1. Volume 4, Issue 1, Pages 157-170 (January 2011)
Efficient Silencing of Endogenous MicroRNAs Using Artificial MicroRNAs in Arabidopsis thaliana 
Eamens Andrew L. , Agius Claire , Smith Neil A. , Waterhouse Peter M. , Wang Ming-Bo  
Molecular Plant 
Volume 4, Issue 1, Pages (January 2011)
DOI: /mp/ssq061
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Schematic Diagrams of pVec8-35Sp and pBlueGreen amiRNA Plant Expression Vectors.
(A) The vector pAth-miR172a contains the Arabidopsis pri-miRNA sequence of miR172a and is used as a template for PCR. Six primers (p1–p6) are required to (1) replace endogenous miR172a and miR172a* sequences with amiRNA and amiRNA* sequences, and (2) flank the full-length PCR product with XhoI 5' and BamHI 3' restriction sites for directional cloning into the similarly digested plant expression vector, pVec8-35Sp. 35Sp, cauliflower mosaic virus 35S promoter; OcsT, octopine synthase terminator; HphR, hygromycin B resistance gene.
(B) The pAth-miR159b vector contains the Arabidopsis pri-miRNA sequence of miR159b and is used as a template for PCR. Universal forward and reverse primers (pF and pR) are used in a PCR amplification step to (1) replace the endogenous miR159b and miR159b* sequences with amiRNA and amiRNA* sequences, and (2) flank the resulting PCR product with SapI restriction sites for cloning into the similarly digested plant expression vector, pBlueGreen. PptR, phosphinothricin resistance gene.
Molecular Plant 2011 4, DOI: ( /mp/ssq061)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Phenotypes and Small RNA Accumulation in Anti-159 Plants.
(A) Phenotypes expressed by 4-week-old wild-type Arabidopsis plants (ecotype Col-0) and amiRNA expression lines. Scale bars = 5 mm.
(B) Whole flowers of Col-0 and anti-159 plants. Scale bars = 500 μm.
(C) Dissected flowers (sepals and petals removed) of Col-0 and anti-159 plants. Scale bars = 500 μm.
(D) Maturing siliques from pollinated flowers of Col-0 and anti-159 plants. Scale bars = 5 mm.
(E) Endogenous and exogenous sRNA species accumulating in wild-type, drb1, and anti-159 plant lines. RNA-blotted filters were hybridized with radiolabeled oligonucleotides specific for endogenous miRNAs, miR159, miR319, and miR173 (internal control) as well as for anti-159 and U6 (loading control) sRNA sequences.
(F) Expression of miR159a and miR159b in anti-159a and drb1 plants assessed by qRT–PCR using miRNA-specific stem-loop RT primers for comparison to Col-0. Error bars represent the standard error of the mean (SEM) between three biological replicates.
Molecular Plant 2011 4, DOI: ( /mp/ssq061)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Artificial MiRNA-Directed RNA Silencing of the MiR159 and MiR319 Families and Expression Deregulation of their Target mRNAs in Anti-159 Plants.
(A) Anti-159-directed cleavage of miR159 primary transcripts.
(B) Anti-159-mediated cleavage of miR319 primary transcripts, PRI-MIR319A, PRI-MIR319B, and PRI-MIR319C.
(C) Relative expression of anti-159-targeted primary miRNA transcripts, PRI-MIR159A, PRI-MIR159B, PRI-MIR319A, and PRI-MIR319B in anti-159a plants and in the miRNA biogenesis mutant drb1 compared to wild-type.
(D) Target transcript expression in anti-159 plants. All transcripts analyzed by qRT–PCR house mRNA sequences complementary to endogenous miRNAs, miR159 and miR319. Error bars ((C) and (D)) represent the standard error of the mean (SEM) between three biological replicates.
Molecular Plant 2011 4, DOI: ( /mp/ssq061)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 Phenotypes and sRNA Accumulation in Anti-164 Plants.
(A) Wild-type (ecotype Col-0).
(B) Anti-164abc amiRNA plant expression line.
(C) Anti-164bus amiRNA plant expression line.
(D) Anti-164bsl amiRNA plant expression line.
(E) Anti-164bds amiRNA plant expression line.
(A–E) The upper row of panels show the aerial tissue phenotypes expressed by 3-week-old plants and the middle and lower rows of panels show the rosette leaf and root phenotypes expressed by 6-week-old plants, respectively. Scale bars (A–E): upper panels: 1 cm; middle panels: 2 cm; lower panels: 1 cm.
(F) The full-length PRI-MIR164B sequence used to design the anti-164bus, anti-164bsl, and anti-164bds amiRNAs targeting the 5' arm of PRI-MIR164B, the stem-loop sequence of PRE-MIR164B and the 3' arm of PRI-MIR164B, respectively. The miR164b and miR164b* sequences are given in red and blue.
(G) Endogenous and exogenous sRNA species accumulating in wild-type, drb1, and anti-164 plant lines. RNA-blotted filters were hybridized with radiolabeled oligonucleotides specific for endogenous miRNAs, miR164 and miR173 (internal control), as well as for anti-164 and U6 (loading control) sRNA sequences.
(H) Combined expression of miR164a and miR164b (miR164ab) in Col-0, drb1, and anti-164 plants assessed by qRT–PCR using miRNA-specific stem-loop RT primers. Error bars represent the standard error of the mean (SEM) between three biological replicates.
Molecular Plant 2011 4, DOI: ( /mp/ssq061)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Artificial MiRNA-Directed Silencing of the Arabidopsis MiR164 Family and Expression Deregulation of their Target mRNAs in Anti-164 Plants.
(A) Anti-164-directed cleavage of miR164 primary transcripts, PRI-MIR164A, PRI-MIR164B, and PRI-MIR164C in anti-164abc plants.
(B) Anti-164-mediated cleavage of the miR164b pri-miRNA transcript in amiRNA plant expression lines, anti-164bus, anti-164bsl, and anti-164bds.
(C) Relative expression of PRI-MIR164A and PRI-MIR164B transcripts in drb1 and anti-164 plant lines compared to wild-type.
(D) Target transcript expression in anti-164 plants. All transcripts analyzed in anti-164abc plants by qRT–PCR house mRNA sequences complementary to miR164. CUC1 and CUC2 expression was also assessed in anti-164bus, anti-164bsl and anti-164bds plants by qRT–PCR. Error bars ((C) and (D)) represent the standard error of the mean (SEM) between three biological replicates.
Molecular Plant 2011 4, DOI: ( /mp/ssq061)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions