Identification of putative interacting proteins of soybean nodulation receptor-like kinase (GmNORK)
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Legume plants establish beneficial symbiosis with nitrogen-fixing soil bacteria, called rhizobia. Root hairs serve as a main entry point for rhizobial invasion of the plant host root. A phosphoproteomic study performed on soybean root hairs in response to its symbiont Bradyrhizobium japonicum revealed significant changes in phosphorylation events at the very early stages of the symbiotic interaction. Nod factors (NF), signaling molecules secreted by rhizobia and perceived by plant hosts, are recognized by the receptor-like kinases (RLK) NFR1 and NFR5 located at the plasma membrane. NF perception triggers downstream signaling cascades, as well as other responses (such as ion changes, calcium oscillation, cytoskeleton reorganization, gene expression, etc), leading to bacterial invasion and nodule formation where nitrogen fixation takes place. Based on our phosphoproteomic data, we designed a 286-synthetic peptide library which we applied in a so-called Kinase-Client Assay (KiC), in order to identify putative interactor candidates and/or substrates of kinases. We used the NORK kinase, which acts downstream of the NF receptors (NFR1 and NFR5), to establish the assay and to validate the peptide library. We identified two putative interaction partners of NORK, putative cytosolic kinases, named as Interactor Candidate of NORK, ICNO1a/b. RNAi-mediated gene silencing of these kinases resulted in a significant reduction in nodule formation on soybean transgenic roots. Based on in silico analysis, both GmICNO1a and GmICNO1b possess an ACT domain (a structural motif in proteins of 70-80 amino acids named from the first letters of three of the proteins, aspartate kinasechorismate mutase-tyrA) at the N-terminus that might suggest the binding of a specific ligand that may regulate their activity and/or interaction with NORK
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