Arief Indrasumunar

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.

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.

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 GmNFR1β) 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 GmNFR1β 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 GmNFR1β within other genotypes had no defects in nodulation, showing that GmNFR1β was redundant. Transgenic overexpression of GmNFR1α, but not of GmNFR1β, 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.

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.

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.

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 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.

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 GmSymRKβ. 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 GmSymRKβ, 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 GmSymRKβ 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.

Population dynamics of soybean root-nodule bacteria in latosol soil used for upland and lowland rice/soybean cropping systems in West Java, Indonesia

Two experiments were established in a latosol soil near Bogor, Indonesia to examine the population dynamics of soybean rhizobia under soybean-upland and -lowland rice management systems. Rice was sown in all plots before sowing the first soybean crop which was inoculated with either the wild-type or antibiotic-resistant mutants of strains CB1809, USDA110 and LRj I1D or left uninoculated. Numbers of soybean rhizobia in the soil before sowing the rice and in rice rhizospheres after 21 and 42 days, before sowing soybeans and in the soybean rhizospheres after 28 and 42 days were estimated by MPN using Glycine soja. In the lowland treatment, numbers of soybean rhizobia in the bulk soil were low before sowing rice (log10 1.86 g−1 soil) but they multiplied in the rhizospheres to ca log10 4.41 g−1 soil. In the upland treatment there were log10 4.45 initially which increased to log10 5.15 at 42 days. Following 2 months fallow and soil preparation for sowing soybeans in the upland treatment, the numbers of soybean rhizobia in the bulk soil were only log10 1.29 g−1 but they multiplied to log10 4.84 g−1 soil at 28 days in the soybean rhizospheres. Numbers in the soybean rhizospheres in the lowland treatment reached log10 5.27 g−1 at 28 days. They again declined rapidly, after the soybeans were harvested, to log10 1.32 and 1.98 g−1 of bulk soil in upland and lowland systems respectively. Inoculation did not affect nodulation, shoot dry weight or grain yeild of soybean despite nodule occupancy by selected strains, of between 52–63% in the lowland and 18–65% in the upland system. The implications of the results are discussed in terms of inoculation and management strategies.

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 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 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 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 GmNFR1promoters. 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 Control in Legumes

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, nodulation reception systems and autoregulation of nodulation (AON; Kinkema et al., 2006). The original discovery of the AON-controlling LRR receptor kinases, GmNARK/ LjHAR1/MtSUNN, which is active in leaf tissue of several legu-mes, now has led to an analysis of the mechanism underlying the signal transduction.