Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor-induced gene expression and for spontaneous nodulation activated by the calcium-and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Ethyl methanesulfonate mutagenesis of the model legume Medicago truncatula has previously identified several genes required for early steps in nodulation. Here, we describe a new mutant that is defective in intermediate steps of nodule differentiation. The lin (lumpy infections) mutant is characterized by a 4-fold reduction in the number of infections, all of which arrest in the root epidermis, and by nodule primordia that initiate normally but fail to mature. Genetic analyses indicate that the symbiotic phenotype is conferred by a single gene that maps to the lower arm of linkage group 1. Transcriptional markers for early Nod factor responses (RIP1 and ENOD40) are induced in lin, as is another early nodulin, ENOD20, a gene expressed during the differentiation of nodule primordia. By contrast, other markers correlated with primordium differentiation (CCS52A), infection progression (MtN6), or nodule morphogenesis (ENOD2 and ENOD8) show reduced or no induction in homozygous lin individuals. Taken together, these results suggest that LIN functions in maintenance of rhizobial infections and differentiation of nodules from nodule primordia.In response to specific soil bacteria called rhizobia, legume roots initiate a unique developmental program that culminates in the formation of nitrogen-fixing root nodules. The highly specialized microenvironment of the nodule provides conditions necessary for the conversion of atmospheric nitrogen to ammonium by the rhizobial nitrogenase enzyme. Nod factors, lipooligosaccharide signal molecules produced by the bacterial partner, are required for the parallel processes of bacterial infection and nodule morphogenesis (Geurts and Bisseling, 2002). In recent years, major advances have been made in defining the host's responses to Nod factors, culminating in the cloning of several plant genes required for Nod factor signal transduction (Schauser et al., 1999;Endre et al., 2002;Stracke et al., 2002;Limpens and Bisseling, 2003;Madsen et al., 2003;Radutoiu et al., 2003;Ané et al., 2004;Lévy et al., 2004). The characterization of these genes and their mutant phenotypes indicates that receptor-like kinases play multiple roles in the perception of Nod factor and that transduction of the signal proceeds via calcium-mediated responses and transcriptional regulation (Cullimore and Dénarié, 2003;Parniske and Downie, 2003;Riely et al., 2004).Current understanding of the plant's contribution to symbiotic development subsequent to Nod factor perception, including rhizobial infection, primordium initiation, and nodule differentiation, comes largely from microscopic examination and characterization of plant genes that are differentially expressed during these processes. Early in the interaction, the bacteria gain access to the root through selective penetration of root hair cells. Invagination of the plant plasma membrane and deposition of cell wall material enable bacterial invasion via infection threads. Some, but not all, infection threads traverse the epidermal cells to invade root cortical cel...