Nodulation factor receptor kinase 1α controls nodule organ number in soybean (Glycine max L. Merr).

Two allelic non-nodulating mutants, nod49 and rj1, were characterized using map-based cloning and candidate gene approaches, and genetic complementation. From our results we propose two highly related lipo-oligochitin LysM-type receptor kinase genes (GmNFR1α and GmNFR1ß) as putative Nod factor receptor components in soybean. Both mutants contained frameshift mutations in GmNFR1α that would yield protein truncations. Both mutants contained a seemingly functional GmNFR1ß homeologue, characterized by a 374-bp deletion in intron 6 and 20-100 times lower transcript levels than GmNFR1α, yet both mutants were unable to form nodules. Mutations in GmNFR1ß within other genotypes had no defects in nodulation, showing that GmNFR1ß was redundant. Transgenic overexpression of GmNFR1α, but not of GmNFR1ß, increased nodule number per plant, plant nitrogen content and the ability to form nodules with restrictive, ultra-low Bradyrhizobium japonicum titres in transgenic roots of both nod49 and rj1. GmNFR1α overexpressing roots also formed nodules in nodulation-restrictive acid soil (pH 4.7). Our results show that: (i) NFR1α expression controls nodule number in soybean, and (ii) acid soil tolerance for nodulation and suppression of nodulation deficiency at low titre can be achieved by overexpression of GmNFR1α.

Glycine max non-nodulation locus rj1: a recombinogenic region encompassing a SNP in a lysine motif receptor-like kinase (GmNFR1α).

The rj1 mutation of soybean is a simple recessive allele in a single line that arose as a spontaneous mutation in a population; it exhibits non-nodulation with virtually all Bradyrhizobium and Sinorhizobium strains. Here, we described fine genetic and physical mapping of the rj1 locus on soybean chromosome 2. The initial mapping of the rj1 locus using public markers indicated that A343.p2, a sequence-based marker that contains sequence similar to a part of the LjNFR1 gene regulating nodule formation as a member of lysin motif-type receptor-like kinase (LYK) family, maps very close to or cosegregates with the rj1 locus. The sequence of A343.p2 is 100% identical to parts of two BAC clone sequences (GM_WBb0002O19 and GM_WBb098N11) that contain three members of the LYK family. We analyzed the sequence contig (262 kbp) of the two BAC clones by resequencing and subsequent fine genetic and physical mapping. The results indicated that rj1 is located in a gene-rich region with a recombination rate of 120 kbp/cM: several fold higher than the genome average. Among the LYK genes, NFR1α is most likely the gene encoded at the Rj1 locus. The non-nodulating rj1 allele was created by a single base-pair deletion that results in a premature stop codon. Taken together, the fine genetic and physical mapping of the Rj1-residing chromosomal region, combined with the unexpected observation of a putative recombination hotspot, allowed us to demonstrate that the Rj1 locus most likely encodes the NFR1α gene.

Inactivation of duplicated nod factor receptor 5 (NFR5) genes in recessive loss-of-function non-nodulation mutants of allotetraploid soybean (Glycine max L. Merr.).

Chemically induced non-nodulating nod139 and nn5 mutants of soybean (Glycine max) show no visible symptoms in response to rhizobial inoculation. Both exhibit recessive Mendelian inheritance suggesting loss of function. By allele determination and genetic complementation in nod139 and nn5, two highly related lipo-oligochitin LysM-type receptor kinase genes in Glycine max were cloned; they are presumed to be the critical nodulation-inducing (Nod) factor receptor similar to those of Lotus japonicus, pea and Medicago truncatula. These duplicated receptor genes were called GmNFR5alpha and GmNFR5beta. Nonsense mutations in GmNFR5alpha and GmNFR5beta were genetically complemented by both wild-type GmNFR5alpha and GmNFR5beta in transgenic roots, indicating that both genes are functional. Both genes lack introns. In cultivar Williams82 GmNFR5alpha is located in chromosome 11 and in tandem with GmLYK7 (a related LysM receptor kinase gene), while GmNFR5beta is in tandem with GmLYK4 in homologous chromosome 1, suggesting ancient synteny and regional segmental duplication. Both genes are wild type in G. soja CPI100070 and Harosoy63; however, a non-functional NFR5beta allele (NFR5beta*) was discovered in parental lines Bragg and Williams, which harbored an identical 1,407 bp retroelement-type insertion. This retroelement (GmRE-1) and related sequences are located in several soybean genome positions. Paradoxically, putatively unrelated soybean cultivars shared the same insertion, suggesting a smaller than anticipated genetic base in this crop. GmNFR5alpha but not GmNFR5beta* was expressed in inoculated and uninoculated tap and lateral root portions at about 10-25% of GmATS1 (ATP synthase subunit 1), but not in trifoliate leaves and shoot tips.

