Preliminary Analysis Of Functional Of The Protein MtHIRs Of Nitrogen-fixing Symbiotic Signaling Pathway In Medicago Truncatula

Legume plants can form a symbiosis relationship with rhizobia bacteria to generate a new plant organ,nodule,and subsequently to obtain fixed nitrogen.In recent years,scientists have identified some key genes involved in the regulation of nitrogen fixing symbiosis in model legume plants.Among them,Medicago truncatula leucine-rich repeat receptor-like kinase DMI2?Does not make infection 2?acts as a co-receptor to percept rhizobia Nod factor signal,and activates downstream infection thread formation and nodule primordia initiation.Therefore receptor-like kinase MtDMI2 plays a critical role in the regulation of the early symbiotic signaling events.To reveal the molecular mechanism of how MtDMI2 helps to recognize Nod factor molecule and to transduct the signal into the palnt cell,previously through mass spectrometric analysis and yeast-two-hybrid assay,we found that MtDMI2 may interact with MtHIRs?Hypersensitive Induced Response Protein?specifically.This research further analyized the interaction between MtDMI2 and MtHIRs,and the functional mechanism of MtHIRs-regulated early symbiotic signal transduction.Investigation into the relationship between MtDMI2 and MtHIRs can have significant implication for the research of nitrogen fixing symbiosis.The detailed results are as follows:1.Bioinformatics analysis revealed that all of the 5 MtHIRs homolog genes we interested in,which belong to the Band 7 protein family,have the SPFH?Stomatin Prohibitin Flotillin HflK/C domain?domain.Sequence alignment analysis of the Medicago truncatula HIRs proteins showed that the MtHIRs family can be devided into two subgroups,one subgroup,including the 5 proteins this study focused on,has conserved sequences between each other.The other group also has conserved sequences between its members.The prediction of MtHIRs protein structures indicated that they are probably lipid binding proteins.Analysis of MtHIRs gene expression showed that MtHIRs genes are highly expressed in both roots and nodules,while the detailed results were not the same among different databases,mostlikely MtHIRs genes have higher expression levels in roots comparing with the nodules.2.To visually investigate and confirm the subcellular localization of MtHIRs proteins before and after the formation of nodules,the constructs expressing MtHIRs::GFP driven by their native promoters were generated.By transforming the Medicago truncatula A17 plants with Argobacterium rhizogene Arqua1 containing the recombinant plasmids,this study observed that MtHIRs proteins mainly localizes at the plasmamembrane of plant root cells,furthermore MtHIR proteins have asymmetrical localization,indicating that the proteins may involve in cell-cell signal communication.3.In order to visually observe the spatio-temporal expression of MtHIRs genes before and after the rhizobia inoculation,this study transformed the Medicago truncatula A17 plants with Argobacterium rhizogene Arqua1 containing the recombinant MtHIRs_pro-pMDC163 plasmids and the results showed that MtHIRs genes were significantly expressed in plant roots.4.To verify the protein interaction between MtDMI2 and MtHIRs,this study generated the luciferase complementation and protoplast BiFc?Bimolecular fluorescence complementation?recombinant plasmids.By expressing the Mt107720-nluc and Mt107730-nluc recombinant constructs in the leaves of Nicothiana benthemiana,we purified the fusion proteins containing Myc tags.Western Blot experiments showed that the fusion proteins could be successfully expressed in tobacco leaves.5.The CRISPR/Cas9 genome editing constructs MtHIR1-pHSE401 and MtHIR2-pKSE401 were successfully generated.The plant transformation experiments are on the way.In summary,this study performed a thorough analysis of the functions of MtHIRs proteins and their relationship with MtDMI2,which paved the way for the full demonstration of the protein interaction between MtHIRs and MtDMI2 and the regulatory roles of MtHIRs at the beginning stage of nitrogen fixing symbiosis.