Transcriptional regulation of NIN expression by IPN2 is required for root nodule symbiosis in Lotus japonicus

Abstract

Expression of Nodule Inception (NIN) is essential for initiation of legume–rhizobial symbiosis. An existing model regarding the regulation of NIN expression involves two GRAS transcription factors – NSP1 (Nodulation Signaling Pathway 1) and NSP2. NSP2 forms a complex with NSP1 to directly bind to NIN promoter. However, rhizobial treatment-induced NIN expression could still be detected in the nsp1 mutant plants, suggesting that other proteins must be involved in the regulation of NIN expression. A combination of molecular, biochemical and genetic analyses was used to investigate the molecular basis of IPN2 in regulating root development and NIN expression in Lotus japonicus. In this study, we identified that IPN2 is a close homolog of Arabidopsis APL (ALTERED PHLOEM DEVELOPMENT) with essential function in root development. However, Lotus IPN2 has a different expression pattern compared with the Arabidopsis APL gene. IPN2 binds to the IPN2-responsive cis element (IPN2-RE) of NIN promoter and activates NIN expression. IPN2, NSP1 and NSP2 form a protein complex to directly target NIN promoter and activate NIN expression in the legume–rhizobial symbiosis. Our data refine the regulatory model of NIN expression that NSP2 works together with NSP1 and IPN2 to activate the NIN gene allowing nodulation in L. japonicus.