Mechanism Of GmNiNa Down-regulating Expression Of GmBAK1e And Promoting Nodulation Of Soybean

Nitrogen is an essential element for plant growth and development.Nitrate nitrogen and ammonium nitrogen are the main forms of nitrogen nutrition obtained from soil by plant roots.Soybean relies on and rhizobia nodule symbiosis as main nitrogen sources,but rhizobia is gram-negative bacteria and can also cause the host's immune response.The previous studies have proved that rhizobial infection triggers the immune response of soybean at the early infection stage which is then gradually reduced with the establishment of the rhizobial infection and symbiotic relationship with host plants.Therefore,inhibition of the immune response of soybean is one of the necessary conditions for the establishment of soybean and rhizobia symbiosis.However,how the immune response is regulated during nodulation and symbiosis still remains largely unknown.The fact that the LiNIN is enriched in the LjBAK1 promoter inspired us to investigate the possible role of NIN in downregulating of BAK and immune response during nodulation using soybean as a model plant.A systematic bioinformatics analysis identified five homologous genes of AtBAK1 in soybean and named as GmBAK1a-e according to their positions on the chromosomes.The eFP expression data showed that GmBAK1d/e are predominately expressed in the underground part of soybean.Finally,the promoter analysis revealed that GmBAK1 e promoter does not have the binding elements for defense response but contains four NIN binding elements in its promoter,suggesting a diverse role of GmBAK1 e,which thereafter was used for further study.Further functional analysis of GmBAK1 e showed that the functional deficiency of GmBAK1 e led to increased nodule formation in soybean,while the overexpression of GmBAK1 e reduced nodule number,indicating that GmBAK1 e was a negative regulatory factor in soybean nodule formation.RT-qPCR and GUS staining results showed that the expression of GmBAK1 e in the transgenic roots overexpressing GmNINa was significantly decreased,suggesting that GmNINa negatively regulates the expression of GmBAK1 e.Finally,the preliminary EMSA assay showed that GmNINa directly bind to the NIN binding element of GmBAK1 e promoter.Together,these results reveal that GmBAK1 e is a negative regulator of symbiotic nodulation and nitrogen fixation in soybean.These results provide novel insights into molecular mechanism of rhizobia-soybean symbiosis and possible link between nodulation and immune response in soybean.