IC4R011-miRNA-2008-18350165

Project Title

• Highly Specific Gene Silencing by Artificial miRNAs in Rice

The Background of This Project

• 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.

Plant Culture & Treatment

• The researchers 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.

Research Findings

• To optimize expression of the amiRNA, we chose to employ an endogenous rice miRNA precursor, a 245 bp fragment of the osaMIR528 locus. Appropriate 21mers to replace the endogenous miRNA and miRNA* of osa-MIR528 were designed based on the TIGR4 rice genome annotation. Modified precursors (Figure 1) were constitutively expressed in two rice varieties, Nipponbare(japonica) and IR64 (indica).

'Figure 1. Secondary Structure of the osa-MIR528 stemloop in pNW55. Depicted is the secondary structure of the 255 bp derived from osaMIR528 (245 bp plus the BamHI and KpNI restriction sites) as predicted by ‘RNAfold’ [33] (Vienna RNA package, http://www.tbi.univie.ac.at/RNA) for 23uC. The sequences replaced in the different transgenes are boxed. The oligo designer in WMD2 [25] (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.doi:10.1371/journal.pone.0001829.g001'

• The method was evaluated by targeting three different rice genes, Phytoene desaturase (Pds,Os03g08570), Spotted leaf 11 (Spl11, Os12g38210), and Eui1(CYP714D1, Os05g40384) (Table 1).

• Transgenic lines containing the amiRNA constructs reproduced the previously described phenotypes for all intended target genes in both varieties (Figure 2, Table 2, Table S1).Phytoene desaturase (PDS) catalyzes an early step in the carotenoid biosynthesis pathway and the absence of protective carotenoids results in bleaching through photo-oxidation of chlorophyll, making it a convenient gene for proof-of-principle applications. PDS is encoded by a single copy gene in the rice genome and silencing of Pds (AF049356) in rice by hpRNAi, using a 470 bp gene fragment, has been reported to cause an albino phenotype. Over 90% (39 of 42) of transgenic Nipponbare plants (NB_pNW78) and 56% (38 of 68) of transgenic IR64 plants(IR64_pNW78) had an albino phenotype (Figure 2, Table 2).

'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.doi:10.1371/journal.pone.0001829.g002

Labs working on this Project

• Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tu ¨ bingen, Germany, 2 International Rice Research Institute (IRRI), Metro Manila,Philippines

Corresponding Author

• Norman Warthmann:norman@warthmann.com & Philippe Hervep:herve@cgiar.org