Short summary: We developed a set of sensors based on the firefly luciferase reporter gene to quantify miRNA activity in vivo, using Arabidopsis mesophyll protoplasts. We show that these sensors are not only responsive to endogenous miRNA activity, but also to exogenous gain-and loss-of-function manipulation of miRNA abundance. As examples, our sensors can be used for studying regulatory mechanisms of miRNA-associated components, for identifying novel factors with miRNA-associated functions, and for assessing the effect of chemicals on miRNA activity, therefore constituting a powerful tool to dissect miRNA pathways. responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this paper, we describe a transient expression system in Arabidopsis mesophyll protoplasts that is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics.We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, either by overexpression or by the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity.Our cell-based transient expression system is fast and easy to set up and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.
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