NF-YAc-stimulated WOX5 expression reprograms cortical cells for nodule primordium initiation in soybean

Abstract Reprogramming of differentiated root cortical cells into proliferative stem cells is the prerequisite for legume nodule organogenesis, yet the molecular trigger that confers stem-cell identity upon these cortical cells remains elusive. Here we demonstrate that, in soybean (Glycine max), the canonical root stem-cell regulator WUSCHEL-RELATED HOMEOBOX gene WOX5 is activated by rhizobia specifically in cortical cells that will give rise to nodule primordia. CRISPR/Cas9-mediated knockout of the three WOX5 homologs, wox5abc mutants reduced nodule number and attenuated nitrogenase activity, attributable to a decrease in primordium density rather than impaired rhizobia infection. Promoter dissection identified a 442 bp legume-specific promoter fragment within the WOX5a promoter that is both necessary and sufficient for primordium-specific expression. Chromatin immunoprecipitation and dual-luciferase assays revealed that this promoter fragment is directly bound by the symbiosis-responsive transcription factor NF-YAc to activate expression of WOX5a. Loss of NF-YAc phenocopied wox5abc, and NF-YAc overexpression failed to rescue nodulation in wox5abc mutants. Collectively, our findings reveal that NF-YAc–mediated activation of WOX5 initiates a de novo stem-cell niche in root cortical cells, providing the critical developmental trigger for nodule primordium initiation in soybean.