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.
Legumes, such as Medicago truncatula, form mutualistic symbiotic relationships with nitrogen-fixing rhizobial bacteria. This occurs within specialized root organs--nodules--that provide the conditions required for nitrogen fixation. A rhizobium-derived signalling molecule, Nod factor, is required to establish the symbiosis. Perception of Nod factor in the plant leads to the induction of Ca2+ oscillations, and the transduction of this Ca2+ signal requires DMI3 (refs 2, 3), which encodes the protein kinase Ca2+/calmodulin-dependent protein kinase (CCaMK). Central to the regulation of CCaMK is an autoinhibitory domain that negatively regulates kinase activity. Here we show that the specific removal of the autoinhibition domain leads to the autoactivation of the nodulation signalling pathway in the plant, with the resultant induction of nodules and nodulation gene expression in the absence of bacterial elicitation. This autoactivation requires nodulation-specific transcriptional regulators in the GRAS family. This work demonstrates that the release of autoinhibition from CCaMK after calmodulin binding is a central switch that is sufficient to activate nodule morphogenesis. The fact that a single regulation event is sufficient to induce nodulation highlights the possibility of transferring this process to non-legumes.
In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule. Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth. A number of plant mutants with defects in the Nod factor signaling pathway have been identified. One such Medicago truncatula mutant, dmi3, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation. On the basis of these data, it is thought that the dmi3 mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking. Additionally, the dmi3 mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses. We report the identification of the DMI3 gene, using a gene cloning method based on transcript abundance. We show that transcriptbased cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants. DMI3 encodes a calcium͞calmodulin-dependent protein kinase. Mutants in pea sym9 have phenotypes similar to dmi3 and have alterations in this gene. The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in Arabidopsis thaliana, which does not participate in the mycorrhizal symbiosis.
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