Molecular analysis of legume nodule development and autoregulation.

Legumes are highly important food, feed and biofuel crops. With few exceptions, they can enter into an intricate symbiotic relationship with specific soil bacteria called rhizobia. This interaction results in the formation of a new root organ called the nodule in which the rhizobia convert atmospheric nitrogen gas into forms of nitrogen that are useable by the plant. The plant tightly controls the number of nodules it forms, via a complex root-to-shoot-to-root signaling loop called autoregulation of nodulation (AON). This regulatory process involves peptide hormones, receptor kinases and small metabolites. Using modern genetic and genomic techniques, many of the components required for nodule formation and AON have now been isolated. This review addresses these recent findings, presents detailed models of the nodulation and AON processes, and identifies gaps in our understanding of these process that have yet to be fully explained.

Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max).

Association between legumes and rhizobia results in the formation of root nodules, where symbiotic nitrogen fixation occurs. The early stages of this association involve a complex of signalling events between the host and microsymbiont. Several genes dealing with early signal transduction have been cloned, and one of them encodes the leucine-rich repeat (LRR) receptor kinase (SymRK; also termed NORK). The Symbiosis Receptor Kinase gene is required by legumes to establish a root endosymbiosis with Rhizobium bacteria as well as mycorrhizal fungi. Using degenerate primer and BAC sequencing, we cloned duplicated SymRK homeologues in soybean called GmSymRKα and GmSymRKß. These duplicated genes have high similarity of nucleotide (96%) and amino acid sequence (95%). Sequence analysis predicted a malectin-like domain within the extracellular domain of both genes. Several putative cis-acting elements were found in promoter regions of GmSymRKα and GmSymRKß, suggesting a participation in lateral root development, cell division and peribacteroid membrane formation. The mutant of SymRK genes is not available in soybean; therefore, to know the functions of these genes, RNA interference (RNAi) of these duplicated genes was performed. For this purpose, RNAi construct of each gene was generated and introduced into the soybean genome by Agrobacterium rhizogenes-mediated hairy root transformation. RNAi of GmSymRKß gene resulted in an increased reduction of nodulation and mycorrhizal infection than RNAi of GmSymRKα, suggesting it has the major activity of the duplicated gene pair. The results from the important crop legume soybean confirm the joint phenotypic action of GmSymRK genes in both mycorrhizal and rhizobial infection seen in model legumes.

The value of biodiversity in legume symbiotic nitrogen fixation and nodulation for biofuel and food production.

Much of modern agriculture is based on immense populations of genetically identical or near-identical varieties, called cultivars. However, advancement of knowledge, and thus experimental utility, is found through biodiversity, whether naturally-found or induced by the experimenter. Globally we are confronted by ever-growing food and energy challenges. Here we demonstrate how such biodiversity from the food legume crop soybean (Glycine max L. Merr) and the bioenergy legume tree Pongamia (Millettia) pinnata is a great value. Legume plants are diverse and are represented by over 18,000 species on this planet. Some, such as soybean, pea and medics are used as food and animal feed crops. Others serve as ornamental (e.g., wisteria), timber (e.g., acacia/wattle) or biofuel (e.g., Pongamia pinnata) resources. Most legumes develop root organs (nodules) after microsymbiont induction that serve as their habitat for biological nitrogen fixation. Through this, nitrogen fertiliser demand is reduced by the efficient symbiosis between soil Rhizobium-type bacteria and the appropriate legume partner. Mechanistic research into the genetics, biochemistry and physiology of legumes is thus strategically essential for future global agriculture. Here we demonstrate how molecular plant science analysis of the genetics of an established food crop (soybean) and an emerging biofuel P. pinnata feedstock contributes to their utility by sustainable production aided by symbiotic nitrogen fixation.

Vermiculite's strong buffer capacity renders it unsuitable for studies of acidity on soybean (Glycine max L.) nodulation and growth.

