Rhizobial bacteria enter a symbiotic association with leguminous plants, resulting in differentiated bacteria enclosed in intracellular compartments called symbiosomes within nodules on the root. The nodules and associated symbiosomes are structured for efficient nitrogen fixation. Although the interaction is beneficial to both partners, it comes with rigid rules that are strictly enforced by the plant. Entry into root cells requires appropriate recognition of the rhizobial Nod factor signaling molecule, and this recognition activates a series of events, including polarized root-hair tip growth, invagination associated with bacterial infection, and the promotion of cell division in the cortex leading to the nodule meristem. The plant's command of the infection process has been highlighted by its enforcement of terminal differentiation upon the bacteria within nodules of some legumes, and this can result in a loss of bacterial viability while permitting effective nitrogen fixation. Here, we review the mechanisms by which the plant allows bacterial infection and promotes the formation of the nodule, as well as the details of how this intimate association plays out inside the cells of the nodule where a complex interchange of metabolites and regulatory peptides force the bacteria into a nitrogen-fixing organelle-like state.

The formation of nitrogen-fixing nodules on legumes requires an integration of infection by rhizobia at the root epidermis and the initiation of cell division in the cortex, several cell layers away from the sites of infection. Several recent developments have added to our understanding of the signaling events in the epidermis associated with the perception of rhizobial nodulation factors and the role of plant hormones in the activation of cell division leading to nodule morphogenesis. This review focuses on the tissue-specific nature of the developmental processes associated with nodulation and the mechanisms by which these processes are coordinated during the formation of a nodule.

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.

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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.