inserted between the 2X 35S promoter and the 35S terminator in a small-sized pOT2 vector (Figure S6). The amiR165 and amiR166 construction was done by two rounds of PCR cloning using a proofreading Taq polymerase and two pairs of long primers (,60–100 nt) engineered to contain a SwaI and a PmeI site, respectively. The two pairs of primers (M0SL-at-miR165- SwaI-PR and M0SL-at-miR165-SwaI-PF or M4SL-at-miR166- PmeI-PR and M4SL-at-miR166-PmeI-PF) covered the entire artificial stem-loop sequences of miR165 or miR166 to minimize errors in the miR165 or 166 duplex regions during the PCR reactions. First, the primers M0SL-at-miR165-SwaI-PR and M0SL-at-miR165-SwaI-PF were used to amplify the pOT2 vector that contains the miR168 backbone. The PCR products were cleaved by SwaI and self-ligated to generate a plasmid that contains pre-miR165. Second, PCR was performed using this plasmid as the template with the primers M4SL-at-miR166-PmeI- PR and M4SL-at-miR166-PmeI-PF. The PCR products were cleaved by PmeI and self-ligated to generate a plasmid containing the tandem pre-miR165 and pre-miR166 sequences. Finally, this plasmid in pOT2 was amplified by a pair of primers (Origin-del- PacI-PF and Origin-del-PacI-PR) that contained PacI sites to delete the plasmid replication origin. The PCR products that contained the amiR165/166 and a chloramphenicol selection marker were introduced into a modified pFGC5941 binary vector through the unique PacI site. Recombinant binary plasmids were verified by DNA sequencing before being used for plant transformation. RNA extraction and realtime RT-PCR Total RNAs were extracted from inflorescences or seedlings using TRI Reagent (Molecular Research Center, Cat# TR118) as per manufacturer’s instructions. Contaminating DNA was re- moved by DNase I (Promega, Cat# M610A; Roche, Cat# 04716728001) digestion. cDNA was synthesized from 2 mg total RNAs using reverse transcriptase (Fermentas, Cat# EP0441) and an oligo-dT primer. Quantitative PCR was performed in triplicates on a Bio-Rad IQ cycler apparatus with the iQ SYBR green supermix (Bio-Rad, Cat# 170-8882). The primers used are listed in Table S2. Small RNA northern blotting Northern blotting to detect small RNAs was performed as described [40,51]. 59-end-labeled (32P) antisense DNA oligonucle- otides were used to detect miRNAs from total RNAs. 59-end- labeled (32P) antisense LNA oligonucleotides were used to detect miRNAs from AGO1 or AGO10 immunoprecipitation samples. The probe sequences are listed in Table S2. Immunoprecipitation FLAG-AGO1 immunoprecipitation was performed as de- scribed [20]. ag-1 pAG::AG-GFP and zll-1 pZLL::YFP-ZLL inflores- cences were ground in liquid nitrogen and homogenized in an equal volume of extraction buffer (20 mM Tris HCl pH 7.5, 300 mM NaCl, 5 mM MgCl2, 5 mM DTT, 1% (v/v) protease inhibitor cocktail (Roche, Cat# 11244800; 1 tablet was dissolved in 1 ml nuclease-free water)). The extracts were centrifuged for 10 min at 16,000 g three times to pellet any debris. The lysates were then precleared with protein-A agarose (Thermo Scientific, Cat# 20333) for 1 hr. Anti-GFP antibodies (Clontech, Cat# 632460) were added to the samples at a 1:200 dilution and the samples were incubated on a rotating shaker for 2 hrs. Protein-A agarose was added at a 1:50 ratio and incubation was continued for another 2 hrs. The immune complexes were washed four times with 1.5 ml of extraction buffer supplied with 0.5% NP-40 and AGO10 and Floral Stem Cell