Highly Specific Gene Silencing by Artificial miRNAs in Rice

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.

[1]  Javier F. Palatnik,et al.  Specific effects of microRNAs on the plant transcriptome. , 2005, Developmental cell.

[2]  C. Llave,et al.  Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA , 2002, Science.

[3]  Eric J Wagner,et al.  Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. , 2002, Molecular cell.

[4]  G. S. Khush,et al.  Green revolution: A mutant gibberellin-synthesis gene in rice , 2002, Nature.

[5]  B. Meyers,et al.  An expression atlas of rice mRNAs and small RNAs , 2007, Nature Biotechnology.

[6]  H. Leung,et al.  Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activityw⃞ , 2004, The Plant Cell Online.

[7]  Jia-Wei Wang,et al.  Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol , 2007, Nature Biotechnology.

[8]  Peter M Waterhouse,et al.  RNA silencing platforms in plants , 2005, FEBS letters.

[9]  M. Kusaba.  RNA interference in crop plants. , 2004, Current opinion in biotechnology.

[10]  Sean R. Eddy,et al.  Rfam: an RNA family database , 2003, Nucleic Acids Res..

[11]  E. Meyerowitz,et al.  Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  G. Droc,et al.  High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. , 2004, The Plant journal : for cell and molecular biology.

[13]  Bin Liu,et al.  Loss of Function of OsDCL1 Affects MicroRNA Accumulation and Causes Developmental Defects in Rice1[w] , 2005, Plant Physiology.

[14]  O. Voinnet,et al.  In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. , 2004, Genes & development.

[15]  N. Chua,et al.  Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance , 2006, Nature Biotechnology.

[16]  T. Komari,et al.  Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens , 2006, Plant Cell, Tissue and Organ Culture.

[17]  I. Longden,et al.  EMBOSS: the European Molecular Biology Open Software Suite. , 2000, Trends in genetics : TIG.

[18]  N. Melamed-Book,et al.  Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance , 2006, Proceedings of the National Academy of Sciences.

[19]  I. Small.  RNAi for revealing and engineering plant gene functions. , 2007, Current opinion in biotechnology.

[20]  Kan Nobuta,et al.  Plant MPSS databases: signature-based transcriptional resources for analyses of mRNA and small RNA , 2005, Nucleic Acids Res..

[21]  K. Shimamoto,et al.  Simple RNAi vectors for stable and transient suppression of gene function in rice. , 2004, Plant & cell physiology.

[22]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[23]  Geert Plaetinck,et al.  Control of coleopteran insect pests through RNA interference , 2007, Nature Biotechnology.

[24]  J. Schelter,et al.  Designing siRNA That Distinguish between Genes That Differ by a Single Nucleotide , 2006, PLoS genetics.

[25]  M. Ellis,et al.  Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Detlef Weigel,et al.  Gene silencing in plants using artificial microRNAs and other small RNAs. , 2008, The Plant journal : for cell and molecular biology.

[27]  Peter F. Stadler,et al.  Thermodynamics of RNA-RNA Binding , 2006, German Conference on Bioinformatics.

[28]  Y. Kamiya,et al.  ELONGATED UPPERMOST INTERNODE Encodes a Cytochrome P450 Monooxygenase That Epoxidizes Gibberellins in a Novel Deactivation Reaction in Rice[W] , 2006, The Plant Cell Online.

[29]  P. Hervé,et al.  Japonica rice varieties (Oryza sativa, Nipponbare, and others). , 2006, Methods in molecular biology.

[30]  Ping Xu,et al.  Computational Estimation and Experimental Verification of Off-Target Silencing during Posttranscriptional Gene Silencing in Plants1[W][OA] , 2006, Plant Physiology.

[31]  Q. Qian,et al.  Genetic and Molecular Analysis of Utility of sd1 Alleles in Rice Breeding , 2007 .

[32]  P. Waterhouse,et al.  Construct design for efficient, effective and high-throughput gene silencing in plants. , 2001, The Plant journal : for cell and molecular biology.

[33]  M. Yano,et al.  Diverse variation of reproductive barriers in three intraspecific rice crosses. , 2002, Genetics.

[34]  Stefan L Ameres,et al.  Molecular Basis for Target RNA Recognition and Cleavage by Human RISC , 2007, Cell.

[35]  Qian Qian,et al.  EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice. , 2006, Plant & cell physiology.

[36]  Detlef Weigel,et al.  Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis[W][OA] , 2006, The Plant Cell Online.

[37]  John A. Hamilton,et al.  The TIGR Rice Genome Annotation Resource: improvements and new features , 2006, Nucleic Acids Res..

[38]  G. Galili,et al.  RNAi and microRNA: breakthrough technologies for the improvement of plant nutritional value and metabolic engineering , 2007, Metabolomics.

[39]  Bruce D. Smith,et al.  The Molecular Genetics of Crop Domestication , 2006, Cell.

[40]  Wei Huang,et al.  A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase , 2007, Nature Genetics.

[41]  E. Blum,et al.  Endogenous and Synthetic MicroRNAs Stimulate Simultaneous, Efficient, and Localized Regulation of Multiple Targets in Diverse Species[W] , 2006, The Plant Cell Online.

[42]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[43]  Helen M Blau,et al.  A brief history of RNAi: the silence of the genes , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[44]  Jing Qu,et al.  Artificial MicroRNA-Mediated Virus Resistance in Plants , 2007, Journal of Virology.

[45]  Michael Kertesz,et al.  The role of site accessibility in microRNA target recognition , 2007, Nature Genetics.