Analysis And Functional Characterization Of NCR Gene Family During Bacteroid Differentiation And Nitrogen Fixation In Astragalus Sinicus

NCR(Nodule specific Cysteine-Rich)are cysteine-rich peptides and have been identified in plants of IRLC(Inverted Repeat-Lacking Clade)and Aeschynomene spp.IRLC legumes form indeterminate nodules with rhizobia.The intrecelluar rhizobia accommodated within nodules undergo irreversible differentiation and this process is mediated by host-derived NCRs.A.sinucus also belongs to IRLC clade,however,the NCR family and their symbiotic function in this legume have not been reported yet.In this study,a big AsNCR gene family was identified,and bioinformatics analysis revealed that there is a new cluster of NCR peptides in A.sinicus characterized by its distinctive properties.The new type NCRs are larger peptides,contain a novel-3-9-5-type cysteine motif showing high similarity with plant defensins,and most of them are cationic.All of these indicate a special evolutionary status of AsNCR family in IRLC species.qRT-PCR analysis showed the specificity of NCR gene expression in nodules of A.sinicus.Cationic and anionic AsNCR peptides exert different impact on free-living rhizibia.The cationic peptides provoke membrane disruption and even cell death at high concentration,however,treatment with anionic AsNCR result in rhizobia proliferation and genome duplication.These reflect the different mode of action(MOA)on NCR-rhizobia interaction,which implies a rather complex process of NCR-drived bacteroid differentiation.Induction of AsNCR083 in M.huakuii 7653R caused significant growth arrest,and transcriptional changes were detected for genes involved in cell division and PG biosynthesis.Constitutive expression of AsNCR083 in M.huakuii elicited Fix-nodules and provoked failure of bacteroid differentiation.These indicate the potential interaction between NCR083 and intracellular rhizobial targets,providing new insights and materials to investigate mechanisms behind bacteroid differentiation and nodule development.By bacterial two-hybrid assay,a M.huakuii GroEL chaperone was identified to interact with AsNCRs.Abolish of GroEL function led to the formation of nitrogen fixation deficient phenotype during symbiosis.A set of AsNCR genes were down-regulated in Fix~-nodules,raising the possibility that interaction of AsNCR peptides with GroEL may be one of the key points in regulation of bacterial differentiation.Our research revealed the speciality of AsNCR evolution,and peptides with distinct structure can function differently.These results would deepen the understanding on evolutionary and functional diversity of NCRs,which further provides new insights and breakthrough to facilitate investigation of NCR-mediated terminal bacteroid differentiation.