Final Report Summary - MEDEPIMIR (Novel si/miRNAs in epigenetic regulation of salt stress responses in M. truncatula) Small RNAs (smRNAs) are important riboregulators in bacteria, fungi, plants and animals, which negatively regulate the expression of specific target genes by base-pairing. In the last decade, smRNA functions have been largely described in plants, both in development and responses to biotic and abiotic interactions (for review, Khraiwesh et al., 2012). Plant smRNAs, 20 to 24 nucleotides (nt) in length, are classically divided into microRNAs (miRNAs) and short-interfering RNAs (siRNAs). Like miRNAs, the natural-antisense siRNAs (natsiRNA) and the trans-acting siRNAs (tasiRNAs) are two classes of siRNAs involved in post-transcriptional gene regulation by degrading and/or inhibiting translation of their mRNA targets. In contrast, the heterochromatin-associated siRNAs (hcsiRNA) are associated to chromatin modifications and transcriptional repression of their target DNA loci. Legumes provide a major source of proteins for humans and other animals. In addition, legumes provide nitrogen for soil improvement, avoiding using nitrogen fertilizers and their damaging effects in the environment as they are able to establish symbiotic interactions with soil Rhizobium developping nitrogen fixing root nodules. Genomic progress has made possible to select two legumes as model plants (Medicago truncatula and Lotus japonicus) and other genomes from diverse legumes are being sequenced (Phaseoulus vulgaris and Glycine max). The model species Medicago truncatula, has a diploid and small genome (2x8 chromosomes), is autogamous and easily transformable. Moreover, it is closely related to extended legumes such as alfalfa, pea, chickpea and bean. In addition to be a model legume, Barrel Medicago’s succeeds in Mediterranean climates, with cool, moist winters and long, hot, dry summers and is well adapted to alkaline soils (over 4.5 million hectares in Australia and in Southern France). Objetive 1.Auxin action is mediated by a complex signalling pathway, which leads to the regulation of auxin responsive genes by transcription factors of the Auxin Response Factor (ARF) family. In Arabidopsis, the microRNA miR160 negatively regulates three ARF genes (ARF10/ARF16/ARF17) and therefore controls several developmental processes, including root growth and lateral organogenesis. In this part of the MEDEPIMIR project, we analysed the role of miR160 in root development and nodulation in the model legume Medicago truncatula. Bioinformatical analyses identified two main mtr-miR160 variants (mtr-miR160abde and mtr-miR160c) and 17 predicted MtARF targets, thus revealing a higher complexity than in Arabidopsis. The miR160-dependent cleavage of 4 predicted targets in roots was confirmed by analysis of PARE data and RACE-PCR experiments. Promoter-GUS analyses for mtr-miR160d and mtr-miR160c genes revealed overlapping but distinct expression profiles during root and nodule development. Composite plants that over-expressed mtr-miR160a presented reduced root length associated to a severe disorganisation of the RAM. Our results suggest that a regulatory loop involving miR160 governs nodule organogenesis in M. truncatula.Objective 2.RNA silencing has been mainly analyzed in Arabidopsis despite that it became a major tool for gene function studies and crop improvement in most plant species. Legumes, the third major crop worldwide, are able to form root nodules through symbiotic interactions with nitrogen-fixing bacteria. In the legume model Medicago truncatula, we identified orthologs of key genes involved in post-transcriptional RNA silencing in Arabidopsis. Mutant alleles were identified through TILLING for SGS3 (SUPPRESSOR OF GENE SILENCING 3) and RDR6 (RNA-DEPENDENT RNA POLYMERASE 6, or SGS2) encoding genes. In contrast to Arabidopsis, Mtsgs3 homozygous mutants were small, sterile and exhibited narrower and very serrated leaves compared to wild-type sibling plants. These mutants were impaired in the biogenesis of TAS3-derived transacting siRNAs (called tasi-ARFs) and accordingly accumulated ARF3 target transcripts, a known regulator of leaf morphology. In contrast, no developmental phenotype was observed in Mtrdr6 plants despite a weak but significant increase in symbiotic nodulation. This genetic background may become a useful tool for transgene expression in M. truncatula as Mtrdr6 roots expressing a GFP transgene, showed reduced post-transcriptional silencing and siRNA production. Genome-wide analysis of siRNA diversity revealed that MtRDR6 is required for the biogenesis of the recently described phasiRNA, involved in silencing of NBS-LRR defense genes, as well as of other novel siRNAs. Our results suggest that RDR6 control diverse siRNA-producing loci, including phasiRNA, that may be involved in symbiotic interactions in legumes.