A Small RNA Deep Seq Approach to Unravel the microRNA Regulations of Root Diseases in the Model Legume Medicago truncatula

MicroRNAs (miRNAs) are small non-coding RNAs of about 21nt and important regulators of gene expression. In Medicago truncatula (Mt), about 600 miRNAs have been identified but the extent to which miRNA pathways contribute to innate immunity during plant-pathogen interactions remains unknown. The bacterium Ralstonia solanacearum (Rs) and the fungus Verticillium albo-atrum (Vaa) are soil-borne pathogens that cause wilt disease in hundreds of plant species including the model legume Mt. To decipher the putative role of microRNAs in the regulation of wilt diseases, we deep-sequenced six small RNAs libraries prepared from roots of compatible (Mt A17/R.s.GMI1000, Mt F83005.5/VaaV32) and incompatible (Mt F83005.5/R.s.GMI1000, Mt A17/VaaV32) interactions and corresponding mock inoculated controls (MirMed Genoscope project). Statistical comparative analysis of the libraries through logistic regression allowed us to identify differential and specific microRNAs. Expression patterns for some new miRNAs have been validated through qRT-PCR. Bacterial wilt disease was subjected to a more detailed analysis. miR393, a miRNA known to be involved in the regulation of both developmental processes and defence against foliar pathogenic bacteria in Arabidopsis thaliana, increases tolerance to Rs: miR393 over-expression in hairy-roots via Agrobacterium rhizogenes transformation induced a significant delay in symptom appearance compared to control plants. Differential expression patterns of this miRNA and its corresponding target Mt-TIR1 in compatible and incompatible interactions with Rs were shown by qRT-PCR analyses. These results indicate a role of miR393 and of auxin in the regulation of basal defence responses towards a soil-borne pathogen in the model legume plant M. truncatula.