The ectomycorrhizal fungus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis.

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus and investigated their expression during mutualistic interaction with Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, , was found to be transported into roots after interaction with Further characterization experiments demonstrate that inhibition of negatively impacts the maintenance of mycorrhizal roots in , while supplementation of led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses.