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.

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.

SUMMARYTwo 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 (GmNFR1a and GmNFR1b) as putative Nod factor receptor components in soybean. Both mutants contained frameshift mutations in GmNFR1a that would yield protein truncations. Both mutants contained a seemingly functional GmNFR1b homeologue, characterized by a 374-bp deletion in intron 6 and 20-100 times lower transcript levels than GmNFR1a, yet both mutants were unable to form nodules. Mutations in GmNFR1b within other genotypes had no defects in nodulation, showing that GmNFR1b was redundant. Transgenic overexpression of GmNFR1a, but not of GmNFR1b, 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. GmNFR1a overexpressing roots also formed nodules in nodulation-restrictive acid soil (pH 4.7). Our results show that: (i) NFR1a 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 GmNFR1a.

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.

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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.

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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 GmNFR5β. Non-sense mutations in GmNFR5α and GmNFR5β were functionally complemented by both wild-type GmNFR5α and GmNFR5β in transgenic roots, indicating that both genes are functional. Both genes are wild-type in some soybean cultivars; however, non-functional NFR5β alleles were discovered in several others, which harbored an identical 1,407 bp retroelement-type insertion. GmNFR5α but not GmNFR5β 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/β transcripts in tap and lateral root portions.