Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Design of, and additional phenotypes induced by miR165bm6. A) Alignment of miR165 and miR165bm6 with the target region of the PHB-like genes. The red marked nucleotides in the gene sequences represent mismatches and cyan, G-U wobbles, with the miR165 sequence. The cyan nucleotide in the miR165m6 sequence signifies the position of the G-U wobble of the miR165bm6 with the target genes. B) The pre miR165b structure showing the miR165 sequence in red. C) The pre miR165bm6 with the miR165bm6 sequence in the red, and the substituted nucleotides underlined. The substituted nucleotide in the complementary sequence is blue. D) Rescue of phv1-d/+ phenotype by ANT>> miR165bm6 (heterozygous for driver and operator construct). Note the radialized leaf in the phv1-d/+ plant on the right (arrow), the lack of radialized leaves in the phv1-d/+ ANT>> miR165bm6 plant on the left and the more wild-type inflorescence and flower morphology (inset) of the phv1-d/+ ANT>> miR165bm6 plants compared with that of the phv1-d/+ plant. E) AP1>> miR165bm6 inflorescence (upper) and flower (lower: a single proximal sepal has been dissected away). The petals can be variably radialized and may be in extra numbers, the anthers of the stamens are frequently reversed and the gynoecium additional carpels. Note the similarity between this phenotype and that of AP1>> miR165b weak lines (Figure 1N), and the contrast with AP1>> miR165b strong lines (Figure 1M).
• Supplemental Figure 2 - Analysis of cleavage products of Arabidopsis ARF genes. RLM-RACE based mapping of cleavage sites in the mRNAs of ARF2 ARF3 and ARF4 of 35S:miR-ARF and wild-type plants. A) Gel image of PCR products of cleaved ETT/ARF3 and ARF2 from wild type and 35S:miR-ARF with amplification of cleavage products at site A (ARF2) and site B (ETT/ARF3) more prevalent in 35S:miR-ARF (Scheme for a and b primers and gel for PCR products of ARF4 cleavage analysis are shown in Figure 2K). B) Chromatograms showing the results of direct sequencing of the PCR product obtained for cleavage analysis in ARF2 (upper), ARF3/ETT (middle) and ARF4 (lower) of 35S:miR-ARF plants. The miR-ARF target region is red, the RNA adapter sequence is in blue. C) Summary of cleavage analysis by sequencing of cloned RLM-RACE products from wild type. The scheme is showing cleavage mapping in ARF2 ARF3 and ARF4 in wild-type plants presumably due to ta-siRNA guided cleavage (the summary of cleavage analysis for 35S:miR-ARF plants is shown in Figure 2L).
• Supplemental Figure 3 - NGA-like gene alignment and cleavage analysis in tomato. A) A phylogenetic tree of the NGA-like proteins, including the two tomato ESTs and their closest Arabidopsis homologs, illustrating that both tomato transcripts fall within the NGAclade. Tree was based on ClustalX alignment shown in B, from the approx 120 AA that constitute the B3 domain. The numbers represent bootstrap percentage from 1000 trials. C) Alignment of cloned, Tomato NGA-like genes and the synthetic miR-NGA. Cyan nucleotides are predicted G-U wobbles. D) Small, redder fruit observed in 35S:miR-NGAa lines relative to wild type. No additional phenotypic alterations were detected. These phenotypes may result from down-regulation of NGA-like/BI934637 and NGA-like/BI93404, although it is possible that miR-NGA targets other gene(s) with greater specificity of which we are not aware E) RLM-RACE detection of tomato NGA-like cleavage products (arrow-heads). The low abundance suggests inefficient cleavage. F) Cleavage point mapping of tomato NGA-like transcripts. The predominant cleavage position in the NGA-like/BI934637 transcript, which has the most mismatches with miR-NGA, apparently shifts from between positions 10 and 11 to positions 9 and 10 relative to that of NGA-like/BI93404 and Arabidopsis NGA1-4 (Figure 3B).
• Supplemental Table 1 - Sequence parameters of pre miRNA.
• Supplemental Table 2 - Primers for PCR-mediated cloning.
• Supplemental Table 3 - Primers for PCR-mediated genotyping of NGATHA2-4 insertion alleles.
• Supplemental Table 4 - Oligonucleotide for miRNA detection by Northern, and primers for RLM-RACE.
• Supplemental Table 5 - Gene-specific primers for RLM-RACE.