Symbioses between arbuscular mycorrhizal (AM) fungi and plants have significant effects on plant biodiversity and carbon cycling, and are therefore of considerable agricultural importance. The discovery that strigolactones, which are secreted by plant roots, are root-recognition signals for AM fungi, raises important questions concerning the mechanisms by which strigolactone signalling is regulated and how these signals are perceived by AM fungi. Experimental evidence indicates that, prior to colonizing plant roots, AM fungi produce one or several diffusible molecules collectively called 'Myc factors'. The roles played by strigolactones and Myc factors are hypothesised to be equivalent to those played by flavonoids and bacterial Nod factors (NFs) in the establishment of the nitrogen fixing symbiosis between legumes and rhizobia, with Myc factors being responsible for recognition by host plants of their symbiotic partners. The mechanisms underlying early mycorrhizal signalling are not well understood, though by testing symbiotic mutants of Medicago truncatula affected in genes that control steps of NF signalling, it was discovered that the three so-called DMI (Doesn't Make Infections) genes also control steps of mycorrhizal signalling, thus defining a common symbiotic pathway. Given that there are many differences between the bacterial and fungal symbiosis, we can hypothesise that there are plant components specific to the AM symbiosis yet to be identified, both upstream and downstream of this pathway. Since projects are underway to characterise Myc factors, it is timely to build on the discovery of the common symbiotic pathway and exploit our pioneering expertise in both NF signalling, and characterisation of plant responses to Myc factors. The objectives of this project are to achieve breakthroughs in understanding whether strigolactones and Myc factor signalling are connected in a regulatory loop and how Myc factors produced by AM fungi activate their host plants for mycorrhization. The originality of the project is that it will use different approaches and tackle different steps of Myc factor signalling. The project is also highly original since we will dissect Myc factor signalling using active fractions, and pure Myc factors as soon as they are available. Throughout, we will focus on a single plant species, Medicago truncatula, on which most of the pioneering analysis of Myc factor signalling has been performed. This will allow us to simultaneously unravel the extent of specificity and overlap between Myc and Nod signalling. To achieve these ambitious aims we will unite the complementary expertise of two partners in a multidisciplinary approach involving molecular genetics, transcriptomics, proteomics and biochemistry. The project will benefit enormously from the unique pioneering experience of the partners in mycorrhizal and NF signalling. The project will also benefit from the recent, considerable development of genetic, genomic and transgenic approaches that have endorsed M. truncatula as a model, and are making functional genomics and cloning projects feasible in short periods of time. To identify new components that could intervene at different stages of the Myc signalling pathway, we will use genetics and transcriptomic approaches. The genetic approach is a forward screen for mycorrhization-deficient mutants that will be tested for responsiveness to Myc factors. Interesting new genes will be cloned by transcript-based cloning. The transcriptome approach will use Affymetrix chips carrying 50,000 M. truncatula sequences, combined with a plant genotype that has a supermycorrhization phenotype and increased responsiveness to mycorrhizal signals. To extend the Myc signalling pathway upstream and downstream of the common signalling pathway, we will target Myc factor perception and the point of divergence of the Nod and Myc signalling pathways at DMI3. Candidate Myc factor receptor genes will be selected from the LysM-RLK gene family of M. truncatula and tested by reverse genetics. Targets of DMI3 will be searched for by a proteomic approach combined with a truncated, constitutively-active form of DMI3. The transcriptomic approach will be combined with plant genetics to determine which genes are involved in the Myc factor-activated signalling pathway that leads to the stimulation of lateral root formation. Characterisation of selected genes will help elucidate the question of how symbiotic signalling intercepts the plant developmental pathway for root development. Finally, we will analyse the molecular dialogue between plant roots and AM fungi, including the use of bio-assays combined with biochemical analyses and relevant mutants to test the hypothesis of a regulation loop linking strigolactone and Myc factor production. Given the known hormonal role of strigolactones in plants, the consequences on plant development of a putative control of their production by Myc factors will also be examined.