Data_Sheet_1_Nodulating Legumes Are Distinguished by a Sensitivity to Cytokinin in the Root Cortex Leading to Pseudonodule Development.docx

Root nodule symbiosis (RNS) is a feature confined to a single clade of plants, the Fabids. Among Fabids capable of RNS, legumes form root cortex-based nodules in symbioses with rhizobia, while actinorhizal species form lateral root-based nodules with actinomycetes. Cytokinin has previously been shown to be sufficient for “pseudonodule” initiation in model legumes. Here, we tested whether this response correlates with the ability to nodulate across a range of plant species. We analyzed the formation of pseudonodules in 17 nodulating and non-nodulating legume species, and 11 non-legumes, including nodulating actinorhizal species, using light and fluorescence microscopy. Cytokinin-induced pseudonodules arising from cortical cell divisions occurred in all nodulating legume species, but not in any of the other species, including non-nodulating legumes. Pseudonodule formation was dependent on the CRE1 cytokinin receptor in Medicago truncatula. Inhibition of root growth by cytokinin occurred across plant groups, indicating that pseudonodule development is the result of a specific cortical cytokinin response unique to nodulating legumes. Lack of a cortical cytokinin response from the Arabidopsis thaliana cytokinin reporter TCSn::GFP supported this hypothesis. Our results suggest that the ability to form cortical cell-derived nodules was gained in nodulating legumes, and likely lost in non-nodulating legumes, due to a specific root cortical response to cytokinin.