BACKGROUND: Vermiculite is the most common soil-free growing substrate used for plants in horticultural and scientific studies due to its high water holding capacity. However, some studies are not suitable to be conducted in it. The described experiments aimed to test the suitability of vermiculite to study the effect of acidity on nodulation and growth of soybean (Glycine max L.). METHODS: Two different nutrient solutions (Broughton & Dilworth, and modified Herridge nutrient solutions) with or without MES buffer addition were used to irrigate soybean grown on vermiculite growth substrates. The pH of nutrient solutions was adjusted to either pH 4.0 or 7.0 prior its use. The nodulation and vegetative growth of soybean plants were assessed at 3 and 4 weeks after inoculation. RESULTS: The unsuitability of presumably inert vermiculite as a physical plant growth substrate for studying the effects of acidity on soybean nodulation and plant growth was illustrated. Nodulation and growth of soybean grown in vermiculite were not affected by irrigation with pH-adjusted nutrient solution either at pH 4.0 or 7.0. This was reasonably caused by the ability of vermiculite to neutralise (buffer) the pH of the supplied nutrient solution (pH 2.0-7.0). CONCLUSIONS: Due to its buffering capacity, vermiculite cannot be used as growth support to study the effect of acidity on nodulation and plant growth.

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

Gene duplication in evolution has long been viewed as a mechanism for functional divergence. We recently cloned two related lipo-oligo-chitin receptor genes (GmNFR1α and GmNFR1ß) 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 GmNFR1αmutants 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 GmNFR1ß 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 GmNFR1promoters. GmNFR1α may encode a high affinity NF receptor responsible for the entire nodulation cascade while GmNFR1ß with lower affinity to NF suffices to induce cell divisions but not early infection events.

pHairyRed: a novel binary vector containing the DsRed2 reporter gene for visual selection of transgenic hairy roots.

We developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated 'hairy-root' transformation. pHairyRed allows easy in planta visualization of transgenic tissue with minimal disturbance to the plant. The DsRed2 reporter gene, encoding a red fluorescent protein, was cloned to yield pHairyRed (harbouring a multiple cloning site), which was used with the highly efficient K599 A. rhizogenes strain to infect soybean (Glycine max L. Merrill) plants. DsRed2 fluorescence was easily detected in planta for the duration of a 5-week study with negligible levels of background autofluorescence. This enabled visual selection of transformed roots and subsequent excission of non-transformed roots. pHairyRed-transformed roots nodulated normally when inoculated with Bradyrhizobium japonicum. Within the nodule, DsRed2 fluorescence was plant-specific, being absent in the bacteroid-dominated nodule infected zone. To test the reliability of pHairyRed as a high-fidelity binary vector reporter system, the gene encoding the soybean Nod factor receptor, GmNFR1α, was cloned into the vector for use in a complementation study with a non-nodulating nfr1α mutant of soybean. Complementation was achieved and, without exception, DsRed2 fluorescence was detected in all hairy roots that successfully formed nodules (100%, n = 34). We anticipate broad application of this reporter system for the further analysis of root-related events in soybean and related legumes.

Duplicated Nod-Factor receptor 5 (NFR5) genes are mutated in soybean.

In symbiosis with Bradyrhizobium japonicum, soybean (Glycine max L.) forms nitrogen-fixing nodules in its roots after mitogenic stimulation from a bacterial lipo-oligosaccharide (the 'Nod-factor'). In our recent paper in Plant and Cell Physiology we utilize two recessive loss-of-function plant mutants with a non-nodulation phenotype, and comparative genomics to clone and functionally analyze relevant soybean genes of the LysM receptor kinase family which are needed for perception of Nod-factor released by its microsymbiont B. japonicum. Two highly related lipo-oligochitin LysM type receptor kinase genes were cloned; they are presumed to be the critical nodulation inducing (Nod) factor receptor. These duplicated receptor genes were called GmNFR5α and GmNFR5ß. Non-sense mutations in GmNFR5α and GmNFR5ß were functionally complemented by both wild-type GmNFR5α and GmNFR5ß in transgenic roots, indicating that both genes are functional. Both genes are wild-type in some soybean cultivars; however, non-functional NFR5ß alleles were discovered in several others, which harbored an identical 1,407 bp retroelement-type insertion. GmNFR5α but not GmNFR5ß was expressed in tap and lateral root portions at about 10-25% of GmATS1 (ATP synthase subunit 1), but not in trifoliate leaves and shoot tips. In general, inoculation treatment down-regulated GmNFR5α/ß transcripts in tap and lateral root portions.

Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.

This protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Young seedlings with unfolded cotyledons are infected at the cotyledonary node and/or hypocotyl with Agrobacterium rhizogenes carrying the gene construct to be tested and the infection sites are kept in an environment of high humidity. When the emerged hairy roots can support the plants, the main roots are removed and the transgenic roots can be tested. Using this method, almost 100% of the infected plants form hairy roots within 1 month from the start of the experiments.