Distribution and Abundance of 21-Nucleotide Small RNAs in TAS1a, TAS1b, TAS1c, and TAS2.
(A) Abundance of Col-0 and rdr2-1 small RNA reads in TAS1a, TAS1b, TAS1c, and TAS2 loci. The top graphs represent the total number of small RNA reads, whereas the bottom graphs shows phase signals from Col-0 and rdr2-1 small RNA reads. Gridlines correspond to 21-nucleotide cycles of small RNAs from the miR173-guided cleavage site. nt, nucleotides.
(B) Comparison of Col-0 and dcl4-2 inflorescence small RNAs. The top graphs represent 20- to 25-nucleotide small RNAs in Col-0 inflorescence versus 20- to 25-nucleotide small RNAs in dcl4-2 inflorescence for TAS1a, TAS1b, TAS1c, and TAS2 loci. The bottom graphs show phase signals from Col-0 inflorescence small RNA reads versus dcl4-2 inflorescence small RNA reads. Small RNA counts were normalized to the size of the dcl4-2 library and also repeat normalized to account for redundant small RNAs in each locus. The schematics below the phase plots represent miR173-guided cleavage and 21-nucleotide small RNA positions in TAS1a, TAS1b, TAS1c, and TAS2 transcripts. The position of the miR173 target site is shown as a thin gray line. Thick black arrows represent 21-nucleotide phase positions from the miR173-guided cleavage site. Colored arrows indicate small RNAs previously validated to target various transcripts. White arrows with colored outlines indicate small RNAs with a predicted target. Arrows with identical colors (solid and outlined) indicate small RNAs within the same family. Red arrows indicate small RNA positions for the ASRP255-like family.

Identification of New Loci and miR390 Target Sites in the TAS3 Family.
(A) The total number of small RNA reads in Col-0 and rdr2-1 libraries for TAS3a and TAS3b and all libraries for TAS3c are shown. The 21-nucleotide cycle positions from the 3′ miR390-guided cleavage site for each locus are indicated below each graph. Predicted and validated miR390 target sequences are indicated by gray boxes, and ASRP2141-like sequences are indicated by red arrows. Asterisks indicate ASRP2141-like sequences.
(B) Phase plots for TAS3a tasiRNAs from Col-0 and rdr2-1 (top) and from normalized reads from Col-0 (bottom, above the x axis) or dcl4-2 (bottom, below the x axis). Vertical gridlines indicate 21-nucleotide cycles.
(C) T-Coffee alignment of ASRP2141-like sequences from TAS3a, TAS3b, and TAS3c. Identical positions are indicated by asterisks in the consensus plot. The numbers of ASRP2141-like tasiRNA reads in Col-0 and rdr2-1 libraries are listed.
(D) Diagrammatic representation of primary TAS3 transcripts and validation of 3′ miR390 cleavage sites by 5′ RACE. ASRP2141-like regions are highlighted in red. The miR390 and target mRNA duplexes are shown in the expanded regions. Bases in bold indicate predicted cleavage sites. Arrowheads indicate positions corresponding to the 5′ end of at least one cloned 5′ RACE PCR product. The 5′ RACE products sequenced for each miR390 target site are indicated in gray and black, respectively. The number of specific 5′ RACE products/total products sequenced at each miR390 target site is indicated. The 3′ miR390 target site for TAS3a was reported by Allen et al. (2005). T-Coffee alignment of 5′ and 3′ miR390 target sites in TAS3a (a), TAS3b (b), and TAS3c (c). Identical positions are indicated by asterisks in the consensus plot.

Flowchart for Identification of RDR6-Dependent, Phased, Small RNA–Generating Loci.

Analysis of PPR-P Clade Genes and siRNAs.
(A) Abundance of small RNA size classes from clade PPR-P transcripts in various tissues and genotypes.
(B) Expression profile of 23 of the 28 PPR-P clade genes in Col-0, Ler, and 10 mutants with defects in silencing factors. Transcripts that yield at least one 21-nucleotide small RNA (red) or no small RNAs (teal) are color-coded. The list of PPR genes in the expression profile is as follows: At4g26800, At1g63070, At1g62670, At1g63130, At1g62930, At1g63080, At1g63320, At1g63150, At1g62590, At1g62910, At1g63400, At5g41170, At3g16710, At5g16640, At1g62720, At1g62680, At1g64580, At1g06580, At1g12700, At1g12620, At1g64100, At1g62860, and At1g63630.
(C) Comparison of small RNA reads and expression level of PPR-P genes that are targeted by miR161, miR400, and/or tasiRNAs from TAS1 or TAS2 loci.

