Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided cleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5' or 3' terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.

Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking. In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter. In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity. TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms.Although much is known about how organs acquire their particular fate, we are only starting to learn how organs are sculpted, even if they are just flat sheets such as wings or leaves. An elegant study recently demonstrated that making a flat organ is not a trivial problem: snapdragon leaves are normally flat, but they become crinkly in plants lacking the CINCINNATA (CIN) gene 1 . In cin mutants, differential regulation of cell division across the leaf is disturbed, causing negative leaf curvature. CIN RNA itself is expressed in a dynamic pattern, in front of and perhaps overlapping the mitotic arrest zone, suggesting a direct role of CIN in regulating leaf morphogenesis.Although it is unknown how expression of CIN, which encodes a TCP transcription factor 2 , is regulated, a specific RNA pattern can result from differential transcription or changes in transcript stability. A post-transcriptional mechanism that has only recently been recognized is that of plant mRNA cleavage initiated by partially or fully complementary microRNAs (miRNAs) 3,4 . The mechanism of cleavage is similar, or identical, to cleavage guided by short interfering RNAs (siRNAs) 5 .The double-stranded ribonucleases Dicer in animals and DICER-LIKE1 (DCL1) in plants process miRNAs-which are usually 21-22 nucleotides long-from longer precursor RNAs with fold-back structure 4,6,7 . Additional factors required for accumulation of miRNAs include members of the Argonaute family and HEN1 protein 8,9 . The importance of miRNAs for plant development is supported by the abnormalities seen in several mutants or transgenic plants with general defects in miRNA accumulation or activity [9][10][11][12] . However, although biochemical studies have demon- Fig. 2b). b, Seedlings, individual leaves and leaf rosettes of Columbia wild-type and jaw-1D plants. Leaves were mounted between glass plates and illuminated from below. Dark green areas indicate overlapping leaf parts after flattening. c, Expression changes of TCP genes in jaw-1D estimated from Affymetrix arrays (grey bars) or from RT-qPCR (black bars). See Supplementary Information for absolute values. NP, termed 'not present' by MAS software. Note th...

Trans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5' adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5' nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps.

The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.