Dongxue Li

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.

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.

Agrobacterium rhizogenes Transformation of the Phaseolus spp.: A Tool for Functional Genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.

Structure–function analysis of the GmRIC1 signal peptide and CLE domain required for nodulation control in soybean

Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptide-encoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) of GmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation.

pGFPGUSPlus, a new binary vector for gene expression studies and optimising transformation systems in plants

A binary vector containing two reporter gene cassettes has been developed. This vector is ideal for optimising new plant transformation systems. Following optimisation, one of the reporter genes can be replaced with a gene of interest; the second can be used as a marker to confirm transgenic lines, and to estimate locus number and determine zygosity. This allows simple, efficient and economical screening for homozygous single-insert lines and azygous controls.

The soybean (Glycine max) nodulation‐suppressive CLE peptide, GmRIC1, functions interspecifically in common white bean (Phaseolus vulgaris), but not in a supernodulating line mutated in the receptor PvNARK

Legume plants regulate the number of nitrogen-fixing root nodules they form via a process called the Autoregulation of Nodulation (AON). Despite being one of the most economically important and abundantly consumed legumes, little is known about the AON pathway of common bean (Phaseolus vulgaris). We used comparative- and functional-genomic approaches to identify central components in the AON pathway of common bean. This includes identifying PvNARK, which encodes a LRR receptor kinase that acts to regulate root nodule numbers. A novel, truncated version of the gene was identified directly upstream of PvNARK, similar to Medicago truncatula, but not seen in Lotus japonicus or soybean. Two mutant alleles of PvNARK were identified that cause a classic shoot-controlled and nitrate-tolerant supernodulation phenotype. Homeologous over-expression of the nodulation-suppressive CLE peptide-encoding soybean gene, GmRIC1, abolished nodulation in wild-type bean, but had no discernible effect on PvNARK-mutant plants. This demonstrates that soybean GmRIC1 can function interspecifically in bean, acting in a PvNARK-dependent manner. Identification of bean PvRIC1, PvRIC2 and PvNIC1, orthologues of the soybean nodulation-suppressive CLE peptides, revealed a high degree of conservation, particularly in the CLE domain. Overall, our work identified four new components of bean nodulation control and a truncated copy of PvNARK, discovered the mutation responsible for two supernodulating bean mutants and demonstrated that soybean GmRIC1 can function in the AON pathway of bean.

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.

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.

Local and Systemic Effect of Cytokinins on Soybean Nodulation and Regulation of Their Isopentenyl Transferase (IPT) Biosynthesis Genes Following Rhizobia Inoculation

Cytokinins are important regulators of cell proliferation and differentiation in plant development. Here, a role for this phytohormone group in soybean nodulation is shown through the exogenous application of cytokinins (6-benzylaminopurine, N6-(Δ2-isopentenyl)-adenine and trans-zeatin) via either root drenching or a petiole feeding technique. Overall, nodule numbers were reduced by treatment with high cytokinin concentrations, but increased with lower concentrations. This was especially evident when feeding the solutions directly into the vasculature via petiole feeding. These findings highlight the importance of cytokinin in nodule development. To further investigate the role of cytokinin in controlling nodule numbers, the IPT gene family involved in cytokinin biosynthesis was characterized in soybean. Bioinformatic analyses identified 17 IPT genes in the soybean genome and homeologous duplicate gene partners were subsequently identified including GmIPT5 and GmIPT6, the orthologs of LjIPT3. Expression of GmIPT5 was upregulated in the shoot in response to nodulation, but this was independent of a functional copy of the autoregulation of nodulation (AON) receptor, GmNARK, which suggests it is unlikely to have a role in the negative feedback system called AON. Legumes also control nodule numbers in the presence of soil nitrogen through nitrate-dependent regulation of nodulation, a locally acting pathway in soybean. Upon nitrate treatment to the root, the tandem duplicates GmIPT3 and GmIPT15 were upregulated in expression indicating a role for these genes in the plant’s response to soil nitrogen, potentially including the nitrate-dependent regulation of legume nodulation pathway. Additional roles for cytokinin and their IPT biosynthetic genes in nodulation and the control of nodule numbers are discussed.