Final Report Summary - SYMBIOSIGNAL (Characterization of Nod factor receptor LYK3 protein-interaction networks during early symbiotic signaling in Medicago truncatula) Proyect overview: Leguminous plants (i.e. pea, soybean, chickpea, clover…) are able to establish nitrogen-fixing symbiosis with soil bacteria collectively referred to as rhizobia. This interaction is based on a very specific signal exchange between plants and bacteria, so that bacteria are let colonize and multiply inside the plant cells in specialized organs named nodules. Bacteria inside are responsible for fixation of atmospheric nitrogen to ammonium that can be readily utilized by the plant as a nitrogen source, while the plant, in turn, provides bacteria with the necessary nutrients and organic compounds required for growth. Although much progress has been made in identifying the components of the signaling pathway in the legume-rhizobium symbiosis, there is a lack of understanding on how bacteria perception at the plasma membrane is connected to the activation of the signaling cascade. Project objectives, main results obtained and perspectives:The project SYMBIOSIGNAL was conceived to identify new proteins/genes involved in early signaling events. To this end, we employed the legume Medicago truncatula, a relative of alfalfa, as a plant model to analyze the early transcriptional events occurring in the roots. To monitor changes in gene expression, we have applied next-generation sequencing techniques (RNAseq) to analyze the changes on the transcriptome of Medicago truncatula roots in very early symbiotic stages (8 time points ranging from 30 min to 48 h after inoculation). To discriminate between Nod-factor and ethylene signaling, we analyzed four plant genotypes: wild type M. truncatula A17 Jemalong, mutants in Nod-factor perception nfp and lyk3, and a mutant in ethylene perception, skl. In total, we have identified almost 11,000 differentially-expressed genes, with more than 8,000 differentially regulated between wild type and skl samples. Some of these genes are co-expressed with previously identified members of the family, which strongly suggest that they are involved in these early symbiotic stages. Among those, numerous genes are involved in ethylene perception, signal transduction and ethylene biosynthesis. Of particular interest, we have found a number of novel transcription factors of the AP2/ethylene-responsive factor (ERF) superfamily involved in very early responses (i.e. 6h) upon inoculation. We selected six candidate transcripts, based on the strength of their transcriptional response, their dependence on ethylene and/or Nod-factor signaling, and their predicted impact on transcription. We are currently analyzing the expression pattern of these genes, as well as their functional role using available mutants and through gene silencing techniques. Results generated during SYMBIOSIGNAL pave the way for future work on the characterization of these and many other novel genes with a likely role in symbiosis.