In silico analysis of fungal small RNA accumulation reveals putative plant mRNA targets in the symbiosis between an arbuscular mycorrhizal fungus and its host plant

Background: Small RNAs (sRNAs) are short non-coding RNA molecules (20–30 nt) that regulate gene expression at transcriptional or post-transcriptional levels in many eukaryotic organisms, through a mechanism known as RNA interference (RNAi). Recent studies have highlighted that they are also involved in cross-kingdom communication: sRNAs can move across the contact surfaces from “donor” to “receiver” organisms and, once in the host cells of the receiver, they can target specific mRNAs, leading to a modulation of host metabolic pathways and defense responses. Very little is known about RNAi mechanism and sRNAs occurrence in Arbuscular Mycorrhizal Fungi (AMF), an important component of the plant root microbiota that provide several benefits to host plants, such as improved mineral uptake and tolerance to biotic and abiotic stress. Results: Taking advantage of the available genomic resources for the AMF Rhizophagus irregularis we described its putative RNAi machinery, which is characterized by a single Dicer-like (DCL) gene and an unusual expansion of Argonautelike (AGO-like) and RNA-dependent RNA polymerase (RdRp) gene families. In silico investigations of previously published transcriptomic data and experimental assays carried out in this work provided evidence of gene expression for most of the identified sequences. Focusing on the symbiosis between R. irregularis and the model plant Medicago truncatula, we characterized the fungal sRNA population, highlighting the occurrence of an active sRNA-generating pathway and the presence of microRNA-like sequences. In silico analyses, supported by host plant degradome data, revealed that several fungal sRNAs have the potential to target M. truncatula transcripts, including some specific mRNA already shown to be modulated in roots upon AMF colonization. Conclusions: The identification of RNAi-related genes, together with the characterization of the sRNAs population, suggest that R. irregularis is equipped with a functional sRNA-generating pathway. Moreover, the in silico analysis predicted 237 plant transcripts as putative targets of specific fungal sRNAs suggesting that cross-kingdom post-transcriptional gene silencing may occur during AMF colonization. Keywords: Arbuscular mycorrhizal fungi, Rhizophagus, Small RNAs, microRNA-like, RNA interference