Molecular Mechanism Of Nodulation Receptor Kinase Regulating Symbiotic Nitrogen Fixation Via Phosphorylation Of NISP1 In Soybean

Symbiotic nitrogen fixation is the most efficient and economical way for legumes to obtain nitrogen.Nod factor is a prerequisite for the symbiotic interaction between legumes and rhizobia to produce nodules.Based on the research in the model legumes,such as Medicago truncatula and Lotus japonicus in the past two decades,the symbiotic signaling pathway has been roughly linearized.NORK（Nodulation Receptor Kinase）（also called Sym RK,Symbiosis receptor kinase or DMI2,Does not make infection 2）as a signal receiver is essential for establishing rhizobia symbiosis in legumes.Although several interacting proteins of NORK have been characterized,most of them could not be phosphorylated by NORK.Hence,it is of great interest to identify the phosphorylation targets of NORK.In this study,we identified that GmNISP1（GmNORKα-interacting small protein 1）is a phosphorylation target of GmNORK.GmNISP1 might function as a secreted peptide hormone that promote nodulation in soybean.The main results are list below:1.Using the kinase domain（KD）of GmNORKα(GmNORKα^KD,591 to 919 a.a.)as a bait in Y2H screening,we identify an unknown small protein that was named GmNISP1.The interaction between GmNISP1 and GmNORK was further verified using yeast two-hybrid assay,in vitro pull-down assay,co-immunoprecipitation assay and luciferase complementation assay in Nicotiana benthamiana leaves.In addition,GmNISP1 can also interact with Lj Sym RK,but not with GmNFR1 and GmNFR5.These results suggest that the interaction between GmNISP1 and GmNORK might be conserved and specific.The full-length c DNA of GmNISP1 has an open reading frame with 273 bp and encodes a protein of 90 a.a.Signa IP-4.1 predicted that GmNISP1 has no signal peptide and other special domains.Sequence alignment indicated that GmNISP1 exists in both legumes and non-legumes.The N-terminal of GmNISP1 protein sequence including a conserved DY motif that is typically required for protein secretion and peptide tyrosine sulfation.2.RT-q PCR analysis found that GmNISP1 was highly expressed in all soybean tissues except stems.Moreover,the levels of GmNISP1 transcripts in roots and nodules was much higher than that in flowers and leaves.We also found that GmNISP1 transcript levels increased markedly during rhizobia infection and nodule development.However,the induction pattern of GmNISP1 gradually disappeared during the nodule mature stage.GUS staining showed that GmNISP1 was highly expressed in rhizobia-infected root hairs,the initiation site of nodule primordia,nodule cortex cells and vascular tissues.These results were consistent with the GmNISP1 transcript levels observed in the RT-q PCR analysis,suggesting that GmNISP1 might be involved in rhizobia infection and the nodule organogenesis initiation.3.GmNISP1 overexpression,RNAi silencing,and CRISPR knockout were used to analyze the phenotype of transgenic hair roots.It was found that GmNISP1 could promote nodulation and induce the downstream symbiotic marker genes transduction,indicating that GmNISP1 is an important regulator in the establishment of symbiotic nitrogen fixation system.4.Biochemical analyses showed that GmNISP1 was directly phosphorylated by GmNORKα.Twelve potential phosphorylation sites of GmNISP1 were identified by mass spectrometry,including S4,Y7,S12,T17,S19,T22,S35,S39,T43,T45,T54and T55.Among them,the mutation of S35A lead to a decrease of GmNISP1phosphorylation signaling in an in vitro kinase assay.We then synthesized a mutated version of GmNISP1 with all 12 potential phosphorylation sites substituted with alanine(GmNISP1^12A)and tested the mutant peptide in the E.coli.In the Duet system,we found that the phosphorylation level of GmNISP1^12Awas significantly decreased.Phenotypic analysis of transgenic hairy roots revealed that phosphorylated GmNISP1can promote symbiotic nodulation.5.Subcellular localization analyses revealed that GmNISP1 was existed apoplast space suggested that GmNISP1 could be transported into the extracellular matrix.In addition,the phosphorylation-mimic GmNISP1^12Dsecreted more into the apoplast space,while the secretion of the phosphorylation-inactive GmNISP1^12Awas attenuated or even disappeared.Exogenous application of purified GmNISP1 or phosphorylated GmNISP1^12Dprotein can promote soybean nodulation.Consistent with this,the nodulation marker genes downstream of symbiosis were also up-regulated.These results indicated that GmNISP1 might function as a potential peptide hormone to promote soybean nodulation.In conclusion,our study established a new regulatory model of receptor kinase-secretory protein in symbiotic signaling transduction.When the nod factor signal is sensed by the co-receptor kinase NORK,it might be activated and subsequently phosphorylated downstream GmNISP1.The phosphorylated GmNISP1 could be secreted to the apoplast space,and involved in soybean nodulation.This model not only enriched the symbiotic signaling transduction pathway,but also provided a theoretical basis for improving soybean nodulation and increasing soybean resources.