GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula.

The symbiotic association of legumes with rhizobia involves bacterially derived Nod factor, which is sufficient to activate the formation of nodules on the roots of the host plant. Perception of Nod factor by root hair cells induces calcium oscillations that are a component of the Nod factor signal transduction pathway. Perception of the calcium oscillations is a function of a calcium- and calmodulin-dependent protein kinase, and this activates nodulation gene expression via two GRAS domain transcriptional regulators, Nodulation Signaling Pathway1 (NSP1) and NSP2, and an ERF transcription factor required for nodulation. Here, we show that NSP1 and NSP2 form a complex that is associated with the promoters of early nodulin genes. We show that NSP1 binds directly to ENOD promoters through the novel cis-element AATTT. While NSP1 shows direct binding to the ENOD11 promoter in vitro, this association in vivo requires NSP2. The NSP1-NSP2 association with the ENOD11 promoter is enhanced following Nod factor elicitation. Mutations in the domain of NSP2 responsible for its interaction with NSP1 highlight the significance of the NSP1-NSP2 heteropolymer for nodulation signaling. Our work reveals direct binding of a GRAS protein complex to DNA and highlights the importance of the NSP1-NSP2 complex for efficient nodulation in the model legume Medicago truncatula.