Highly specific gene silencing by artificial microRNAs in Arabidopsis

Abstract

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

Figure 1.

Engineering of amiRNAs. Site-directed mutagenesis on precursors of endogenous miRNAs was performed using overlapping PCR. Oligonucleotide primers I to IV were used to replace miRNA and miRNA* regions (blue) with artificial sequences (red). Primers A and B were based on template plasmid sequence. Regeneration of functional miRNA precursors was achieved by combining PCR products A-IV, II-III, and I-B in a single reaction with primers A and B.

Figure 2.

Expression of amiRNAs and Cleavage of Predicted Targets. (A) RNA gel blot analysis of amiRNA overexpressers using a mixture of probes for all amiRNAs. The outermost lanes contain two standards. miR156a and miR172a overexpressers were included as controls. Lane 1, amiR-mads-2 (MIR319a backbone); lane 2, amiR-mads-1 (MIR172a backbone); lane 3, amiR-trichome (MIR319a backbone); lane 4, amiR-lfy-2 (MIR319a backbone); lane 5, amiR-lfy-2 (MIR172a backbone); lane 6, amiR-lfy-1 (MIR172a backbone); lane 7, amiR-yabby-2 (MIR319a backbone). M, size marker; nt, nucleotides. (B) Mapping of target cleavage products by rapid amplification of cDNA ends using PCR. Fraction of sequenced clones with particular 5′ end indicated on top. In the case of LEAFY (LFY), only one clone had a 5′ end at the expected position, opposite nucleotides 10 to 11 of the intended amiRNA. The 5′ end of most clones was offset by two nucleotides, suggesting that most of the amiRNAs were offset as well. The sequence predicted from the aberrant processing is indicated in gray.

Figure 3.

Phenotypes of amiRNA Overexpressers. (A) Inflorescences. From left to right: the wild type, lfy-12, and amiR-lfy-1 (MIR172a backbone) overexpresser. (B) Seedlings. From left to right: the wild type, gun4-1, and amiR-white-1 (MIR172a backbone) overexpresser. Bleaching of cotyledons is more pronounced in the amiR-white plants than in gun4-1, consistent with the more severe molecular profile of the amiR-white overexpressers. (C) Adult plants sown on the same day. From left to right: the wild type, ft-10, and amiR-ft-2 (MIR172a backbone). (D) Leaf rosettes. From left to right: the wild type, try cpc double mutants, and amiR-trichome (MIR319a backbone) overexpresser. Clustered trichomes are evident even at low magnification. (E) Flowers. From left to right: the wild type, weak amiR-mads-2 (MIR319a backbone) overexpresser, and strong amiR-mads-2 (MIR319a backbone) overexpresser. In both amiR-mads overexpressers, outer whorls are transformed into leaf-like structures. In the strong line, secondary inflorescences replace the central gynoecium. (F) Flowering plants. Left, the wild type; right, amiR-mads-1 (MIR172a backbone) overexpresser with increased number of cauline leaves (arrowheads). (G) Rosette leaves of the wild type (left) and amiR-yabby-1 (MIR172a backbone) overexpressers. Abaxial side is at the left. (H) Cauline leaves of the wild type (left) and amiR-yabby-2 (MIR319a backbone) overexpressers (right) with polarity defects.

Figure 4.

Expression Analyses of amiRNA Overexpressers. (A) Microarray profiles of LFY and some of its direct downstream targets in inflorescences of the wild type (Columbia [Col-0]), lfy-12 mutants, and amiR-lfy-1 (MIR172a backbone) overexpressers. (B) Microarray profiles of predicted amiR-mads-2 targets in inflorescences of the wild type (Col-0) and weak and strong amiR-mads-2 (MIR319a backbone) overexpressers. (C) Microarray profiles of predicted amiR-mads-1 targets in the wild type (Col-0) and amiR-mads-1–overexpressing (MIR172a backbone) inflorescences. (D) RT-PCR analysis of amiR-yabby overexpressers (inflorescence tissue). Reactions were stopped in the linear phase of amplification. Lane 1, amiR-yabby-1 (MIR172a backbone); lane 2, amiR-yabby-2 (MIR319a backbone). (E) Overlap of significantly downregulated genes using the Linear Model for Microarray Data (LIMMA) package (Smyth et al., 2005) or logit-T (in parentheses; Lemon et al., 2003) in lfy-12 and amiR-lfy-1 plants indicates a very similar molecular phenotype, with amiR-lfy-1 plants being on average weaker than lfy-12 plants. Only genes called as present by Affymetrix algorithms in wild-type controls were considered. (F) Distributions of Smith-Waterman scores (Smith and Waterman, 1981) are similar between genes that are significantly downregulated in response to amiRNA overexpression (light gray bars) and all genes present in the control (dark gray bars). Predicted targets have been removed.

Figure 5.

Inducible and Tissue-Specific Expression of amiRNAs. Uninduced or wild-type controls are shown at the left. (A) Ethanol-induced ubiquitous expression of amiR-white-1 3 and 5 d after induction. After 3 d, young leaves are all yellow; after 5 d, the youngest leaves are green again. (B) Ethanol-induced ubiquitous expression of amiR-trichome (right) 3 d after induction. Clustered trichomes appear as white covering of youngest leaves (arrowhead). (C) Inflorescences of plants expressing amiR-white-1 from the AP1 promoter (middle) are pale yellow. Strong lines expressing amiR-mads-2 from the AP1 promoter (right) resemble ap1 cal double mutants. (D) Expression of amiR-lfy-1 from the LFY promoter (1) results in flowers resembling lfy mutants. amiR-mads-2 expressed from AG regulatory elements in the center of the flower (2) produces organ transformations in the central two whorls. Outer whorls remained unaffected. An opposite phenotype was seen after expression of amiR-mads-2 from the AP1 promoter (3; weaker line), which didn't affect inner whorls but resulted in secondary flowers, resembling ap1 mutants. (E) Epidermal expression of amiR-white-1 from the MERISTEM LAYER1 (ML1) promoter resulted in pale plants.