Benefits of hairpinRNAi in plants
The search for new genes for valuable yield and quality traits has been accelerated by hairpinRNAi. Not only is hairpinRNAi a valuable technology for the identification of trait genes in plants, but it also delivers crucial information about gene function. Knowing and validating gene function is essential before any gene becomes the focus of a plant breeding program.
HairpinRNAi has the following benefits.
Precise
CSIRO has developed design rules to ensure gene specific silencing. These rules are especially important when trying to target one gene amongst a family of related genes.
For example, CSIRO targeted the phytochrome gene family in the model plant Arabidopsis (Figure 1a). Phytochromes enable the plants to perceive light and exist as a family of five closely related members PhyA to PhyE. Each phytochrome performs a different function that can be distinguished by growing plants under specific wavelengths of light.
The nucleotide sequences are highly similar – yet, single members can be precisely targeted by altering the design of the hairpinRNAi construct. From these kinds of experiments CSIRO has developed hairpinRNAi design rules to ensure that only the target gene is silenced without unwanted cross silencing of related genes.
After testing on various plants and silencing different genes no off target effects have been identified. Whilst there may be reduction of other RNAs based on feed back loops, the destruction of non-target RNA through RNAi has not been observed (Figure 2).
Figure 1. (a) Phytochromes exist as a family of genes with similar nucleotide sequences; (b) Arabidopsis plants – wild type on the left and PhyB knock out on the right
Figure 2. Microarray analysis of CHS knockout plants. None of the other differentially expressed genes shared any sequence homology with CHS
Efficient
HairpinRNAi constructs targeting a number of genes in a range of plants have proven to be very efficient. The majority of transgenic plants show a silencing phenotype.
Typical results in the table show that 70 – 100 per cent of independent transformation events show a phenotype resulting from silencing of the target gene.
Figure 3. Efficiency of hairpinRNAi with different genes in a variety of plants
Click to enlarge
User friendly
HairpinRNAi technology is made user friendly with CSIRO’s vectors.
The target sequence is amplified with PCR, cloned into generic vector
either by using restriction enzymes or through Gateway™ recombination
(Invitrogen).
(Link to Hannibal, Hellsgate)
High-throughput
High throughput vectors based on the Gateway™ (Invitrogen) technology are designed to make hairpinRNAi constructs for large sets of genes (eg. members of a gene family, or pathway, or even whole genomes).
The combination of fewer steps and automation make the technology very cost-effective. CSIRO has been able to assemble hundreds of constructs within a week.
pHELLSGATE and pWATERGATE provide constitutive silencing in dicot plants and pSTARGATE in monocots.
Genome wide knockout plants in Arabidopsis
Several projects are underway to produce the resources that are required for hairpinRNAi to be used as a tool for high throughput plant genomic research.
The CATMA group (Complete Arabidopsis Transcriptome MicroArray) is generating a set of PCR products called gene sequence tags (GSTs) that represent each Arabidopsis gene and have been designed to hybridise in a gene specific manner on Arabidopsis cDNA microarrays. The primers that are used to generate these PCR products have 5' extensions that enable them to be re-amplified and cloned into GATEWAY vectors by a BP clonase reaction.
AGRIKOLA (Arabidopsis Genomic RNAi Knock-Out Line Analysis) is using this set of PCR products to generate hairpinRNAi constructs for every gene using a vector derived from pHELLSGATE.
Stable
HairpinRNAi has been shown to be stably inherited over several generations. For example, the targeted gene FAD2 is responsible for the production fatty acid desaturase activity in Arabidopsis. The high degree of gene silencing was stably inherited over five generations.
The stability of hairpinRNAi. Distribution of oleic desaturation proportion (ODP) values for hairpinRNAi (full circles) T1 plant population and the T2, T3, T4, and T5 progeny of a selected highly silenced T1 plant, in comparison with Columbia (empty circles) control plants grown at the same time
Figure 4. Stoutjesdijk PA et al (2002) Plant Physiology, 129: 1723-1731
Flexible
Individual or multiple genes can be silenced
The tools and design rules of CSIRO’s technology permit silencing of one, some, or all members of a gene family using unique or shared target sequences. This is a powerful technology to study the actions and interactions of members of gene families.
