Abstract
Symbiotic nodulation is initiated in the roots of legumes in response to low nitrogen and rhizobial signal molecules and is dynamically regulated by a complex regulatory network that coordinates rhizobial infection and nodule organogenesis. It has been shown that the miR156-SPL module mediates nodulation in legumes; however, conclusive evidence of how this module exerts its function during nodulation remains elusive. Here, we report that the miR156b-GmSPL9d module regulates symbiotic nodulation by targeting multiple key regulatory genes in the nodulation signalling pathway of soybean. miR156 family members are differentially expressed during nodulation, and miR156b negatively regulates nodulation by mainly targeting soybean SQUAMOSA promoter-binding protein-like 9d (GmSPL9d), a positive regulator of soybean nodulation. GmSPL9d directly binds to the miR172c promoter and activates its expression, suggesting a conserved role of GmSPL9d. Furthermore, GmSPL9d was coexpressed with the soybean nodulation marker genes nodule inception a (GmNINa) and GmENOD40-1 during nodule formation and development. Intriguingly, GmSPL9d can bind to the promoters of GmNINa and GmENOD40-1 and regulate their expression. Our data demonstrate that the miR156b-GmSPL9d module acts as an upstream master regulator of soybean nodulation, which coordinates multiple marker genes involved in soybean nodulation.