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MicroRNAs (miRNAs) control expression of endogenous target genes through transcript cleavage or translational inhibition. Legume plants can form a specialized organ, the nodule, through interaction with nitrogen fixing soil bacteria. To understand the regulatory roles of miRNAs in the nodulation process, we functionally validated gma-miR171o and gma-miR171q and their target genes in soybean. These two miRNA sequences are identical in sequence but their miRNA genes are divergent and show unique, tissue-specific expression patterns. The expression levels of the two miRNAs are negatively correlated with that of their target genes. Ectopic expression of these miRNAs in transgenic hairy roots resulted in a significant reduction in nodule formation. Both gma-miR171o and gma-miR171q target members of the GRAS transcription factor superfamily, namely  GmSCL-6  and  GmNSP2 . Transient interaction of miRNAs and their target genes in tobacco cells further confirmed their cleavage activity. The results suggest that gma-miR171o and gma-miR171q regulate  GmSCL-6  and  GmNSP2 , which in turn, influence expression of the  Nodule inception  ( NIN ),  Early Nodulin 40  ( ENOD40 ), and  Ethylene Response Factor Required for Nodulation  ( ERN ) genes during the  Bradyrhizobium japonicum -soybean nodulation process. Collectively, our data suggest a role for two miRNAs, gma-miR171o and gma-miR171q, in regulating the spatial and temporal aspects of soybean nodulation.

Characterization of the Spatial and Temporal Expression of Two Soybean miRNAs Identifies SCL6 as a Novel Regulator of Soybean Nodulation