Functional Analysis Of Nodulation Signaling Pathway 2 Interaction Protein IPN2 In Legume

The interaction of Rhizobium bacteria and legumes results in the formation of root nodules, that provide plant available nitrogen source. The unique and complex interactions between legume roots and Rhizobia are tightly controlled by the host in the various steps of infection, the formation of nodule primordium and the development of the N2-fixing root nodule. Therefore, the genes of the host plant must have an important role in the regulation of all these steps. Studies in the model legumes Lotus japonicus and Medicago truncatula by Genetic and genomic method have identified a set of genes, which play an essential role in the perception and signal transduction of Rhizobial Nod factors.Among them, the proteins of NSP1(Nodulation Signaling Pathway 1) and NSP2 have homology to transcriptional regulators of the GRAS-type protein family, which are the significant signal proteins in the symbiotic signaling pathway. In order to study the regulatory mechanism of these transcription factors on the formation of the root nodules, base on the protein Lj IPN2, which interacts with Lj NSP2 and was identificated from Lotus japonicus, we do the reseach on its homologous protein in Medicago truncatula, refer as Mt IPN2 and Mt ILK, the results are as follows:In this study, with the Lj IPN2 amino acid sequence, we do BLAST comparison analysis appraisal in http://www.medicagohapmap.org/ website(Medicago truncatula genome database), two homologous proteins found in Medicago truncatula, named Mt IPN2 and Mt ILK(IPN2- like) respectively. They belong to MYB-like transcription factor family, containing a single MYB repeat domain binds to DNA and a predicted class of coiled-coil dimerization domain.The Y2 H assay showed that the GRAS domain of Mt NSP2 was required for interacting with Mt IPN2 and Mt ILK, while the MYB and coiled-coil domain of Mt IPN2 and Mt ILK were sufficient for interacting with Mt NSP2. Moreover, Mt IPN2 showed strong transcriptional activation activity in yeast cells, that was different from Mt ILK. These interactions were confirmed by Bi FC assay in Nicotiana benthamiana. The mc Bi FC assay showed that Mt NSP2, Mt NSP1 and Mt IPN2 or Mt ILK form ternary complexes in a cell of Nicotiana benthamiana. Mt IPN2 and Mt ILK were localized in the nucleus of Medicago truncatula root. We performed EMSA to prove that Lj IPN2 binds to the Lj NIN promoter(-436 to-406) and Lj NSP1 binds to the Lj NIN promoter(-500 to 1) in vitro.