Termination two times with RISC (RNA-induced silencing complex) buffer (40 mM Hepes pH 7.4, 100 mM KOAc, 5 mM MgOAc, 4 mM DTT). Finally, the immune complexes were resuspended into 100 ml RISC buffer. In vitro transcription and RNA cleavage assay The PHB and PHBm cDNAs were amplified by PCR with primers PHBprobeF and PHBprobeR (see Table S2 for sequence information) from the wild type PHB and the miR165/166- resistant PHB G202G [30] templates, respectively. The PCR products were cloned into pGEM-T Easy (Promega, Cat#A1360) and selected clones were sequenced to ensure the absence of unwanted mutations. The clones were linearized with SpeI (NEB, Cat# R0133L) and the linearized DNA was gel purified. In vitro transcription and RNA cleavage assays were carried out as described [20]. In vitro transcription was performed by incubating 800 ng DNA in a 25 ml reaction with T7 RNA polymerase (Promega, Cat# P2077) and a-P32-UTP at 37uC for 1.5 hrs. Labeled PHB and PHBm probes were gel purified and dissolved in 50 ml nuclease-free water. 3 ml probes were used in a 25 ml cleavage reaction mix containing 20 ml AGO1 or AGO10 immune complexes in RISC buffer, 1 ml 25 mM ATP and 1 ml RNase inhibitor (Fermentas, Cat# EO0381). The reaction mix was incubated at 37uC for 1.5 hrs. The RNAs were resolved in an 8 M Urea/5% polyacrylamide gel and detected with a Typhoon Phosphorimager. Protein extraction and western blotting To extract proteins from plants, 25 mg inflorescence material was ground in liquid nitrogen and homogenized with 150 ml 1XSDS loading buffer supplied with 0.5% (v/v) 2-mercaptoeth- anol and 1% (v/v) protease inhibitor cocktail mix. The extracts were boiled for 5 min, followed by centrifugation at 16,000 g at 4uC for 5 min. 15 ml supernatant was resolved in a 10% SDS- PAGE gel. To assay the AGO1 or AGO10 immunoprecipitation samples, 8 ml immune complexes were boiled with an equal volume of 2XSDS loading buffer with 1% (v/v) 2-mercaptoeth- anol and 2% protease inhibitor cocktail mix. The proteins were resolved in a 7.5% SDS-PAGE gel and a monoclonal anti-GFP antibody (Abcam, Cat# ab3277) was used for immunodetection. Supporting Information Figure S1 Diagrams of AG, AGO10, and PHV genes and multiple sequence alignments of Arabidopsis AGO proteins. (A) Diagrams of AG, AGO10, and PHV. In ag-10, the replacement of the guanine at nucleotide 3618 (position 1 being the ‘‘A’’ in the ACG start codon) by adenine causes an E-to-K substitution. ago10-12 has a C-to-T transition at nucleotide 2991 (position 1 being the ‘‘A’’ in the ATG start codon), which results in an L-to-F substitution at amino acid 674 in the protein. ago10-13 has a G-to-A transition at nucleotide 833, which introduces a premature stop codon in the second exon. ago10-14 is a G-to-A mutation at nucleotide 3323 causing a D-to- N substitution. phv-5d contains a G-to-A transition at nucleotide 1410 (position 1 being the ‘‘A’’ in the ATG start codon), causing an G-to-D substitution. (B) Multiple sequence alignment of all Arabidopsis AGO proteins indicates that 674 L is conserved. (C) A diagram showing that the phv-5d mutation disrupts the binding site for miR165/166. Found at: doi:10.1371/journal.pgen.1001358.s001 (12.36 MB TIF) Figure S2 The accumulation of miRNAs, ta-siRNAs and their target mRNAs in ago10 mutants. (A) Northern blotting to detect six miRNAs and three ta-siRNAs. The ago10-13 mutation resulted in PLoS Genetics | www.plosgenetics.org 12 March 2011 | Volume 7 | Issue 3 | e1001358