Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species

Abstract

Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic transformations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for regulated expression of multiple related genes in diverse species.

Figure 1.

miRNAs Can Quantitatively Regulate Multiple Transcripts Simultaneously and Phenocopy Their Combined Loss of Function. (A) A scheme of an endogenous pre-miRNA. The red and blue fragments will be cleaved by DICER-LIKE1 (DCL1) to generate the miRNA and miRNA*, respectively. (B) A 10-d-old wild-type seedling. (C) The promoter of PHB drives GFP expression throughout the shoot apex in wild-type heart-stage embryos. (D) Arabidopsis cuc1 cuc2 double mutant seedlings. (E) F1 seedlings of OP:miR164b transactivated by PHB:LhG4. (F) A monocot-like phv phb rev triple mutant seedling. (G) A monocot-like PHB≫miR165b seedling of comparable age. (H) Whole shoot and flower (inset) of 35S:miR167a plant next to same age wild type display identical alterations found in arf6 arf8 double mutants (cf. with Nagpal et al., 2005). (I) Scanning electron micrograph of wild-type flowering apex. (J) A cross section through AP1≫HP-GFP flowering apex with expression throughout emerging flower meristems. (K) Wild-type flower. (L) Fused sepals and absent petals in AP1≫miR164b flower. (M) Flowering apex and filamentous flowers (inset) of strong AP1≫miR165b plant. (N) Normal sepals, radial petals, distorted stamens, and multiple carpels in a weak AP1≫miR165b flower. (O) Sequence alignment of the wild type and phv-1d mutant with corresponding miR165b and miR165bm6. (P) Seedling expressing ANT≫miR165b#4 results in radialized cotyledons and aborted meristem. (Q) and (R) Adaxial surface of wild-type (Q) and ANT≫miR165bm6 (R) leaves. Note the adaxial outgrowths of the transgenic leaf (arrow). (S) Normal sepals, distorted stamens, and multiple carpels in a strong ANT≫miR165bm6 flower. FM, flower meristem; IM, inflorescence meristem; P, petal. Bars = 3 mm in (B), (F), and (G) and 20 μm in (C), (I), and (J).

Figure 2.

Custom-Designed Synthetic miRNAs Efficiently Regulate ta-siRNA Targets. (A) Sequence alignment of the ta-siRNA binding sites in Arabidopsis ARF2, ARF3, and ARF4, the endogenous ta-siRNAs, and a designed miR-ARF sequence with better homology to all target sites. The A and B sites are designated according to Allen et al. (2005). Mismatches are marked red and G-U wobbles cyan. (B) Predicted folding and dicing of the pre miR164b backbone before (left) and after (right) replacement of miR164 with the miR-ARF sequence. (C) Abaxial side of wild-type (a), ett-1 arf4-1 (b), and 35S:miR-ARF (c) leaves. (D) Bolting shoot of ett-1 arf4-1 plant. (E) Bolting shoot of 35S:miR-ARF plant. (F) to (H) Flowers of wild-type (F), ett-1 arf4-1 (G), and 35S:miR-ARF plants (H). Note the gradual increase in the number of sepals, stamen radialization, and decrease in petal width. sp, sepal; p, petal; st, stamen. (I) Detection of miR-ARF and miR164 in wild-type and 35S:miR-ARF plants by RNA gel blot analysis. Both miRNAs are the same ∼21 nucleotides. (J) Reduced levels of full-length transcripts of the three ARF genes in 35S:miR-ARF plants. (K) A scheme of ARF4 cDNA with primers used for RLM-RACE detection. Gel images showing RLM-RACE–detected wild-type and 35S:miR-ARF (miR-ARF) cleavage products at sites A and B detected using either primer a or b, where A is the expected gel position for a product cleaved at site A, and B is the expected gel position of product cleaved at site B. In 35S:miR-ARF, amplification products are more prevalent relative to the wild type. In addition, amplification of a product cleaved at site A could only be obtained with the a primer (gels for PCR products of ARF2 and ARF3 cleavage analysis are shown in Supplemental Figure 2 online). (L) Summary of cleavage analysis by direct sequencing of RLM-RACE products (see Supplemental Figure 2 for details) and product cloning of the three ARF genes. Cleavage analysis for 35S:miR-ARF plants is shown, and the dispersed cleavage products of the wild type are shown in Supplemental Figure 2 online and in Allen et al. (2005). DS, direct sequencing.

Figure 3.

