Reverse Genetics Approaches to Characterize CLE Peptide Induced Regulation of Nodulation

The ability of legumes to enter into a symbiotic relationship with nitrogen fixing bacteria is one of their defining characteristics. The host plant retains control of this symbiosis in response to external and internal stimuli, including environmental nitrate availability, and via a systemic autoregulation of nodulation (AON) mechanism maintained by the Nodulation Autoregulation Receptor Kinase in soybean (GmNARK). Due to the high degree of molecular similarity between GmNARK and the LRR receptor-like kinase CLAVATA1 in Arabidopsis, it has been proposed that GmNARK-dependent regulation may be induced by a similar ligand/s to that interacting with CLAVATA1, a family of small peptides called CLEs (CLV3-related peptides). We have identified candidate CLEs that respond to inoculation and/or nitrate treatment and which inhibit nodulation in a GmNARK-dependent manner when expressed in the root. The nodule inhibition response resulting from over-expression of these CLEs provides a model system to assess the relative importance of specific domains and motifs within these CLE peptides. To identify the key components contributing to the functional specificity of these putative ligands we are using reverse genetics approaches including site-directed mutagenesis and domain-swap experiments to identify critical residues and functional domains.