For example, phytochromes exist as a small family of five members in Arabidopsis (Figure 1a). Phytochrome B and D are very similar as compared with the others. To determine the function of Phytochrome B, which is 80 per cent similar to D, very specific silencing is needed – this was achieved by designing hairpinRNAi constructs using unique sequences in the untranslated regions (UTRs).
All seven members of the codeinone reductase (COR) gene family in opium poppy have been silenced using one hairpinRNAi construct. COR converts codeinone to codeine, which is demethylated to morphine. Transgenic plants displayed varying degrees of diminished morphine production, from 25 to 100 per cent along with compensatory accumulation of the morphine precursor reticuline which lies eight steps upstream from morphine (Allen et al., 2004).
Figure 5. hairpinRNAi construct to silence multiple genes
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Silence multiple genes
CSIRO hairpinRNAi technology enables multiple genes to be silenced with one single construct. CSIRO experiments have shown effective silencing of two genes from one hairpinRNAi construct and have indicated the possibility of silencing more.
Allen et al., 2004, Nature Biotechnology 22: 1559 – 1566.
Used in non model plants
Since hairpinRNAi works with equal efficiency both in model and non model plants, the knowledge generated from the functional genomics of model plants can easily be extended to non model plants.
Gene replacement possible
The precision of hairpinRNAi technology can be exploited to specifically target an unwanted gene which can then be replaced by a transgene with the desired properties. Altering catalytic properties of an enzyme is one such application, as was done to create the blue rose.
Tissue-specific silencing
Marc De Block and colleagues at Bayer CropScience used hairpinRNAi to prevent the development of petals in Arabidopsis and canola flowers. Although the target gene is required for the development of both petals and stamens, the use of a promoter expressed only in sepals and petals gave organ-specific silencing in petals (Byzova et al., 2004).
Byzova et al., 2004, Planta 218: 379 – 387.
Figure 6. Tissue specific silencing using
hairpinRNAi
Morphological features of Brassica napus flowers: mature wild-type
flower (left), mature flower of a transgenic
plant (right). The second-whorl organs
of a transgenic flower are yellowish-green sepaloid petals (arrow).
Inducible silencing
CSIRO has developed a vector system that is capable of inducing silencing
when required. The production of hairpinRNAi from this system can be turned
on and off by the application and removal of the inducing substance. The
inducibility of this system will be very useful in helping to identify
the functions of genes which when constitutively silenced give embryo
lethality or complex phenotypes. A slightly modified version of this system
can also be used for tissue-specific hairpinRNAi expression.
CSIRO chose phytoene desaturase (PDS) gene of Arabidopsis to test the
ability of the inducible RNAi system to silence endogenous genes. The
PDS gene was selected because loss of the phytoene desaturase enzyme blocks
carotenoid synthesis culminating in a photobleaching phenotype because
of photo-oxidation of chlorophylls.
Silencing was highly effective 24 hours after application of dexamethasone, appeared to be stably maintained by the presence of the hormone, and was significantly released 24 hours after its removal.
Figure 7. Inducible silencing with hairpinRNAi
Individual T1 plants of Arabidopsis ecotype Col plants transformed with
pOpOff2(hyg)::PDS growing (A) on standard media and (B) on media supplemented
with dexamethasone, plants photographed 7 days after transfer onto dexamethasone
media. Plants on dexamthasone show clear photobleaching.
Better than antisense
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With CSIRO’s hairpinRNAi it is possible to obtain plants where the target gene activity is totally silenced – similar to a ‘null mutant’. However, it also produces plants with gene activity reduced to different levels. These plants are similar to an ‘allelic series’ and are extremely valuable in studying the function of genes that are vital for the survival of the plant. The ‘knock down plants’ are useful when a complete knockout is lethal to the plant.
As an example, CSIRO silenced a gene called chalcone synthase responsible for anthocyanin production targeted by our vector system. A gradation in the seed colour was observed – the stronger the silencing, the paler the colour (Wesley et al., 2001).
In another example when the FLC gene was silenced, which controls flowering time, plants were obtained that flowered at varying times – the strongest silenced plant flowered the earliest – in 18 days – just like the null mutant. The weakest silenced plant flowered in 29 days – only a couple of days earlier than the wild type plant which flowered at 33 days (Wesley et al., 2001)
The highest silencing obtained with an antisense construct was only as good as the least silenced plant with hairpinRNAi.