Highly specific gene silencing by artificial miRNAs in rice

Abstract

Background: Endogenous microRNAs (miRNAs) are potent negative regulators of gene expression in plants and animals. Artificial miRNAs (amiRNAs)-designed to target one or several genes of interest-provide a new and highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants.

Methodology: We devised an amiRNA-based strategy for both japonica and indica type strains of cultivated rice, Oryza sativa. Using an endogenous rice miRNA precursor and customized 21mers, we designed amiRNA constructs targeting three different genes (Pds, Spl11, and Eui1/CYP714D1). Upon constitutive expression of these amiRNAs in the varieties Nipponbare (japonica) and IR64 (indica), the targeted genes are down-regulated by amiRNA-guided cleavage of the transcripts, resulting in the expected mutant phenotypes. The effects are highly specific to the target gene, the transgenes are stably inherited and they remain effective in the progeny.

Conclusion/significance: Our results not only show that amiRNAs can efficiently trigger gene silencing in a monocot crop, but also that amiRNAs can effectively modulate agronomically important traits in varieties used in modern breeding programs. We provide all software tools and a protocol for the design of rice amiRNA constructs, which can be easily adapted to other crops. The approach is suited for candidate gene validation, comparative functional genomics between different varieties, and for improvement of agronomic performance and nutritional value.

Figure 1. Secondary Structure of the osa-MIR528 stemloop in pNW55.

Depicted is the secondary structure of the 255 bp derived from osa-MIR528 (245 bp plus the BamHI and KpNI restriction sites) as predicted by ‘RNAfold’ (Vienna RNA package, http://www.tbi.univie.ac.at/RNA) for 23°C. The sequences replaced in the different transgenes are boxed. The oligo designer in WMD2 (http://wmd2.weigelworld.org) designs the amRNA* sequence (black) such that it pairs to the respective miRNA (red) in the same way as in osa-MIR528.

Figure 2. Phenotypes of plants overexpressing amiRNAs.

The empty vector control is always on the left. (A–D) pNW76, construct targeting Spl11. (A) Nipponbare whole plants, (B) Nipponbare leaves, (C) IR64 whole plants, (D) IR64 leaves at maximum tillering stage. (E–H) pNW78, construct targeting Pds. (E) Nipponbare whole plants, (F) Nipponbare leaves, (G) IR64 whole plants, (H) IR64 leaves at tillering stage. (I–L) pNW81, construct targeting Eui1. (I) Nipponbare whole plants, (J) Nipponbare uppermost internode, (K) IR64 whole plants, (L) IR64 uppermost internode. The ruler in (J) and (L) is 60 cm long.

Figure 3. Molecular characterization of transgenic plants.

Cleavage site mapping was performed on mRNA from one transgenic plant for each transgene in both varieties (Nipponbare and IR64). Numbers above the arrows denote the number of clones ending at the particular position, with the total number of clones in parentheses. The binding energy (ΔG) of the RNA-RNA duplex between target (denoted by TIGR locus identifier) and amiRNA is given in kcal/mol and as a fraction of the calculated binding energy for a perfect match to the target site. Total RNA from two transgenic plants for each construct (leaf tissue for SPl11 and Pds, young panicles for Eui1/CYP714D1) was used for RT-PCR for the target (histograms, top right), and small RNA blots (bottom right). Gel images are provided as loading control for small RNA blots. Comparison was to an empty vector control (IRS-154). Expression was normalized to the respective empty vector control. Error bars indicate the variation between technical replicates (range).

Figure 4. Predicted amiRNA-mRNA interactions.

The program ‘RNAup’ was used to assess the thermodynamics of RNA-RNA binding considering 80 bp surrounding the target site at 23°C. The predicted cleavage sites (between positions 10 and 11 of the miRNA) are highlighted in grey. ‘RNAup’ calculates the effective free hybridization energy (ΔG) as sum of the energies necessary to open the target site in the target mRNA (ΔGu_l), the internal folding of the amiRNA (ΔGu_s), and the energy gained from hybridization of amiRNA and target mRNA (ΔGint).