The Interaction Between Symbiosis And Immunity During Legume Nodulation

Legumes establish a symbiotic relationship with nitrogen-fixing bacteria.The establishment of this relationship relies on the signal exchange between legumes and bacteria to initiate mutual recognition.Legumes need to accurately identify microbes-"friends or foes"to regulate the innate immune response,promoting the establishment of a symbiotic relationship with rhizobia and inhibiting the spread of pathogens.This study reveals the mechanism of NAD1 involved in the regulation of the balance between symbiosis and immunity in legumes-rhizobia interaction.1.NAD1 gene from the model legume Medicago truncatula and Lotus japonicus is transcriptionally activated both in response to rhizobial bacteria and throughout the cortical division of nodule development.This study describes the characterization of cis-regulatory elements that regulate NF-dependent and cortical specific gene transcription in Lotus japonicus.To identify the regulatory sequences involved in LjNAD1 expression,a series of promoter deletions fusion with the beta-glucuronidase reporter gene were analyzed in transgenic Lotus japonicus root nodules.These results have revealed that distinct regulatory regions are involved in rhizobial infection-related NAD1 expression during nodule development.In particular,the region 400-200bp promoter sequence immediately upstream from the start codon is sufficient for NAD1 expression in cortical cell.Using Partial sequence alignment of legume NAD1 promoter,three conserved nodulation signaling responsive regulatory units（NS-box1/2/3）are identified.Detailed analysis of the LjNAD1 promoter using deletion,mutation constructs identified the NS-box1 and NS-box3 cis motifs indispensable to the NAD1 expression in Lotus nodule.However,either NS-box1 or NS-box3 is sufficient to activate the expression of NAD1 in Medicago nodules.2.Suppression of immunity in host cells is essential for successful rhizobial symbiosis during nodulation in legumes.In the nad1 plants,the very early defense response was activated as bacterial intracellular colonization and terminal differentiation.The rhizobia,along with their symbiotic plant cells become necrotic after rhizobia were released from infection threads into symbiotic cells of nodules.However,the mechanism of activated defence in nad1 mutant nodules is largely unknown.This study shows that the plant plasma membrane-localized NADPH oxidases,known as repiratory burst oxidase homologues（RBOHs）,are involved in plant immunity during nodulation.The early development of bacteroids is strongly repressed in nad1 mutant nodules compared with WT nodules at the early stage.Simultaneously,the defense responses including induction of defense-related genes expression and accumulation of reactive oxygen species（ROS）are significantly induced in nad1 mutant nodules compared with WT nodules.Among the selected defense-related genes,the Rboh homologue Rboh C is highly induced in nad1 early nodules.It is predicted that Rboh-mediated ROS production is required for evoking a strong defence response in the nad1 mutant nodules.Interestingly,knocking out Rboh family in the nad1 mutant via utlizing CRIPSR/CAS9 technology could fully restore the normal nodule phenotypes:nitrogen fixation and nodule development.Moreover,knocking out Rboh B or Rboh D gene in hairy root rescued nitrogen fixation and normal infection cells in the nad1 mutant nodules.Knocking out Rboh C gene significantly reduces defence symptom in the nad1 mutant nodules.In addition,genes encoding group I CDPKs were strongly expressed in nodules.CDPK5 interacts with Rboh family by co-immunoprecipitation and bimolecular fluorescence complementation assays.Furthermore,the constitutively active form CDPK5^VK induces the cell death enhanced by co-expression of Rboh family in Nicotiana benthamiana leaves.Moreover,knocking out of CDPK5 could partially rescue the normal phenotype in the nad1mutant nodules and CDPK5^VK over-expression lines in the wild type display ineffecitive nodules,which is reminiscent of the nad1 mutants nodules.Interestingly,CDPK5 may form the complex with the upstream Lys M-PRR receptors in Nicotiana benthamiana leaves.Morevoer,knock-out of Lys M-PRR receptors also patially rescued nitrogen fixation and normal infection cells in the nad1-1 mutant.Remarkably,ulvan recognized by Lys M-PRR receptors possibly triggers immunity in nodules.NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules.NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum.Functional complementation analysis showed that the two conserved TM domains were critical for NAD1 function.A conserved CFCF motif in TM1 and an FVV motif in TM2 were also shown to be indispensable.Co-immunoprecipitation and bimolecular fluorescence complementation assays indicated that NAD1 formed a homodimer or homooligomer.These results suggest that the symbiotic gene NAD1suppresses of plant innate immunity mediated by the complex of Lys M-PRR/CDPK-Rboh possibly via recognition of extrinsic factor ulvan or intrinsic factor during nodulation...