Analysis of Small Non-coding RNAs As Signaling Intermediates of Environmentally Integrated Responses to Abiotic Stress

Research to date on abiotic stress responses in plants has been largely focused on the plant itself, but current knowledge indicates that microorganisms can interact with and help plants during periods of abiotic stress. In our research, we aim to investigate the interkingdom communication between the plant root and the rhizo-microbiota. Our investigation showed that miRNA plays a pivotal role in this interkingdom communication. Here, we describe a protocol for the analysis of miRNA secreted by the plant root, which includes all of the steps from the isolation of the miRNA to the bioinformatics analysis. Because of their short nucleotide length, Next Generation Sequencing (NGS) library preparation from miRNAs can be challenging due to the presence of dimer adapter contaminants. Therefore, we highlight some strategies we adopt to inhibit the generation of dimer adapters during library preparation. Current screens of miRNA targets mostly focus on the identification of targets present in the same organism expressing the miRNA. Our bioinformatics analysis challenges the barrier of evolutionary divergent organisms to identify candidate sequences of the microbiota targeted by the miRNA of plant roots. This protocol should be of interest to researchers investigating interkingdom RNA-based communication between plants and their associated microorganisms, particularly in the context of holobiont responses to abiotic stresses.