Legumes form symbiotic associations with either nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. Formation of these two symbioses is regulated by a common set of signalling components that act downstream of recognition of rhizobia or mycorrhizae by host plants. Central to these pathways is the calcium and calmodulin-dependent protein kinase (CCaMK)–IPD3 complex which initiates nodule organogenesis following calcium oscillations in the host nucleus. However, downstream signalling events are not fully understood. Here we show that Medicago truncatula DELLA proteins, which are the central regulators of gibberellic acid signalling, positively regulate rhizobial symbiosis. Rhizobia colonization is impaired in della mutants and we provide evidence that DELLAs can promote CCaMK–IPD3 complex formation and increase the phosphorylation state of IPD3. DELLAs can also interact with NSP2–NSP1 and enhance the expression of Nod-factor-inducible genes in protoplasts. We show that DELLA is able to bridge a protein complex containing IPD3 and NSP2. Our results suggest a transcriptional framework for regulation of root nodule symbiosis.

The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis.

Summary: The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen‐fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA‐sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense‐associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.