Functions And Regulation Mechanisms Of Proteins Involved In Nodulation Signaling Pathway In Lotus Japonicus

Nitrogen is one of the limiting factors required for plant growth. The root nodule symbiosis (RNS) between legume plants and rhizobia is the most efficient and productive source of fixed nitrogen, and has critical importance in agriculture. But this endosymbiosis is specific for host plant and is restricted to legume plants and occasionally some nonlegume plants. Engineering nitrogen-fixing cereals like rice, wheat or maize is a highly topic challenge, and more efforts will further be required to reveal the molecular mechanisms of nitrogen fixing.The formation of nitrogen-fixing nodules requires the coordinated development of rhizobial infection and nodule organogenisis, which can be separated genetically. During the past decade, a lot of genes have been cloned from various symbiotic mutants of the two model legumes:Lotus japonicus and Medicago truncatula. A model of early symbiotic signaling cascade has been built based on these studies. Evidence is emerging that CCaMK, NSP1 and NSP2 form a complex on DNA, which need to be further confirmed. Here, we got some results as follows:1. Using the kinase domain of CCaMK as a bait in yeast two-hybrid screening, we identify a novel protein, CIP73 (CCaMK interacting protein of approximately 73 kDa), that interacts with CCaMK. CIP73 contains a Scythe_N ubiquitin-like domain and belongs to the large ubiquitin superfamily. Deletion and mutagenesis analysis demonstrate that CIP73 could only interact with CCaMK when the calmodulin-binding domain and three EF hand motifs are removed from the kinase domain. The N terminus 80 amino acid residues (80-160) of CCaMK are required for interacting with CIP73 in yeast cells. On the other hand, protein pull down assay and BiFC assay in Nicotiana benthamiana show that the full-length CCaMK could interact with CIP73 in vitro and in planta. Importantly, CCaMK phosphorylates the N-terminus of CIP73 in a Ca2+/calmodulin dependent manner in vitro. CIP73 transcripts are preferentially expressed in roots, and very low expression is detected in leaves, stems and nodules. The expression in roots is significantly decreased after inoculation of Mesorhizobium loti. RNA interference (RNAi) knockdown of CIP 73 expression by hairy root transformation in Lotus japonicus led to decreased nodule formation, suggesting that CIP73 performed an essential role in nodulation.2. In L. japonicus, snf1, a gain-of-function CCaMK (T265I) point mutation, which resides in the autophosphorylation site of the kinase domain, develops spontaneous nodules in the absence of rhizobia. The rice ortholog OsCCaMK mutant has defect in arbuscular mycorrhiza (AM) symbiosis. Moreover, molecular transfection of OsCCaMK could fully complement symbiosis defects in the corresponding Lotus mutant lines for both the AM and RN symbioses. To explore the symbiotic effects of CCaMK in rice, we transformed the OsCCaMK (T263A) or LjCCaMK (T265I) point mutation to rice, which was drived by the CaMV 35S or their native promoter respectively. The transgenic plants were identified by PCR amplification and GUS staining, but the symbiotic effects need to be further tested in the future.3. Using yeast two-hybrid (Y2H) screening, we identified a novel MYB coiled-coil transcription factor, referred as IPN2, which interacts with NSP2 in Lotus japonicus. This interaction was confirmed by protein pull-down assay and BiFC assay in Nicotiana benthamiana. Moreover, IPN2 and NSP2 were co-localized in nuclei of L. japonicus hairy roots. The Y2H assay showed that the GRAS domain of NSP2 was required for interacting with IPN2, while the coiled-coil domain of IPN2 was necessary and sufficient for interacting with NSP2. IPN2 showed strong transcriptional activation activity in yeast cells, and it bound to the NIN promoter in vivo and in vitro. IPN2 was widely expressed in various organs, and showed phloem-specific expression within the vasculature of transgenic hairy roots. Overexpression of IPN2 promoted nodulation, while RNA interference (RNAi) knockdown of IPN2 expression led to decreased nodule formation in L. japonicus. Take together, our results strongly suggest that IPN2 plays an essential role in nodulation.