Targeting of the PPR Gene Family by miRNAs and tasiRNAs.
miR161.1, miR161.2, miR400, and tasiRNAs from TAS1 and TAS2 are predicted to target several PPR-P clade transcripts from two gene clusters in chromosome 1.
(A) A schematic representation of PPR targeting by miR161.1, miR161.2, miR400, TAS1a 3′D9(−) (also named TAS1a siR9 by Yoshikawa et al. [2005]), TAS1b 3′D4(−) (siR4), TAS1c 3′D10(−) (siR10), and TAS2 3′D6(−), 3′D9(−), 3′D11(−), and 3′D12(−) (siR6, siR9, siR11, and siR12). Red and gray arrows indicate experimentally validated and predicted-only targets, respectively. Solid arrows indicate targeting events that direct phasing of small RNAs from the PPR-P transcript. The peak P score is listed under each gene with phased small RNAs. PPR genes are color-coded based on repeat normalized abundance of small RNAs from Col-0 and rdr2-1 libraries. The PPR gene clusters are shown relative to genomic position on chromosome 1. From left to right, the PPR-P genes are as follows: At1g12300, At1g12620, At1g12700, At1g12775, At1g62590, At1g62670, At1g62680, At1g62720, At1g62860, At1g62910, At1g62930, At1g63070, At1g63080, At1g63130, At1g63150, At1g63230, At1g63320, At1g63330, At1g63400, At1g63630, and At1g64100.
(B) Schematic representation of PPR motifs within four PPR-P genes and the location of small RNA target sites relative to the four genes illustrated that they collectively contain targets for all 11 PPR-targeting miRNAs and tasiRNAs. Numbers above the diagram indicate the PPR motif. Identical colors for miRNA or tasiRNA target sites represent members of the same family.
(C) PPR domain conservation within four Arabidopsis PPR genes and one Populus PPR gene. PPR domains listed are those that collectively contain target sites for all 11 miRNA or tasiRNA predicted targets from Arabidopsis and two known miRNA target sites from Populus. Each PPR domain consists of 105 nucleotides. Colored boxes indicate the position of the target sites within each PPR gene and correspond to colors in (B). A consensus PPR motif, with conservation highlighted by a heat map, is shown.

Phase Transform Plots for Nine Arabidopsis PPR Genes.
Phased cycles from miR161.1, miR161.2, and TAS2 3′D6(−) target sites are indicated by color coding. Arrows below each plot indicate the miRNA and tasiRNA target sites within each PPR transcript.

Primary and Secondary Targeting of PPR-P Transcripts.
(A) miRNA target prediction for randomly selected miRNA family members (one/family). Target predictions were done with 10 sets of either miRNAs or sequence-shuffled RNAs, and the mean (±sd) target score distribution was plotted. miRNA target hits for a validated gene family (blue) or a nonvalidated gene family (green) are grouped according to target score. miRNA families used for target prediction were miR156/157, miR158, miR159/319, miR160, miR161, miR162, miR163, miR164, miR166/165, miR167, miR168, miR169, miR171/170, miR172, miR173, miR390/391, miR393, miR394, miR395, miR396, miR397, miR398, miR399, miR400, miR402, miR403, miR408, miR447, miR773, miR775, miR824, miR827, miR842, miR844, miR856, miR857, and miR858.
(B) Secondary siRNA target prediction for all antisense 21-nucleotide small RNAs arising from all clade PPR-P transcripts. Clade-derived siRNAs predicted to target the clade PPR-Ps (blue), nonclade PPRs (red), or non-PPRs (green) were plotted by target score. The total number of genes in each group is listed in parenthesis.
(C) The percentage of clade PPR-P (blue), nonclade PPR (red), and non-PPR (green) genes that have transcripts targeted by clade-derived siRNAs. Target hits in (A) to (C) were grouped according to target score.
(D) Repeat normalized abundance of small RNAs with identity to each PPR-P transcript. Dots indicate PPR-P transcripts with predicted target sites for miR161, miR400, TAS2 3′D6(−), or other tasiRNAs. Transcripts that are singly (blue) or multiply (red) targeted by one or more clade-derived siRNAs are color-coded within the phylogenetic tree. PPR amino acid sequences were aligned using ClustalW with the BLOSUM matrix series. A tree that only differed in the placement of PPR proteins outside of the clade was obtained using the GONNET matrix series (data not shown).