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Expression of a microRNA-resistant target transgene misrepresents the functional significance of the endogenous microRNA: target gene relationship.

Dear Editor, A current major challenge in microRNA (miRNA) biology is the elucidation of their functions. A key first step in this pursuit is the identification of functionally relevant miRNA:target gene relationships. Although the molecular verification of miRNA targets with degradome or 5’-end RACE approaches has been very successful in plants, the detection of miRNAguided cleavage products may not be reflective of functional importance (Allen et al., 2010), which can only be definitively defined by genetic approaches. This remains challenging, as isolation of loss-of-function mutations in miRNA genes has been rare, due to both their small size and the fact that many exist in multi-gene families. Consequently, much of our current understanding of miRNA function has relied heavily on gain-of-function approaches, such as the transgenic expression of miRNA-resistant target genes, where multiple synonymous mutations are created within the miRNA binding site of a putative target gene, conferring the gene resistant to miRNA regulation (Garcia, 2008). By comparing the phenotypic impact of the expression of this miRNA-resistant target to its wild-type counterpart, functionally significant miRNAmediated regulation may be elucidated. Here we have used this miRNA-resistant transgene approach to understand the functional role of miR159 in Arabidopsis. MiR159 is bioinformatically predicted to target approximately 20 genes, 10 of which are validated targets (Allen et al., 2010). However, only the regulation of two redundant GAMYB-like genes, MYB33 and MYB65, appears to be functionally relevant, as the severe pleiotropic rosette defects displayed by a strong loss-of-function mir159a/mir159b double mutant (mir159ab) are fully suppressed in a mir159ab.myb33.myb65 quadruple mutant (Allen et al., 2007). This raises the question of what is the functional significance of miR159 regulation for these other target genes. One such gene is MYB120, another member of the GAMYBlike family which has a highly conserved miR159 binding site. It is cleaved by miR159 in vivo and overexpression of miR159 results in down-regulation of MYB120 transcript levels (Schwab et al., 2005; Allen et al., 2010). As determined by online microarray data, MYB120 is predominantly transcribed in anthers and pollen, but negligibly in rosettes (Winter et al., 2007). To investigate what functional impact miR159 regulation has on MYB120, Arabidopsis plants expressing either a MYB120 transgene or its miR159-resistant version, mMYB120, were generated. Both transgenes were made from a large genomic clone of MYB120 containing extensive 5’ and 3’ flanking regions to maximize the chance of faithfully reproducing endogenous expression, with only the seven synonymous mutations introduced into the miR159 target site of mMYB120 being the distinguishing feature of the two transgenes (Figure 1B). The phenotype of all 25 MYB120 transformants generated was indistinguishable from wild-type (Figure 1C and D, and Supplemental Figure 1). Of the 34 mMYB120 lines obtained, all had flowers, anthers, and pollen that were indistinguishable from wild-type (Supplemental Figure 1), but, paradoxically, 21 mMYB120 lines had altered rosette development similar to that of mir159ab, including dwarfed rosettes with upwardly curled leaves (Figure 1E and F). This result suggests that miR159 regulation of MYB120 is important for rosette development, despite the mir159ab.myb33.myb65 mutant demonstrating otherwise (Allen et al., 2007). To investigate this paradox, we first examined MYB120 mRNA levels in rosettes of wild-type and mir159ab plants by qRT– PCR. Consistently with online microarray data, the MYB120 mRNA level is very low in wild-type rosettes; however, an approximately sevenfold increase was seen in the mir159ab mutant implying miR159 regulation (Figure 1A). Similar foldlevel changes of mMYB120 transcripts did not result in any obvious phenotypic impact, as the mMYB120 line m2 that had a sixfold increase in transcript levels was indistinguishable from wild-type (Figure 1G). Conversely, in mMYB120 plants displaying morphological abnormalities, the fold-level changes of mMYB120 mRNA levels were found to be several orders of magnitude higher (Figure 1G). This demonstrates