Final Report Summary - GRAS NOD SIGNALLING (NSP1 and NSP2: two GRAS proteins at the interplay of Nod Factor and Cytokinin signalling during symbiosis in Medicago truncatula) Modern agricultural practices are heavily reliant on inorganic nitrogen fertilisers. While the use of nitrogen fertiliser supports efficient food production, it comes at a heavy environmental and economic cost. Biological nitrogen fixation provides an attractive alternative to inorganic nitrogen fertilisers, but a significant investment in the research underpinning our understanding of this process is required if nitrogen-fixing non-leguminous crop plants is to become a reality. Our long-term objective is to dissect the molecular mechanisms that enable plant legumes to establish a symbiosis with bacteria conducting to the formation of a new root organ, where bacteria can fix nitrogen.Plant perception of the bacterial signalling molecule Nod factor (NF) is sufficient to initiate an organogenesis program in the root cortex. NF perception leads to the activation of calcium oscillations in root epidermis, which are likely decoded by CCaMK, whose function requires two GRAS transcription factors, NSP1 and NSP2. Moreover, activation in the root cortex of the cytokinin (CK) receptor CRE1 alone is sufficient to induce nodule formation and also requires NSP1 and NSP2.The overall objective of this proposal is to build a model of the CK pathway and its interaction with the NF pathway during symbiosis through the study of the transcription factors NSP1 and NSP2. The main questions that we addressed in this proposal are the following:1. Are epidermal and cortical symbiotic programs truly independent? 2. Are NSP1 and NSP2 components or targets of the CK signalling pathway? 3. Do NSP1 and NSP2 move to the root cortex or are they produced in the cortex? 4. Do NSP1 and NSP2 play a direct role in the cortex by activating cortical nodulation targets? To achieve these goals, we combined multiple analyses on the plant model Medicago truncatula. Thanks to promoter GUS analyses and to the use of tissue specific promoters, we confirmed that both NSP genes are expressed and required in both root type tissues: epidermis and cortex. We then conducted a significant microarray analysis comparing roots treated with Nod factor or Cytokinin. The use of nsp1, nsp2, and cre1 mutants enabled us to decipher the importance of those genes in these two pathways happening in the different root tissues. Moreover, a phylogenetic study coupled with protein-interactions tests gave us initial clues on cytokinin components involved during symbiosis. We identified a protein of phosphotransfer (MtHP1) interacting specifically with cytokinin components induced during symbiosis: the receptor MtCRE1 and the response regulators MtRR1 and MtRR4. Interestingly, we showed that this protein is also interacting with NSP2 in yeast two hybrid and bimolecular fluorescence complementation assays.We have shown that at least 70 % of the genes regulated by NF are dependent on the CK signalling pathway, confirming the essential role played by CK during symbiosis establishment. We confirmed that NSP1 and NSP2 are essential to NF responses and demonstrated for the first time that they are also essential to CK responses in legume roots. We showed that NSP1 and NSP2 are essential in both epidermal and cortical root tissues by regulating two main set of genes, one independently and one dependently of the CK pathway. We gave also first evidences that nonetheless being a target of the CK pathway, NSP2 is also directly interacting with it. The characterisation of the different targets CK dependent and non CK dependent identified in this study should give clues in a near future to the understanding of the key question of symbiotic programmes coordination, an essential knowledge to engineer nodulation in non legumes in the future.