The <i>Medicago truncatula</i> lncRNA <i>ENOD40</i> is a mediator of <i>microRNA169</i> -controlled <i>NF-YA</i> activity in nodule initiation

The hallmark of the legume lncRNA <i>EARLY NODULIN40</i> (<i>ENOD40</i>), involved in rhizobium-induced nodulation, is the presence of a highly conserved stretch of 24 nucleotides, designated box2, preceded by a small open-reading-frame (sORF) coding for a peptide of 12 to 13 amino acids. Although there is a well-established link between <i>ENOD40</i> and nodulation, it is not fully clear by which mechanism <i>ENOD40</i> functions in this process. Here, we show that a region harboring box2 can complement nodule formation in an <i>ENOD40</i> knock-out mutant (<i>enod40-1-2/1</i>). The sequence of box2 bears the characteristics of a <i>miR169</i> target mimic. We show that the artificial target mimic <i>MIM169defg</i> can indeed complement the reduced capability of nodule formation in <i>enod40-1-2/1</i>, and that box2 exhibits target mimic activity in a transient luciferase assay. In addition, the introduction of a <i>miR169</i>-resistant form of <i>MtNF-YA1</i> also elevates the capacity to form nodules in <i>enod40-1-2/1.</i> We conclude that <i>ENOD40</i> effectuates nodule initiation by posttranscriptional upregulation of the <i>miR169</i> target <i>NF-YA1</i>, which encodes an essential transcription factor in this step of the nodulation process.