Custom-Designed Synthetic miRNA Efficiently and Specifically Codownregulate Non-Native Small RNA Targets. (A) A phylogenetic tree of the NGA-like proteins and their closest Arabidopsis homologs. Tree was constructed with the ∼120 amino acids that constitute the B3 domain as shown in Supplemental Figure 3 online. ETT was included as an outgroup, and numbers represent bootstrap percentage from 1000 trials. (B) A general scheme of NGA1/2/3/4 transcripts, outlining the position of a consensus sequence aligned. A synthetic miRNA has 0 to 2 mismatches with all four, and its conceptual dicing from pre miR164b backbone is illustrated. The arrow above pileup denotes cleavage point as described below. (C) Predicted folding and dicing of the pre miR164a backbone before (left) and after (right) replacement of miR164 with the miR-NGA sequence. (D) to (F) Young seedling of wild-type (D), nga1-1 nga2-1 nga3-1 nga4-1 quadruple mutant (E), and 35S:miR-NGAa (F) plants. Note the angular leaf blade of the mutants compared with the round leaf blade of the wild type. cd, cotyledons. (G) to (I) Inflorescence and pre-anthesis flower (insets) of wild-type (G), nga1-1 nga2-1 nga3-1 nga4-1 quadruple mutant (H), and 35S:miR-NGAa (I) plants. Note the broad yellowish petals and the protruding distal portion of the unfused gynoecium (gy). (J) Cotransactivation of NGA1 and miR-NGAa by CAB3 promoter eliminates dwarfism induced by ectopic NGA1 with the same promoter line (inset). (K) Detection of miR-NGA in wild-type and 35S:miR-NGAa plants by RNA gel blot analysis. (L) RLM-RACE detection of cleaved products of the four NGA-like transcripts in 35S:miR-NGAa (miR-NGA) plants but not wild-type plants. (M) RNA gel blot analysis reveals differential reduction in RNA levels of NGA1 and NGA3 in wild-type and 35S:miR-NGAa plants.

Figure 4.

Arabidopsis Pre-miRNA Backbones Induce Homologous Mutant Phenotypes Heterologously. (A) Wild-type and F1 tomato seedlings of OP:miR164b transactivated by 35S:LhG4. The top and bottom insets are close-ups of the upper portion of the seedling. (B) and (C) Upper part of wild-type (B) and FIL≫miR165b (C) tomato shoots. Leaves in (C) are short and radial, and floral organs are narrow. l, leaf; f, flower; rl, radialized leaf. (D) Abaxial side of wild-type and 35S:miR-ARF leaves. A close-up of 35S:miR-ARF leaf at the right illustrates the abaxial-specific outgrowths found along the veins. (E) to (H) Flowers ([E] and [F]) and carpels ([G] and [H]) of wild-type ([E] and [G]) and 35S:miR-ARF ([F] and [H]) tomato plants. Note the thin sepals and petals, the short style, and the thickened green stigma of the transformant. st, style; sg, stigma. (I) to (K) Comparison of leaves (I), flowers (J), and carpels (K) of wild-type (left) and 35S:miR-ARF tobacco plants. Gradual effects from weak to strong (right) are notable in independent T1 plants. As in tomato, abaxial leaf outgrowths are evident along the veins. Corolla outgrowths are external (abaxial) only. (L) and (M) Gynoecium of wild-type (L) and ett-1 (M) Arabidopsis plants. As in tomato and tobacco, stigmatic tissue in the mutant is expanded basally while style length is reduced.

Figure 5.

Induction of Solanaceae Target Cleavage by Arabidopsis-Based Synthetic Pre-miRNAs. (A) Sequence alignment of tomato ARF2/3/4, tobacco ETT/ARF3, and miR-ARF. The nucleotides not in gray are predicted wobbles. (B) Gel images showing RLM-RACE detection of tomato and tobacco ARF3 cleavage products from wild-type and 35S:miR-ARF (miR-ARF) plants. Primer position follows the ARF4 design in Figure 2K. Cleavage products at sites A and B were detected using either primer a or b, where A is the expected gel position for a product cleaved at site A, and B is the expected gel position of product cleaved at site B. (C) Cleavage point mapping of tomato and tobacco ARF3. Arrows mark cleavage sites in sequenced, cloned products, where arrow size corresponds with frequency of clones obtained (also given). Arrows above each sequence are for clones obtained from the wild type, and those below are from 35S:miR-ARF plants. The number of clones matching the predicted target region out of total number of sequenced clones is shown in parentheses.

Figure 6.

miRNA Activities Are Spatially Restricted and Do Not Cross Graft Joints. (A) Transverse section of an Arabidopsis flower showing promoter AP3-mediated expression of GFP limited to petals and stamen. (B) Longitudinal section of same age Arabidopsis flower showing that promoter CRC-mediated expression of GFP is limited to carpels. (C) Flower of AP3≫miR165b plant with radial petals and stamens but normal gynoecium. (D) Close-up of radial stamens with normally abaxial-restricted guard cells scattered all around (arrows). (E) Flower of CRC≫miR165b plant with thin gynoecium and normal stamens. (F) A graft of 35S:miR-ARF on wild-type tomato. Picture was taken 4 months after grafting and after two rounds of defoliation (arrows) of wild-type leaves. Newly initiating shoots remain normal. White box shows graft union. (G) to (I) A graft of the wild type on 35S:miR-ARF tobacco (G). Here, too, defoliation (arrows) of wild-type leaves did not stimulate miRNA-derived phenotype on wild-type shoot. The white box shows the graft union. Upon flowering, wild-type acceptor shoots were normal (H) even though 35S:miR-ARF donor flowers are highly distinct (I). sp, sepal; p, petal; st, stamen; gy, gynoecium; rp, radial petal; rst, radial stamen. Bars = 50 μm in (A) and (B) and 100 μm in (C) and (D).