Integrated miRNA–mRNA analysis reveals candidate miRNA family regulating arbuscular mycorrhizal symbiosis of Poncirus trifoliata.

Over 70% land plants live in mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, and maintenance of symbiosis requires transcriptional and post‐transcriptional regulation. The former has been widely studied, whereas the latter mediated by symbiotic microRNAs (miRNAs) remains obscure, especially in woody plants. Here, we performed high‐throughput sequencing of the perennial woody citrus plant Poncirus trifoliata and identified 3750 differentially expressed genes (DEGs) and 42 miRNAs (DEmiRs) upon AM fungal colonization. By analyzing cis‐regulatory elements in the promoters of the DEGs, we predicted 329 key AM transcription factors (TFs). A miRNA‐mRNA regulatory network was then constructed by integrating these data. Several candidate miRNA families of P. trifoliata were identified whose members target known symbiotic genes, such as miR167h‐AMT2;3 and miR156e‐EXO70I, or key TFs, such as miR164d‐NAC and miR477a‐GRAS, thus are involved in AM symbiotic processes of fungal colonization, arbuscule development, nutrient exchange and phytohormone signaling. Finally, analysis of selected miRNA family revealed that a miR159b conserved in mycorrhizal plant species and a Poncirus‐specific miR477a regulate AM symbiosis. The role of miR477a was likely to target GRAS family gene RAD1 in citrus plants. Our results not only revealed that miRNA‐mRNA network analysis, especially miRNA‐TF analysis, is effective in identifying miRNA family regulating AM symbiosis, but also shed light on miRNA‐mediated post‐transcriptional regulation of AM symbiosis in woody citrus plants. Summary statement: In our study, we integrated miRNA with mRNA analysis, especially specific miRNA‐TF co‐expression node analysis, and attempted to reveal the regulatory links between transcriptional and post‐transcriptional regulatory mechanisms involved in AM symbiosis of woody citrus plants. [ABSTRACT FROM AUTHOR]