ago10-13 vs. wild type by western blotting (data not shown). This is likely attributable to insufficient sensitivity of the assay in the detection of small differences. Since the levels of miR172 were not affected by ago10-13 (Figure S2A), it is likely that miR172- mediated repression of AP2 requires AGO10. AGO10 is associated with miRNAs in vivo Although our genetic evidence indicates that AGO10 mediates the activities of miR172 and previous genetic studies implicate AGO10 in mediating the translational repression of target mRNAs by multiple miRNAs [26], molecular evidence of AGO10 acting directly with miRNAs has been lacking. We sought to determine whether AGO10 was associated with miRNAs in vivo. We immunoprecipitated (IP) YFP-ZLL from inflorescences of zll-1 pZLL::YFP-ZLL plants [36] with anti-GFP antibodies. pAG::AG- GFP and Ler (wild type) were used as a positive and a negative control, respectively, for the IP. Proteins of the expected sizes from the pZLL::YFP-ZLL and pAG::AG-GFP IP samples were detected by western blotting (Figure 5A). We also included pAGO1::FLAG- AGO1 [20] and Col (a control for pAGO1::FLAG-AGO1) for comparison. We extracted RNAs from the IP samples and performed small RNA northern blotting to detect selected miRNAs and siRNAs. AGO10 bound miRNAs such as miR165/166, miR172, miR173, miR319 and miR168 (Figure 5B), but not miR390 (Figure S4), which specifically associates with AGO7 [18]. Northern blotting failed to detect signals for 24 nt endogenous siRNAs from several loci from the IP samples (data not shown). The AGO10-associated miRNAs are also associated with AGO1 in vivo (Figure 5B; [17,20]). AGO10 has ‘‘slicer’’ activity Because AGO10 contains the DDH catalytic residues that are critical for the ‘‘slicer’’ activity of AGO proteins, we decided to determine whether AGO10 is catalytically active in vitro. We performed a ‘‘slicer’’ activity assay on the miR165/166 target PHABULOSA (PHB). A portion of the PHB transcript containing the miR165/166 binding site was generated through in vitro transcription and incubated with IP samples from pAGO1::FLAG- AGO1 and pZLL::YFP-ZLL plants as well as the corresponding negative controls Col and pAG::AG-GFP, respectively. A miR165/ 166-resistant PHB transcript, PHBm, was also included as a negative control. The PHB transcript was cleaved into two fragments of expected sizes by both FLAG-AGO1 and YFP-ZLL but not by the corresponding control IPs (Figure 5C). As expected, the PHBm transcript failed to be cleaved by either FLAG-AGO1 or YFP-ZLL (Figure 5C). This shows that AGO10, like AGO1, can cause the cleavage of miRNA-targeted mRNAs. Given that AGO10 has ‘‘slicer’’ activity, we next examined the levels of small RNA-targeted mRNAs in ago10 mutants. The levels of most examined mRNAs were not obviously different between ago10-13 and wild type (Figure S2B), consistent with findings from a previous study [26]. A small elevation in the levels of CUC1 mRNA, which is targeted by miR164, was consistently detected in ago10-13 (Figure S2B). Interestingly, a small reduction in the levels of miR164 in ago10 mutants was consistently observed (Figure S2A). The miR164-resistant CUC1 transgene (pCUC1::CUC1m- GFP; [41]) did not enhance the determinacy defects of ag-10 (data not shown), suggesting the ag-10 ago10-13 floral determinacy defects were unlikely attributable to de-repressed CUC1 expres- sion. There are several explanations for the lack of differences in miRNA target mRNA levels between ago10 and wild type. First, changes are masked by the target mRNAs outside of the highly restricted expression domains of AGO10. Second, AGO1 is sufficient to regulate most miRNA-targeted mRNAs at the AGO10 and Floral Stem Cell Termination transcript level, and AGO10 acts primarily through translational inhibition in