The symbiotic nitrogen fixation process between legume roots and rhizobia initiates at the root hairs. Rhizobia initially colonize the tip of the root hairs and induce its curling to become entrapped. However, the specific molecular mechanisms underlying root hair deformation and curling in response to rhizobial infection remain unclear. In this study, transcriptome analysis of wild-type JiMa389 and nodulation-deficient mutant jima61 of Melilotus albus, the Rho-like small GTPase MaROP10. Our results show that MaROP10 functions as an interacts with the Nod factor receptor NFR5 to regulate rhizobia-induced root hair deformation and infection thread formation during the early stages of rhizobial infection. To elucidate the mechanism of MaROP10, we further identified its downstream potential effector protein, MaRIC6, which positively regulates root hair deformation and infection thread formation. Taken together, MaROP10 likely integrates signals from the symbiotic receptor NFR5 to regulate downstream signaling pathways through its effector MaRIC6, thereby coordinating root hair deformation and infection thread development during the early stages of rhizobial infection.

The plant natural product scopolin, a coumarin secondary metabolite, has been extensively exploited in flavor, cosmetic, medicine, and other industrial fields. Melilotus albus, a leguminous rotation crop, contains high concentrations of coumarin. The transcriptional regulatory network that controls the flow through the scopolin biosynthesis pipeline in M. albus remains poorly understood. MabHLH11 encodes a basic helix–loop–helix (bHLH) transcription factor whose transcription is positively associated with scopolin accumulation and with the expression of MaMYB4, the bHLH partner of the MYB-bHLH complex. Phylogenetic analysis grouped MabHLH11 in the TRANSPARENT TESTA 8 (TT8) clade of the bHLH IIIf subgroup. The MabHLH11 protein contained an MYB-interacting region and physically interacted with MaMYB4 in yeast and tobacco leaves. Co-overexpression of MabHLH11 with MaMYB4 in M. albus additively increased the expression of UDP-glucosyltransferase (MaUGT79) and induced more scopolin accumulation than occurred under the expression of MabHLH11 alone. MabHLH11 directly targeted the promoter of MaUGT79 and the activation of MabHLH11 was strengthened by the presence of MaMYB4. Thus, MaMYB4 enhanced the function of MabHLH11 in upregulating scopolin biosynthesis in M. albus, providing a theoretical basis for scalable production of a high-value plant natural product.