Role of microRNAs in plant innate immunity

Summary Small RNAs (sRNAs) are short non-coding RNAs that guide gene silencing in most eukaryotes. Plants have two main classes of sRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are distinguished by their mode of biogenesis and mechanisms of action. In this day and age, crop losses due to pathogens and pests are estimated from 50% to 80%, factors limiting crop production and causing important economical losses. There is then an imperative need to improve our knowledge in defense mechanisms and to develop novel strategies for crop protection. To improve the understanding in this field, we carried out studies in Arabidopsis and rice plants, the two model systems used for functional genomic studies in dicot and monocot plant species. In the first chapter, we analyzed alterations on the accumulation of smRNAs in response to elicitor treatment, including miRNAs, in Arabidopsis plants. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA in the miRBase registry. We demonstrated that this smRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. In the second chapter, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection. In the third chapter, we used a combination of bioinformatic tools and experimental analyses for the discovery of new polycistronic miRNAs in rice, revealing 23 loci with the ability to form the typical hairpin structure of miRNA precursors in which two or more mature miRNAs mapped along the same structure. Evidence is presented on the polycistronic nature of 7 miRNA precursors containing homologous or non-homologous miRNA species. We also demonstrated a pattern of conservation in the genome of rice (Oryza sativa) species that have an AA genome, but not in primitive rice species. Collectivelly, results obtained in this work support the notion that miRNAs might be considered as components of the plant response to pathogen infection, possible acting as regulatory nodes of different physiological processes during plant adaptation to infection conditions.