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

Leguminous plants interacting with rhizobia establish a symbiotic relationship and form a highly specialized organ-nodule.The establishment of root nodule symbiosis depends on accurate signal exchange between leguminous plants and rhizobia.Leguminous plants secrected Nod Factor was perceived by rhizobia,then activates common symbiosis signalling pathway and promotes rhizobial infection and nodule organogenesis.Legumes respose to Nod Factor signal and activate Ca²⁺oscillation.CCa MK decodes Ca²⁺signal and activates phosphorylation itself and the substrate phosphorylation,thus induces the downstream gene expression.Symbiotic signal transmission involves multiple genes,but the complex regulatory mechanism remains to be revealed.This paper uses the model legume plant Lotus japonicus as material to explore the multiple gene function and regulation mechanism of molecular chaperone HIP and Myb transcription factor IPN2 in nodule symbiosis based on the following technological methods:gene expression analysis,protein-protein interactions,and genetic analysis,etc.1.The CCa MK-interacting protein of 73 k Da（CIP73）phosphorylated by calcium/calmodulin-dependent protein kinase CCa MK to involve in root nodule symbiosis.However,the functional mechanism of CIP73 in Lotus remains unclear.In this study,the screen analysis identified a HSP70 interacting protein（HIP）with CIP73（414 to 691 aa）through yeast two-hybrid screen in a L.japonicus c DNA library.Pull-down assay and Bi FC assay further indicated that HIP interacts with CIP73 dependent on the C-terminal St I1-like domain and forms homodimer itself.Further evidence indicated that HIP located in cytoplasm and nucleus and colocalizated with CIP73 in nuclei.In addition,the protein complexes CIP73/HIP and CIP73/CCa MK could take place simultaneously in the same nuclei.Spatio-temporal expression analysis revealed that the transcript level of HIP was slightly elevated in roots 1 day postinoculation（dpi）and soon returned to its original level at 2 dpi and then dropped drastically between 8 and 25 dpi after inoculation with rhizobia.The HIP promoter activity was mainly detected in the root tip,vascular bundles,and lateral root primordia.the HIP promoter activity was also observed at the nodule primordia and was down-regulated drastically in matured nodules after inoculation with rhizobia.Nodule number was significant increased compared to control at 16 dpi through RNAi-mediated knockdown of HIP expression.However,the difference was no longer significant at 32 dpi.These data suggested that HIP may play a negative regulatory role in the early stages of nodulation.2.Legume-rhizobium symbiotic signaling pathway is associated with complicated transcriptional regulation.A MYB coiled-coil transcription factor IPN2 was previously found to interact with NSP2 and bind to the NIN promoter.However,the biological functions of IPN2 in Lotus japonicus remain unclear.This study reveals that the ipn2-1 mutant exhibits phloem developmental defects which can be partially restored by IPN2 and Arabidopsis APL.Interestingly,the promoter activity of legume IPN2 homologous genes in root hair,cortex and vascular tissue is different from that of the phloem-specific gene APL.The infection thread formation is completely blocked in the ipn2-1 mutant.In addition,cortical expression of IPN2 in wild-type plants results in a significant increase in nodule number.This study also reveals that IPN2 is essential for the expression of NIN,NPL and ERN1.Transactivation and EMSA analyses indicate that IPN2 specifically binds to the IPN2 responsive cis element（IPN2-RE）of NIN promoter to activate its expression.Strikingly,NSP1 competes against IPN2 for the IPN2-RE to inhibit the activation of NIN promoter by IPN2 while NSP2 enhances the IPN2-mediated NIN activation.Collectively,these results demonstrate that IPN2 is a functional analogue of Arabidopsis APL and acts as a symbiotic regulator to directly regulate the expression of the major symbiotic genes in nodulation signaling pathway.