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Exploring plant genomes by RNA-induced gene silencing

Key Points

  • Although genome sequence data are available for model dicotyledon (Arabidopsis) and monocotyledon (rice) plant species, the functions of many proteins encoded by these genomes are unknown. Finding the function of these proteins is a major challenge for plant biology.

  • Loss of a gene's expression can give insight into its function. RNA-induced gene silencing (or RNA interference, RNAi) is a good way to remove gene function, because it is driven by RNA hybridization, so genes can be targeted specifically and directly, as can gene families; it can be used in a wide variety of plant species and it reduces gene expression to varying degrees.

  • RNAi can be induced in plants by double-stranded RNA (dsRNA), by self-complementary 'hairpin' RNA (hpRNA) or by viral RNA. A key trigger of RNA-induced gene silencing is the production of dsRNA, which leads to the production of small interfering RNAs (siRNAs) of 21 nucleotides that are used to target of degradation RNA with complementary sequence.

  • RNAi can be delivered by transient methods (such as particle bombardment, Agrobacterium infiltration or viral infection) or by stable ones (such as the introduction of hpRNA or amplicon transgenes). The features of these methods are summarized in this review.

  • Virus-induced gene silencing (VIGS) and hpRNA transgenes have features that make them the most likely to be useful for genomics projects.

Abstract

The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms — for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.

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Figure 1: The current model of RNA-mediated gene silencing in plants.
Figure 2: DNA constructs for RNA-mediated gene silencing.
Figure 3: Tobacco plant phenotypes after infection with a satellite-virus-induced silencing system.
Figure 4: The current model of hairpin-RNA-directed transcriptional gene silencing in plants.
Figure 5: Degrees of silencing produced by hairpin-RNA-encoding transgenes.

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Acknowledgements

We thank M. Metzlaff and S. Dinesh-Kumar for access to figures and manuscripts in press.

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Correspondence to Peter M. Waterhouse.

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DATABASES

FAD2

PDS

FURTHER INFORMATION

AGRIKOLA

Arabidopsis Gauntlet Project

Arabidopsis Stock Centre

CATMA

Chris Helliwell's and Peter Waterhouse's laboratories

Construction of an Arabidopsis open reading frame library

CropDesign

David Baulcombe's laboratory

Gateway Technology

Monto Kumagai's laboratory

Salk Collection at TAIR

S. Dinesh-Kumar's laboratory

Glossary

CO-SUPPRESSION

The silencing of an endogenous gene due to the presence of a homologous transgene or virus. Co-suppression can occur at the transcriptional or post-transcriptional level.

AGROBACTERIUM TUMEFACIENS

A gram-negative soil bacterium that is used to transfer DNA into plant cells by a process similar to bacterial conjugation. The transferred DNA (T-DNA) randomly integrates into the plant genome to produce stably transformed plants.

MONOCOTYLEDON

(monocot). One of the two classes of flowering plants that is characterized by one embryonic leaf (cotyledon). Maize, rice and other grasses are common monocots.

β-GLUCURONIDASE

(GUS). An easily visualized reporter gene that is used in plant research.

STOMATA

Natural openings in the epidermis of a stem or leaf of a plant that are surrounded by specialized guard cells, and allow gas exchange with the air.

CAMV 35S PROMOTER

A promoter derived from cauliflower mosaic virus that has been widely used in transgenic plants because of its ability to direct high-level constitutive transcription.

GEMINIVIRUSES

A specific group of viruses that have genomes composed of single-stranded DNA.

MERISTEM

The undifferentiated tissue at the tips of stems and roots in which new cell division is concentrated.

SATELLITE VIRUS RNA

A specific parasitic RNA that depends on a virus for its replication.

DICOTYLEDON

(dicot). One of the two principal classes of flowering plant that is characterized by two cotyledons (primitive leaves) in the embryonic plant. Tomatoes, maple trees and mustard are common dicots.

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Waterhouse, P., Helliwell, C. Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 4, 29–38 (2003). https://doi.org/10.1038/nrg982

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