The transcriptional repressor LjTIE1 constrains cytokinin signaling to balance infection and organogenesis during root nodule symbiosis

Summary Cytokinin (CK) is a key regulator of root system architecture including primary root (PR) and lateral root (LR) development. During root nodule symbiosis in legumes, CK promotes nodule organogenesis in the root cortex while simultaneously suppressing infection thread (IT) formation in the epidermis, yet the molecular mechanisms enabling this spatial specificity remain incompletely understood. TCP INTERACTOR CONTAINING EAR MOTIF PROTEIN (TIE) proteins negatively regulate CK signaling in Arabidopsis roots, but whether legume orthologs modulate symbiotic CK responses remains unknown. We characterized Lotus tie1 loss-of-function mutants through root and nodulation assays, transcriptomics, confocal microscopy of CK signaling reporter TCSn, and ethylene quantification. Lj TIE1 expression was induced by CK and Nod-factor, tie1 mutants exhibited reduced PR length, LR density, IT formation, and nodule number alongside elevated ethylene emission - phenotypes fully rescued by ethylene biosynthesis inhibition. Despite enhanced CK signaling (confirmed via TCSn reporter and transcriptomics), tie1 roots formed no spontaneous nodules but displayed hypernodulation under exogenous CK treatment. LjTIE1 provides temporal control of symbiosis by dampening CK signaling after nodule initiation, revealing a regulatory layer that prevents constitutive nodulation despite elevated CK perception.