AGO1 recruitment of small RNAs. (A) Immunoprecipitation of FLAG-AGO1. FLAG-AGO1 was immunoprecipitated from crude inflorescence extract as described in Materials and Methods. Input, crude extract before immunoprecipitation; unbound, supernatant after immunoprecipitation; IP, immunoprecipitate. AGO1(+), FLAG-AGO1 transgenic extracts; AGO(-), WT extracts. (B) miRNAs are recruited by AGO1. sRNA blots were hybridized with DNA oligonucleotide probes complementary to the indicated miRNAs. RNAs were either extracted directly from plant material (RNAtot) or from α-FLAG immunoprecipitate (αFLAG IP). (C) AGO1-recruited miRNAs are methylated at the 3′ terminus. A synthetic unmethylated GFP 21-nt RNA oligomer mixed with the immunoprecipitated RNAs (Upper) displays an increased electrophoretic mobility after a β-elimination reaction (β-El +) whereas miR167 (Lower) does not, indicating that its 3′ end is protected by methylation. Both GFP and miR167 hybridization were done on the same blot after stripping. (D) ta-siRNAs are recruited by AGO1. (E) Endogenous chromatin siRNAs are not recruited by AGO1. In B, D, and E, the IP samples were derived from 7-fold more tissue than directly extracted RNA. (F) Transgene-derived siRNAs are associated with AGO1. FLAG-AGO1 plants were crossed with plants coexpressing the GFP together with a GF inverted repeat silencer construct (GF-IR line) or with plants coexpressing the GFP together with a Potato Virus X-GFP silencer construct (GFP-Amp). Blots show GFP-derived siRNAs (Top) or miR167 (Middle) from parent and F₁ plants. The IP samples were derived from 20-fold more tissue than directly extracted RNA. (Bottom) A Western blot of FLAG-AGO1. (G) Viral siRNAs are not associated with AGO1. FLAG-AGO1 or WT plants were infected with Cucumber Mosaic Virus strain I17F (CMV), Turnip Crinkle Virus (TCV), and Cruciferae Tobacco Mosaic Virus (CrTMV). siRNAs (Upper) were detected by hybridization with an in vitro transcribed sense probe corresponding to the coat protein sequence of the virus (CMV and TCV) or the full-length cDNA (TMV). miR167 (Lower) was detected with a complementary DNA oligonucleotide probe on the same blot after stripping. The IP samples were derived from 10-fold more tissue than directly extracted RNA.

Mutations of conserved residues of the AGO1 PIWI domain affect Slicer activity. (A) Alignment of the catalytic center of the Arabidopsis and human ARGONAUTE PIWI domains. The positions of the Mg²⁺ coordinating residues in the DDH catalytic triad are indicated above the alignment (arrows), as well as the mutations introduced in FLAG-AGO1 (*). (B and C) Slicer activity of mutant AGO1 proteins. FLAG-AGO1, FLAG-AGO1_G758S, and FLAG-AGO1_H798P were expressed transiently in N. benthamiana by Agrobacterium infiltration, and Slicer activity of the immunoprecipitated proteins was assayed in vitro (B top). FLAG-AGO1_D760A could not be assayed because it was unstable in the transient assay, but it did accumulate and its PHV Slicer activity could be assayed in extracts of transgenic Arabidopsis (C Top). Recruitment of miR165 was verified by Northern blotting of sRNAs extracted from immunoprecipitates (Bottom). The level of immunopurified FLAG-AGO1 proteins was tested by Western blotting (Middle).

Generation of epitope-tagged AGO1 transgenic Arabidopsis. (A) Diagram of the FLAG-AGO1 construct. Positions of the restriction sites used for the cloning are given relative to the start codon. The sequence and position of the FLAG epitope is indicated. Thick lines, regions encoding the PAZ and PIWI domains; thin broken arrow, translation start; black dot, translation stop. (B) Diagram of the AGO1 genomic locus. Gray boxes, exons; triangle, T-DNA insertion in ago1 mutant Salk_087076 line (ago1-36) with left border (Lb) and right border (Rb) orientation. Other symbols as in A.(C) FLAG-AGO1 complements the ago1-36 phenotype. Photographs are taken 2 weeks postgermination. (D) PCR genotyping of the FLAG-AGO1 line. The ago1-36 (Upper) and not the WT allele (Lower) is amplified from the selected FLAG-AGO1 transgenic line. (E) Expression of FLAG AGO1 transcripts. ago1-36 mutants produce a truncated transcript comprising the sequence 5′ (Middle) but not 3′ (Bottom) of the T-DNA insertion. Expression of a full-length AGO1 transcript is restored in the selected FLAG-AGO1 line. Actin primers (Top) were used to confirm equal loading, and reactions without reverse transcriptase were performed to exclude DNA contamination. DNA, control PCR with genomic DNA.

AGO1 copurifies with Slicer activity. In vitro-labeled WT PHV or mutant G → A phv target RNAs were incubated with immunoprecipitates from FLAG-AGO1 and WT plants or with wheat germ extracts (Wg) as positive control. The sizes of the predicted 5′ and 3′ PHV RNA cleavage products are indicated.

FLAG-AGO1 Slicer is present in low molecular weight complexes. FLAG-AGO1 was immunopurified and eluted from α-FLAG M2 agarose beads by competition with 3XFLAG peptides. The concentrated eluate was fractionated on a Superpose 6 column, and the fractions were tested for cleavage of PHV target RNA (Upper) and for presence of AGO1 by Western blotting (Lower). The elution profile of the molecular weight markers is indicated. V_o, void volume; In, input; Wg, wheat germ.