Host regulation of the symbiosis between legume plants and nitrogen fixing bacteria ensures optimal plant growth is matched with environmental conditions and plant resources. The symbiosis results in the development of new root organs known as nodules and provides cost and environmental advantages to legume crops. Soybean (Glycine max) is the most widely cultivated legume and the availability of a complete genome sequence makes it a model crop for understanding nodulation. The regulation of nodulation occurs systemically and requires a key component in the shoot, the Nodulation Autoregulation Receptor Kinase in soybean (GmNARK). The systemic autoregulation of nodulation (AON) pathway involves a root-derived signal (Q) that is perceived by GmNARK. This perception triggers the production of a shoot-derived inhibitor (SDI) that prevents further nodule development. The chemical nature of both the root- and shoot-derived signals in AON remains unknown. This thesis aimed to identify the root-derived fact
Due to the similarity of GmNARK to AtCLAVATA1, peptides similar to CLV3 (CLE peptides) which directly binds CLV1 have been proposed as possible candidates for the Q signal in AON. Three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) were identified in soybean that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to B. japonicum produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by B. japonicum inoculation and, unlike the other two GmCLE genes, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas B. japonicum induced regulation was contingent on GmNARK function in the shoot. These B. japonicum induced peptides are now candidates for the root-derived mobile signals that act as ligands for GmNARK.
The nodule inhibition response resulting from expression of these CLE peptide encoding genes provides a model system to assess the relative importance of each domain within these CLE peptides. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain of GmRIC1. This approach identified the arginine-1, alanine-3, proline-4, glycine-6, proline-7, aspartic acid-8, histidine-11 and asparagine-12 residues as critical to GmRIC1 activity. In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised suppression activity. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each peptide domain to the suppression differences that exist between the two peptides. We found that the signal peptide and CLE peptide regions of GmRIC1 could significantly enhance activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the activity of GmRIC1. The identification of critical residues and domains allows prediction of CLE peptides likely to possess nodule inhibition activity in legumes and confirms a role for the signal peptide in the initiation of systemic nodule inhibition by CLE peptides.
In order to determine whether the CLE peptide candidates are transported systemically to act as the Q signal, a bioassay was developed. This bioassay detects root-derived signalling molecules in xylem sap of soybean plants which may function in AON. The bioassay involves feeding of xylem extracts via the cut hypocotyl of soybean seedlings and monitoring of molecular markers of AON in the leaf. Transcript abundance changes occurring in the leaf in response to feeding were used to determine the biological activity of the extracts. To identify transcript abundance changes that occur during AON, which may also be used in the bioassay, we used an RNA-seq-based transcriptomics approach. We identified changes in the leaves of bioassay plants fed with xylem extracts derived from either B. japonicum-inoculated or uninoculated plants. Differential expression responses were detected for genes involved in Jasmonic acid metabolism, pathogenesis and receptor kinase signalling. We identified an inoculation- and GmNARK-dependent candidate gene (GmUFD1a) that responds in both the bioassay and intact, inoculated plants. GmUFD1a is a component of the ubiquitin-dependent protein degradation pathway and provides new insight into the molecular responses occurring during AON. It may now also be used in our feeding bioassay as a molecular marker to assist in identifying the factors contributing to the systemic regulation of nodulation.