The Interaction Of Nodulation Signaling Pathway NSP1 And NSP2 Proteins In Lotus Japonicus

The leguminous plants can specifically establish a symbiosis "nodule" with rhizobia. The establishment of the symbiotic relationship is involved in the complex process incloding signal perception, gene expression and regulation, the interactions of intermolecules between microbes and host plants. Lotus japonicus is one of the model legumes plants for studying the system on the symbiotic nitrogen fixation. The research of the Nod factors signaling pathway and the mechanism of intermolecular interaction in Lotus japonicus will uncover the secret about the establishment of symbiotic relationship and the formation of the symbiosis.In Lotus japonicus, the proteins of NSP1 (Nodulation Signaling Pathway 1) and NSP2 are belonged to the GRAS family transcription factors, which are the pivotal signal proteins in the symbiotic signaling pathway, and in the downstream of the CCaMK. In this study, we focus on the protein-protein interactions between NSP1 and NSP2, characterization of interaction domain, and identification of the role of the NSP1 binding to the promoter of taget genes. The results are as follows:1. Constructing a series of deletion mutants of NSP1 and NSP2, we assessed the interactions between NSP1 and NSP2 using yeast two-hybrid approach. The results indicate that NSP1 can interact with NSP2 and the LHRI domain alone (105-186aa) of NSP2 is necessary for the interaction. This suggests that NSP1 and NSP2 may form heteropolymers for performing Nod factors signaling in L. japonicus.2. A series of full and truncated proteins of NSP1 and NSP2 were expressed and purfitied from E. coli, which contains different domains. The protein-protein interactions in vitro were performed using pull-down and detected with western blotting. The results showed that the NSP1 in the absence of LHR1 and VHIID could be interacted with NSP2, and the interaction was abolished without the domain of the LHRII. This reveals that the LHRII of NSP1 is involved in the interaction between NSP1 and NSP2 in L. japonicus.3. Using the electrophoretic mobility shift analysis (EMSA), we tried to test whether NSP1 could directly bind to the NIN gene promoter which is a initial nodulation gene. The preliminarily results just showed some DNA smear on the EMSA gel and need to be further demonstrated.