Inhibiting plant microRNA activity: molecular SPONGEs, target MIMICs and STTMs all display variable efficacies...

Elucidation of microRNA (miRNA) function through a loss-of-function approach has proven difficult due to extensive genetic redundancy among most plant and animal miRNA families. Consequently, miRNA decoy technologies such as target MIMICs (MIMs) and short tandem target MIMICs (STTMs) in plants or molecular SPONGEs (SPs) in animals have been developed to generate loss-of-function phenotypes by perturbing endogenous miRNA activity. To test whether SPs can inhibit plant miRNA activity, synthetic SP transgenes containing multiple miRNA binding sites targeting different Arabidopsis miRNA families were generated. Additionally, their silencing efficacies were compared to the corresponding MIM and STTM transgenes via scoring the frequency and severity of phenotypic abnormalities elicited by each transgene. While SPs with wild-type miRNA binding sites have no apparent impact, SPs containing miRNA binding sites with two central mismatches (cmSPs) can generate strong loss-of-function phenotypes. However, their efficacy varied dramatically, from inducing strong loss-of-function phenotypes to failing to produce any phenotypic impact. Variability was also observed when MIMs and STTMs were compared to cmSPs. While cmSP165/166 and STTM165/166 showed a stronger efficacy than MIM165/166, MIM159 was stronger than cmSP159 and STTM159. Although increasing the number of miRNA binding sites or strengthening the free energy of the miRNA binding site interaction can improve decoy efficacy, clearly additional unknown overriding factors are at play. In conclusion, we demonstrate that no one approach guarantees the strongest miRNA inhibition, but rather distinct miRNA families respond differently to the various approaches, suggesting that multiple approaches may need to be taken to generate the desired loss-of-function outcome.