Study On Transduction And Action Mechanisms Of Symbiosis Signals Of Rhizobium In Lotus Japonicus

The rhizobia develop root nodules with legumes,the symbiotic relationship is specified. Establishing this symbiosis requires a response of molecular signals,gene expression and nutrient exchange between the plant and the bacteria.The first step in the molecular dialogue between the plant and the bacteria is the detection by rhizobia of FLAVANOIDS and related molecules that are secreted from the legume roots.Production of Nod factor molecule is activated by the release of plant predominantly flavonoids,into the rhizosphere,where they activate Nod factor production through induction of a set of nod genes in the appropriate rhizobial strain.Nod factors induce root hair deformation,formation of infection threads and expression of early nodulin genes(ENOD) till nodulation was formation.However,we did not clear the mechanism of the signal transduction pathway which activated by Nod Factor in host plant.To determine this mechanism of the signal transduction pathway and explore the unknown molecules in this cascade we study the model legume Lotus japonicus by method of biological chemistry and molecular biology.In current work,we have obtained these results as follows:1.The Nod factor receptor kinase 1(NFR1),Nod factor receptor kinase 5(NFR5), Symbiosis Receptor-like Kinase(SymRK) and CASTOR genes were identification from Lotus japonicus which were key factor in the Nod Factor signal transduction pathway.We check the interaction of LjNFR1,LjNFR5,LjSymRK and LjCASTOR proteins by yeast two hybrid systems.The result indicates that proteins could not interact with each other.2.Cultivate Lotus japonicus for the plant tissue;harvest the root of Lotus japonicus after inoculation by 2d,4d,6d,8d and 12d.Combine all the tissues and extract the total RNA Synthesize the ds cDNA by RT-PCR.We constructed a yeast two hybrid(Y2H) AD-cDNA library with root tissue of Lotus japonicus.The library capacity is 2.5×10~6 transformants/3 ug DNA.The PCR result of random chosen library colonies shows that the average length of cDNA inserts is 0.5 kb to 2.0kb.3.Lotus SymRK gene comprises the classical hallmarks of an RLK;a signal peptide, three leucine-rich repeats(LRRs) of the extracellular type are found in the predicted extracellular domain,a transmembrane domain(TM) and an intracellular protein kinase domain(PK).We screened the library using SymRK extracellular domain(LRR) and protein kinase domain(PK) as the bait proteins.The result indicates that 15 types of genes were characterization.We describe here a novel DNA-binding protein from Lotus japonicus, referred to as SIP1,because it was identified as a SymRK-interacting protein 1.BLAST analysis of SIP1 protein contained conserved AT Rich Interaction Domain(ARID)(residues 126-217).The predicted 3-D model of the ARID domain of SIP1 consists of 8α-helices,twoβ-strands,and four structure-undefined loops.4.The N-terminal half of SIP1(SIP1N) containing the N-terminus and the ARID domain was not found to interact with SymRK-PK.The C-terminus of SIP1(SIP1C) was found to be responsible for its interaction with the kinase domain of SymRK。The interactions were also detected when the C-terminus of SIP1 was used SIP1C with SIP1;SIP1C with SIP1C,suggesting that the C-terminal 184 residues are responsible for SIP1 dimerization in the absence was not required for SIP1 dimerization.In contrast,the N-terminus(223 residues) of SIP1,which contains the ARID domain,was not required for SIP1 dimerization.5.SIP1 specifically binds to the promoter of LjNIN,but not to that of LjCBP1(a calcium-binding protein gene),both of which are known to be inducible by Nod factors.SIP 1 recognizes two of the three AT-rich domains present in the NIN gene promoter.Deletion of one of the AT-rich domain at the NIN promoter diminishes the binding of SIP1 to the NIN promoter.The sequence -59 to -70 is the most important site for SIP1 Bingding with NIN promoter.6.Using quantitative PCR,we examined the SIP1 and NIN mRNA levels in different tissues of L.japonicus.The SIP1 gene is expressed constitutively in leaves and the uninfected roots,and its expression levels are elevated after infection by Mesorhizobium loti.This expression pattern was distinct from that of NIN,which exhibited significant induction 5 h after rhizobial inoculation and maintained a high expression level in inoculated roots.It is proposed that SIP1 may be required for the expression of NIN and involved in the initial communications between the rhizobia and the host root cells.The protein is localized to the nucler when expressed as a red fluorescence fusion protein in the onion epidermal cells. Using the kinase assay,we confirmed that SIP1 protein was not the phosphorylation substrate of SymRK protein.