Function And Regulation Mechanisms Of MPK6 In Root Nodule Symbiosis In Lotus Japonicus

The Rhizobium-legume symbiosis is studied extensively,to establish this mutualistic relationship,the hosts and the rhizobia have to continually communicate with each other by responding to molecular signals secreted from each partner,then rhizobia could enter into root cells via ITs to trigger cortical cell division resulting in the formation of nodule structures.In a functional nodule,the rhizobia can gain a steady carbon and amino acid supply in a protected cellular environment,while the plants gain fixed nitrogen from metabolism of rhizobia.In recent years,a series of genes associated with symbiosis were identified in model plants Medicago truncatula and Lotus japonicus,and a model of early symbiosis transduction signaling was established.SymRK?symbiosis receptor-like kinase?has important function in nodule symbiosis(root nodule symbiosis?RNS?,which can regulate rhizobia infection,but the regulatory mechanism remains unknown.In order to identify target proteins,a yeast two hybrid was carried out using SymRK as a bait to screen a L.japonicus AD-cDNA library.A Lotus mitogen-activated protein kinase kinase?MAPKK?named SIP2 was identified to interact with SymRK and involved in the early symbiosis signaling and nodule organogenesis,but the molecular mechanism is still unclear.In this study,we found the target protein of SIP2,and studied on the mechanism of MAPK cascade involved in nodule symbiosis.1.Previous report AtMPK6 is a phosphorylation target of SIP2,therefore,we found the homologous protein of AtMPK6 in Lotus japonicus database via sequence alignment,named LjMPK6.SIP2 specificlly interaction with LjMPK6 in yeast,and the interaction depend on the activity of SIP2.Moreover,LjMPK6 was shown to be phosphorylated by SIP2 in vitro kinase assay.2.The expression of LjMPK6 was detected in all the tissues and the expression levels were significantly increased after inoculated with M.loti and nod factor treatment.The results of promoter-GUS analyses and in situ immunolocalization showed the localization of LjMPK6 in root hair along infection thread,nodule primordia,and parenchyma and vasculature in the mature nodule.3.To study the function of LjMPK6 in nodule formation,we obtained the knockout mutant of LjMPK6 from Lotus LORE1 retrotransposon insertion collection,however,homozygous mutant seedlings were unable to be identified from progenies of LjMPK6^+/-plants.Then we tried the CRISPR/Cas9 approach to knock out LjMPK6,but no LjMPK6 knock-out mutant plants could be obtained.Finally,we switched to knock down the expression of LjMPK6 under the control of the nodule-specific LjNAD1 promoter and two T1 independent stable transgenic plants were obtained.The nodule density and nodule primordia number in the LjMPK6 RNAi plants were significantly reduced as compared with that in the control plants.To further confirm the positive role of LjMPK6 involved in nodule symbiosis,we generated stable transgenic plants with overexpression of LjMPK6 under the control of Ljubiquitin promoter.However,the LjMPK6 overexpression stable transgenic plants were unable to be obtained.Thus,we used maize ubiquitin promoter to generated LjMPK6 overexpression plants and two T2 independent stable transgenic plants were obtained.The stable transgenic plants overexpressing LjMPK6 had larger nodule density and the numbers of infection threads and nodule primordia were significantly increased than control plants.4.Yeast two hybrid was carried out using LjMPK6 as a bait to screen a L.japonicus AD-cDNA library,LHK1 was identified as an LjMPK6-interacting protein.The interaction between LjMPK6 and LHK1 was confirmed via Y2H and CoIP,and the interaction depend on the activation of LHK1 receiver domain.5.A stable transgenic line carrying the cytokinin two-component output sensor ?TCS?:GUS was used to monitor the cytokinin response during nodulation whenLjMPK6 was overexpressed,the GUS activity was significantly reduced compared with the control.We also detected the expression of cytokinin-induced ACS genes,the results showed that LjMPK6 could partly negatively regulate expression of LjACS1 and LjACS2,and this inhibition required the cytokinin receptor LHK1.We then detected the ethylene levels in L.japonicus.After rhizobial infection,ethylene production was significantly decreased in LjMPK6-overexpressing stable transgenic plants and was significantly increased in LjMPK6-RNAi stable transgenic plants.6.In vitro protein competition assay showed LjMPK6 could partly inhibit LHP1 binding to the LHK1 receiver domain.In vitro kinase assay showed that LjMPK6 could partly inhibit the phosphorylation of LHP1 by LHK1.