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.

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11TGUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

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.

Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.

The symbiotic association between plants and arbuscular mycorrhizal fungi is almost ubiquitous within the plant kingdom, and the early stages of the association are controlled by plant-derived strigolactones acting as a signal to the fungus in the rhizosphere and lipochito-oligosaccharides acting as fungal signals to the plant. Hyphopodia form at the root surface, allowing the initial invasion, and this is analogous to appressoria, infection structures of pathogenic fungi and oomycetes. Here, we characterize RAM2, a gene of Medicago truncatula required for colonization of the root by mycorrhizal fungi, which is necessary for appropriate hyphopodia and arbuscule formation. RAM2 encodes a glycerol-3-phosphate acyl transferase (GPAT) and is involved in the production of cutin monomers. Plants defective in RAM2 are unable to be colonized by arbuscular mycorrhizal fungi but also show defects in colonization by an oomycete pathogen, with the absence of appressoria formation. RAM2 defines a direct signaling function, because exogenous addition of the C16 aliphatic fatty acids associated with cutin are sufficient to promote hyphopodia/appressoria formation. Thus, cutin monomers act as plant signals that promote colonization by arbuscular mycorrhizal fungi, and this signaling function has been recruited by pathogenic oomycetes to facilitate their own invasion.

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.

The formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. A screen for supernodulating Medicago truncatula mutants defective in this regulatory behavior yielded loss-of-function alleles of a gene designated ROOT DETERMINED NODULATION1 (RDN1). Grafting experiments demonstrated that RDN1 regulatory function occurs in the roots, not the shoots, and is essential for normal nodule number regulation. The RDN1 gene, Medtr5g089520, was identified by genetic mapping, transcript profiling, and phenotypic rescue by expression of the wild-type gene in rdn1 mutants. A mutation in a putative RDN1 ortholog was also identified in the supernodulating nod3 mutant of pea (Pisum sativum). RDN1 is predicted to encode a 357-amino acid protein of unknown function. The RDN1 promoter drives expression in the vascular cylinder, suggesting RDN1 may be involved in initiating, responding to, or transporting vascular signals. RDN1 is a member of a small, uncharacterized, highly conserved gene family unique to green plants, including algae, that we have named the RDN family.

SummaryArbuscular mycorrhizas (AM), mutualistic plant-fungus symbioses formed on the majority of land plants, appear to play an important role in plant mineral nutrition, plant health, and might influence the structure of plant communities. Progress in understanding the genetic and molecular basis of this important symbiotic association has been hampered by the obligate biotrophy of the fungal partner, and by the lack of mycorrhiza formation on the plant model species Arabidopsis thaliana . Recently, legume plants, such as Medicago truncatula and Lotus japonicus , have been chosen as experimental systems for research of plant-microbe symbioses. The application of both reverse-and traditional forward-genetic approaches, in conjunction with genomics initiatives in model legumes, will rapidly increase our understanding of the plant-encoded genetic determinants of the arbuscular mycorrhiza. An essential step in this process has been the generation, isolation and analysis of mycorrhizal mutants. This review provides an introduction to existing plant mutants affected in AM formation.© New Phytologist (2001) 150 : 525 -532