In the model legume Medicago truncatula, we identified a new transcription factor of the CCAAT-binding family, MtHAP2-1, for which RNA interference (RNAi) and in situ hybridization experiments indicate a key role during nodule development, possibly by controlling nodule meristem function. We could also show that MtHAP2-1 is regulated by microRNA169, whose overexpression leads to the same nodule developmental block as MtHAP2-1 RNAi constructs. The complementary expression pattern of miR169 and MtHAP2-1 and the phenotype of miR169-resistant MtHAP2-1 nodules strongly suggest, in addition, that the miR169-mediated restriction of MtHAP2-1 expression to the nodule meristematic zone is essential for the differentiation of nodule cells.Supplemental material is available at http://www.genesdev.org.

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.

In this study, we describe a large-scale expression-profiling approach to identify genes differentially regulated during the symbiotic interaction between the model legume Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti. Macro-and microarrays containing about 6,000 probes were generated on the basis of three cDNA libraries dedicated to the study of root symbiotic interactions. The experiments performed on wild-type and symbiotic mutant material led us to identify a set of 756 genes either up-or down-regulated at different stages of the nodulation process. Among these, 41 known nodulation marker genes were up-regulated as expected, suggesting that we have identified hundreds of new nodulation marker genes. We discuss the possible involvement of this wide range of genes in various aspects of the symbiotic interaction, such as bacterial infection, nodule formation and functioning, and defense responses. Importantly, we found at least 13 genes that are good candidates to play a role in the regulation of the symbiotic program. This represents substantial progress toward a better understanding of this complex developmental program.Legume plants have the unique capacity to enter a nitrogen-fixing endosymbiosis with prokaryotes of the genera Rhizobium, Sinorhizobium, Mesorhizobium, and Bradyrhizobium (collectively termed rhizobia). In exchange for plant photosynthates, the endosymbiotic rhizobia convert dinitrogen to ammonia that is supplied to the plant for incorporation into amino acids and ultimately proteins. Symbiotic nitrogen fixation thus allows legumes to grow and produce protein-rich seeds even on nitrogen-depleted soil.Endosymbiotic interactions represent a particular case of biotrophic interactions (Parniske, 2000) where the microorganism is enclosed in a host-derived membrane within transient organelles, termed symbiosomes. These are harbored in a specific organ that differentiates from root tissues, the root nodule. Nodule formation and bacterial infection are strictly controlled by the plant (Schultze and Kondorosi, 1998;Stougaard, 2000). First of all, in wild-type legumes, nodulation is possible only when alternative sources of assimilable nitrogen (nitrate or ammonium) are not available. Second, legumes allow invasion of a very limited range of bacteria species producing highly specific signals, the Nod factors (chitolipooligosaccharidic molecules whose perception is essential to trigger the plant symbiotic program), and proper cell wall components (notably exopolysaccharides and lipopolysaccharides). Finally, nodules and infection threads (tubular structures of plant origin) develop in defined places and limited numbers. This is regulated by the plant via a locally operating mechanism that involves the plant hormone ethylene and a systemically operating mechanism, with a mobile signal of as yet unknown nature . In our experimental system, the differentiation of a nitrogen-fixing nodule takes about 1 week. Such a functional nodule consists of central tissues (the distal meris...