The CCAAT-box is a ubiquitous cis-acting regulatory element found in approximately 30% of eukaryotic promoters. The major protein complex binding this motif is the CCAAT-box binding complex (CBF), also called HAP (for Heme activator protein) in yeast and plants and nuclear factor-Y (NF-Y) in animals. The HAP complex was extensively studied in animal systems where it was shown to be composed of three subunits: HAP2 or NFY-A, HAP3 or NF-YB and HAP5 or NF-YC. In mammals the HAP complex is required to activate developmentally-regulated genes, in particular during the cell cycle and was shown to be a central regulator of cell proliferation and early development. In addition a major characteristic of NF-Y is its ability to interact with other key transcriptional regulators thereby controlling specific developmental pathways. In plants, although evidence for the importance of HAP protein complexes is gradually accumulating, much less is known about the structure and function of the HAP complex as well as of the individual transcription factors belonging to the CBF family. Like their homologues in mammalian systems, plant genes coding for HAPs can be classified in three groups, namely HAP2, HAP3 and HAP5. In contrast to their animal counterparts however, which are encoded by a single gene, the plant HAP genes have diversified and are encoded by small gene families of around 10 members. With so many individual HAP genes, plants have the potential to generate large numbers of HAP trimer combinations that have potentially been recruited into a wide range of processes including plant specific pathways. Indeed HAP genes have been implicated in early embryo development but also in flowering time control, drought tolerance, blue light and ABA signalling and chloroplast biogenesis. Our work provided the first evidence for a role of a HAP gene during the symbiotic interaction between soil bacteria known as Rhizobia and leguminous plants. This interaction results in the formation of a new organ called nodule on the root of its host plant and inside which atmospheric nitrogen is fixed for the benefit of the plant. We identified MtHAP2a by a transcriptome analysis as strongly and specifically expressed during early stages of nodule development and showed that, in mature nodules, Mt HAP2a expression is specifically restricted to the nodule meristematic zone. We established that MtHAP2a is by far the major HAP2 gene expressed in nodules. However several other MtHAP2 genes are also expressed in nodules. Using both RNAi and mutant analysis we have then shown that MtHAP2a plays a central role during both early symbiotic signalling and nodule meristem functioning and maintenance. This project aims at understanding the regulatory network by which MtHAP2a controls several important steps of the symbiotic rhizobium-legume interaction by indentifying both its partners inside protein complexes and the target genes whose expression is regulated by this transcription factor. For this purpose the present project is structured into three synergistic tasks. Task1: We will characterise the Medicago truncatula HAP genes expressed during nodulation. First an in situ expression analysis will unravel their tissular expression patterns. By looking for genes co-expressed with MtHAP2a we will identify HAP2 genes potentially playing complementary and/or additive roles, but also HAP3 and HAP5 genes coding for potential interactors inside symbiotic heterotrimeric HAP complexes. A functional analysis using RNAi and mutants will then reveal the potential role of co-expressed HAP genes. Task2: We aim at identifying and characterising the protein partners of MtHAP2a both the HAP3 and HAP5 partners and other transcriptional regulators. Screenings and tests will be performed using the yeast two hybrid system. For validation of interactors we will use a combination of bi-molecular fluorescence complementation (biFC), Fluorescence Resonance Energy Transfer technology (FRET), and co-localisation experiments using immunocytochemistry. Task 3: To identify target genes we will compare the transcriptome of wild type, Mthap2a-1 KO mutants and of plants overexpressing MtHAP2a using a Dexamethazone-inducible system. This will be complemented by PCR-assisted binding site selection (Selex) to determine the specific binding sites of this TF, combined with a bioinformatic approach. Validation of targets will be performed using electrophoretic mobility shift assays (EMSA) and immunoprecipitation (ChIP) assays. In addition to revealing the function of a nodal symbiotic regulator this project should, by understanding how MtHAP2a regulates nodule meristems, give insights into fundamental aspects of cell cycle control and cell differentiation during organogenesis. The present project should also enable us to show that, beyond nodulation, HAP2 proteins function in plants as regulatory HUBS connecting several signalling and developmental pathways.