Lateral root formation is stimulated by common symbiosis genes and NIN in Lotus japonicus

Lateral roots (LR) and the root nodules (RN) of legumes are structurally related and the decision processes leading to RN formation involve signal exchange with the shoot. In order to disentangle these processes, we established a quantitative assay for LR formation in hairy root liquid cultures (HRLC) for the legume Lotus japonicus. In HRLC, ectopic expression of SymRK, or deregulated, auto-active versions of CCaMK and Cyclops stimulated LR formation in a NIN-dependent manner, but spontaneous RN were never observed. It appears that the previously described spontaneous RN formation induced by these versions requires the presence of the shoot. Interestingly, CCaMKT265D increased LR number in a cyclops mutant, revealing the presence of additional CCaMK targets mediating LR formation. Constitutive and ectopic expression of NIN under the ubiquitin promoter resulted in a significant increase in LR number. We compared the responsiveness of two Rosaceae that have either retained NIN (Dryas drummondii) or lost it (Fragaria vesca) to stimulation with the constitutively active variant CCaMK1-314. Intriguingly, CCaMK1-314 was able to increase LR formation in Dryas but not in Fragaria, pointing to consequences of the evolutionary loss of NIN on root architecture. Taken together our data provide evidence for NIN as a molecular link between symbiosis-signaling and LR formation. Non-inoculated nsp1 and nsp2 mutant plants as well as HRLC of these mutants exhibited increased LR densities that were no further increased by expression of CCaMK1-314. We propose a model in which LR density is balanced by the activation of NIN expression by SymRK and CCaMK and the LR suppressing activity of NSP1 and NSP2.