprint patterns were detected allowing the prediction of two contiguous regions in the soybean genome. As K411 and A343 markers exist in two soybean chromosomes, we propose that the two BAC defined regions represent these loci. PCR amplification of BACs in the two contigs indicated two types of products, one smaller by 374 bp. Sequencing of representative products revealed two highly related sequences belonging to the LysM receptor kinase family. These are labeled GmNFR1α and GmNFR1β. The genomic region of BAC54B21 and BAC55N1 was sequenced to reveal the entire genes and their promoters.

The gene structure and sequence of GmNFR1α and β are nearly identical (92%). Intron 6 GmNFR1β is 374 bp shorter than that of GmNFR1α. Likewise, the soybean genes are closely related to the LotusNFR1 gene. Identity in the kinase domain is highest, but notable sequence divergence occurs in exon 1, 3 and 4, which are possible ligand specificity determinants.

The genomic DNA of nod49, rj1 and Bragg was repeatedly sequenced to determine the site of the mutations causing the non-nodulation phenotype. nod49 is mutated in exon 5 of GmNFR1α through a T deletion (T986Δ) leading to a reading frame shift and protein termination within 5 amino acids. The resultant protein, if stable, would lack the entire protein kinase domain. rj1 is mutated in exon 4 by an A deletion (A769Δ) of GmNFR1α leading to protein termination within 51 amino acids. Like nod49, most of the kinase domain would be absent. Wild-type Bragg, nod49 and rj1 have wild-type GmNFR1β sequences.

During sequencing it was discovered that the GmNFR1β of BAC PI437.654 differed from that of Bragg (and thus nod49 and rj1) through a SNP in exon 10 that leads to a non-sense mutation (K512*). The SNP was confirmed in genomic DNA of cultivar PI437.654, which retained a wild-type GmNFR1α. Interestingly, PI437.654 was chosen as a reference cultivar for the BAC library because of its strong soybean cyst nematode resistance.²⁰ Nematode interaction with legumes is known to involve mechanisms similar to Rhizobium-legume signaling.²¹ We do not know whether the GmNFR1β mutation in PI437.654 is causally connected to its nematode resistance phenotype. Nodulation tests showed that PI437.654 is Nod⁺ and Fix⁺.

Genetic complementation via hairy root transformation with Agrobacterium rhizogenes strain K599,²² confirms that the sequenced SNPs in GmNFR1α were causative for the non-nodulation seen in mutants nod49 and rj1. Roots formed on nod49 and rj1 transformed with GmNFR1α nodulated effectively, while empty vector controls or uninoculated controls failed to nodulate. Transformed roots expressing the GmNFR1α gene from the 35S promoter tended to have more nodules 2–3 times that of the empty vector control, often clustered in the upper root regions, suggesting that nodulation success rate in part is controlled by the abundance of the GmNFR1α protein.

Model of Nod Factor Perceptionin Soybean

Unlike model legumes L. japonicus and M. truncatula which have only one copy of Nod factor receptor (NFR), soybean has duplicated copy of NFR. The here-described work represents the first cloning and confirmation of the putative soybean NF receptors. The ancestral genome duplication in soybean permitted divergence in function of the two receptor kinases (Fig. 2).

Figure 2.

Model of nodulation factor (NF) perception in soybean. NF perceptionis required at several stages of the nodule ontogeny with early infection events responding differently than cortical, and presumably pericycle, cell divisions.

GmNFR1α, presumably in partnership with GmNFR5, is capable of fulfilling all functions and is thus similar to LjNFR1. GmNFR1β lacks the ability to perceive NF at low Bradyrhizobium titers, yet suffices for the induction of cortical cell divisions (CCDs). Actual infections are combinations of successful infection threads and CCDs. Infections mediated by GmNFR1α allow the enrichment of rhizobia and NF leading to subsequent maintenance of CCDs and concomitant pericycle cell divisions. “Low” and “High Nod factor” refers to presumed local concentrations. Grey shaded boxes are symbiotic stages achieved in mutants nod49 and rj1.²³ nod139 and nn5, which are mutated in both GmNFR5α and β fail to show any morphological changes.⁴

As shown by the nod49 and rj1 mutants, GmNFR1β alone in combination with GmNFR5α/β cannot efficiently recognize Nod factor in epidermal and root hair cells to induce root hair deformation, curling and infection thread formation. In contrast GmNFR1α was able to facilitate these steps as shown by functional nodulation in the GmNFR1β Q513* mutant of PI437.654. GmNFR1β, however, sufficed to induce subepidermal cell divisions in response to NF perception in the epidermis by itself. Since infection threads formation is needed to allow early cell divisions in the cortex to progress, mutations in GmNFR1α result in non-nodulation. However, if GmNFR1α mutants are exposed to high Nod factor (not achieved in a normal soil environment) they form nodules via a normal infection pathway, showing that GmNFR1β suffices for all early steps, when the NF concentration is high. Increased partial nodulation success per plant on nod49 by elevated rhizobial concentration⁵ and addition of purified Nod Factor (NodV:MeFuc; 10 nM),⁵ support this hypothesis. This indicates that the receptor containing GmNFR1β recognizes, though inefficiently Nod Factor. Accordingly we propose that GmNFR1α and β are distinguished by their affinity towards the NF ligand.

Addendum to: Indrasumunar A, Searle I, Lin MH, Kereszt A, Men A, Carroll BJ, et al. Nodulation factor receptor kinase 1α controls nodule organ number in Soybean (Glycine max L. Merr.) Plant J. 2011;65:39–50. doi: 10.1111/j.1365-313X.2010.04398.x.