vivo. miR165/166 and its targets, the HD-Zip genes, are crucial players in floral determinacy The fact that the ap2-2 mutation failed to completely suppress hua1 hua2 ago10-12 or ag-10 ago10-13 suggests that AGO10 regulates floral stem cell termination also through other miRNAs. Because AGO10 associates with miR165/166 and the HD-Zip genes PHB, PHAVOLUTA (PHV), and REVOLUTA (REV) targeted by this miRNA strongly influence the formation of the SAM [29], we tested whether proper regulation of PHB by miR165/166 was crucial for floral stem cell termination. We crossed the miR165/ 166-resistant form of PHB, phb-1d [42], with ag-10 ago10-13. Both ag-10 phb-1d/+ and ag-10 ago10-13/+ phb-1d/+, but not phb-1d/+ flowers had bulged gynoecia with ectopic floral organs inside (Figure 4F; Figure S3Q,S3R). Sections of ag-10 phb-1d/+ flowers revealed indeterminate floral meristems inside the primary carpels (Figure 4H). In addition, in the genetic screen in the ag-10 background, we isolated a semi-dominant phv allele, phv-5d, as an ag-10 enhancer. The ag-10 phv-5d double mutant exhibited bulged siliques throughout the plant (Figure 4G) whereas most but not all siliques in ag-10 phv-5d/+ plants were bulged (Figure S3S). phv-5d contained a G-to-A mutation in the miR165/166 binding site (Figure S1A,S1C) and this lesion was identical to those of the previously characterized phv-1d, -2d, -3d, and -4d alleles [29]. Although the mutation resulted in a glycine-to-aspartic acid substitution, extensive studies of an identical mutation in PHB (phb- 3d) showed that it was the disruption of miR165/166 targeting rather than the amino acid change that caused the developmental defects in phb-3d [30]. Collectively, these results demonstrate that miR165/166-mediated regulation of HD-Zip genes is necessary for floral stem cell termination. Having shown that the repression of PHB and PHV by miR165/ 166 is important for floral stem cell termination, we sought to determine whether ago1 or ago10 mutations resulted in defects in miR165/166-mediated repression of HD-Zip genes. We first examined the accumulation of miR165/166 in ago1-11, ago1-11 ago10-13/+, ago10-13, and ago1-11/+ ago10-13 inflorescences. Intriguingly, miR165/166 levels were reduced in ago1-11 but increased in ago10-13 (Figure 5D). The increase in miR165/166 levels was consistently observed in all ago10 alleles tested (ago10-12, ago10-13, and pnh-1; Figure S2A and data not shown), and was also previously observed in ago10 seedlings [43]. Next, we examined the levels of HD-Zip mRNAs in Ler, ago10- 13, ago1-11, ag-10, and ag-10 ago10-13 flowers by realtime RT- PCR. The levels of PHB, PHV and CORONA (CNA) mRNAs were reduced in ago10 genotypes as compared to controls (Figure 5E,5F), which correlated with the increased abundance of miR165/166. This indicated that AGO1 was sufficient to control the mRNA levels of the HD-Zip genes in the absence of AGO10. In ago1-11, in which miR165/166 levels were low, PHB and PHV mRNA levels were slightly higher than wild type (Figure 5E), consistent with AGO1 being the major slicer of PHB and PHV in vivo. Therefore, ago1 and ago10 mutations have opposite effects on miR165/166 accumulation and expression of the HD-Zip genes. ago10 mutants were previously found to have increased levels of miR165/166 in seedlings and the increase in miR165/166 and the resulting decrease in HD-Zip expression were responsible for the SAM and leaf polarity defects of ago10 mutants [43]. Therefore, it is possible that reduced HD-Zip expression caused by elevated levels of miR165/166 also underlies the floral determinacy defects of ago10 mutants. However, it is also possible that AGO10 regulates the HD-Zip genes through translational repression since it is PLoS Genetics | www.plosgenetics.org 8 March 2011 | Volume 7 | Issue 3 | e1001358