WOX5 expression stimulated by the transcription factor NF-YAc reprograms cortical cells for nodule primordium initiation in soybean

Reprogramming of differentiated root cortical cells into proliferative stem cells is a 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 rhizobial 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.