This section was contributed by George Coupland (John lnnes Centre) and Andy Greenland (AstraZeneca).
The ale regulon is a self-contained system that, in Aspergillus nidulans, controls the cellular response to ethanol (Felenbok 1991). In A. nidulans, the AlcA gene, which encodes alcohol dehydrogenase I, is regulated by the pathway-specific AlcR transcription factor. Binding of ethanol to AlcR induces a conformational change that permits binding of the complex to the AlcA promoter, resulting in high levels of gene expression. The Ale system has been adapted for use in higher plants by expressing AlcR from the constitutive CaMV 35S promoter, allowing the expression of any gene fused to the AlcA promoter to be induced in plant tissues treated with ethanol (Caddick et al. 1998).
One of the benefits of the Ale gene switch is the convenience of the inducing agent. Ethanol is a powerful inducer that is readily available, it has negligible phytotoxicity at the concentrations required for induction, and it can be applied to plants easily using a variety of techniques. The method chosen for application will depend on the desired effect (whole plant or localized induction) and the size and/or stage of development of the plant itself.
We recommend that the dose response and time course of induction be determined for each plant species, and each Ale promoter and gene combination. In our hands, reporter gene expression in tobacco and Ara-bidopsis, using either chloramphenicol acetyltransferase (CAT) or |3-glu-curonidase (GUS), can be detected 2 hours after ethanol treatment, with the peak of expression occurring at 48 hours (Salter et al. 1998). The duration of induced expression will depend on the presence of ethanol and the properties of the protein encoded by the target gene. Since ethanol is both volatile and metabolized by plants, a pulse of gene expression can be achieved following single doses. Sustained gene expression may require repeated dosing, depending on the application and approach taken (see below). The level of gene expression achieved can be titrated through use of a range of ethanol concentrations. At the concentrations required to achieve induction, there are few, if any, visible phytotoxic effects of ethanol. As noted below, care must be taken when combining ethanol with a surfactant, such as Silwet L-77, to improve uptake, as this may cause phytotoxic effects.
In intact plants, and in the absence of ethanol, the Ale switch is tightly regulated and background expression levels are low (Caddick et al. 1998; Salter et al. 1998). However, as ethanol vapor is an effective inducer, noninduced experimental controls must be well separated from treated plants to avoid background expression. In several experiments, we have detected background expression during transformation and tissue culture, which may lead to counterselection if the gene product is harmful at this stage. Background expression should be less of a problem when using vacuum infiltration for Arabidopsis transformation, because the plants do not go through a callus phase, which is known to induce many genes unspecifically.
• Plants in soil: Root drench. For tobacco growing in 7.5-cm diameter (200 ml) pots, apply 50 ml of 1-5% ethanol (v/v) to the soil as part of the normal watering regime. For Arabidopsis, apply 0.1-5% ethanol (v/v). Use proportional volumes for different size pots. Make sure that the soil is slightly moist before drenching. Further enhance induced protein levels by applying ethanol 1-2 days after the first application. Collect tissue samples on Day 3.
• Plants in soil: Vapor induction. Uniform induction in aerial organs can be achieved by enclosing plants with a source of ethanol vapor. The conditions for induction using this approach should be determined for each experiment, as temperature and light intensity will affect the concentration of ethanol vapor achieved. High concentrations of vapor result -n plant damage. As a general guide, nsert 30 1.5-ml microcentrifuge tubes (caps open) at regular intervals across the surface of a 25 x 35-cm propagation tray. Pipette 1 ml of 10-100% ethanol (v/v) into each tube. Seal the whole tray in a transparent plastic bag (approximate volume 20 liters) for 24 hours. Then remove the plastic bag, centrifuge the tubes and surplus ethanol, and harvest the tissue samples at an appropriate time after the initial treatment.
• Plants in soil: Foliar spray. The application of ethanol by spraying is generally the least robust of all the methods described, but under certain circumstances, it can be appropriate. Use a hand sprayer to apply a 5-10% ethanol solution until run-off occurs (Caddick etal. 1998). Use a surfactant such as Silwet L-77 at concentrations up to 0.2% (v/v) to improve the level of induction (be careful, concentrations exceeding this level are detrimental to the plant).
• Hydroponic growth. For plants growing hydroponically, add ethanol to the growing media to a final concentration of 0.1% (v/v). The ethanol-growing media can be kept for an appropriate period of prolonged induction or exchanged for fresh, noninducing media to achieve a pulse of induction.
• Induction of detached leaves. The induction of single detached leaves is useful as a rapid early screen for transgenic plants expressing an induced target gene. Remove the leaves and expose them to ethanol vapor for 48 hours by placing them on damp filter paper in a sealed plastic box (approximate volume 2 liters) containing a beaker with 40 ml of 0.1% (v/v) ethanol.
Glucocorticoid Inducible Control of Gene Expression
This section was contributed by Yiji Xia (Salk Institute).
The GVG glucocorticoid-mediated transcriptional induction system was developed by Aoyama and Chua (1997). The system consists of a chimeric transcription factor "GVG," which 15 a fusion of the yeast
GAL4 DNA-binding domain, the ?r¿ms-activation domain of the herpesvirus protein VP 16, the ligand-binding domain (LBD) of the rat glucocorticoid receptor, along with a responder gene driven by a minimal promoter linked to GAL4-binding sites (UAS). In the original system, based on the pTA7002 vector, the activator and responder are carried on the same T-DNA. in pTA7002, GVG is under the control of the CaMV 35S promoter.
In the absence of a ligand, the LBD interferes with the function of GVG by keeping the fusion protein in the cytoplasm. Upon binding of the LBD to a ligand such as dexamethasone (DEX), a synthetic glucocorticoid, the chimeric protein moves to the nucleus, where it binds to the UAS and causes activation of the downstream gene. There have been reports that DEX causes phenotypic defects in some GVG lines (Kang et al. 1999), but several others report successful use of this system. As a precaution, it is best to have the GVG and the responder on different T-DNAs, such that any activator lines that do show phenotypic effects in the absence of the responder can be identified and discarded.
In pTA7002, the gene of interest can be subcloned into the XboVSpel sites, downstream from the UAS promoter. Select plasmids in E. coli and Agrobacterium using kanamycin (50 mg/liter). Where necessary, the CaMV 35S promoter for the GVG gene can be replaced with a tissue-specific promoter.
Select transgenic lines on MS plates (see Chapter 1) using hygromycin (20 mg/liter) and test individual transgenic lines by northern or western blotting. Identify the lines that show a low basal level of responder gene expression in the absence of inducer and strong induction in its presence.
Prepare a 30 mM stock of DEX by resuspending 118.2 mg of DEX in 10 ml of absolute ethanol. The stock is stable for up to 1 month when stored at -20°C.
To induce GVG activity, grow plants on MS medium containing 0.2-10 pM DEX (add DEX after MS agar has been autoclaved and the medium has cooled to ~50°C). Alternatively, spray the plants with an aqueous solution containing 0.5-30 pM DEX and 0.01% Tween-20. The optimal concentrat on of DEX depends on individual transgenic lines and the desired mduction level of the gene of interest. Gene induction can be detected wirhm 30 m mutes after DEX treatment and reaches its highest level between 10 and 24 hours after treatment (Aoyama and Chua 1997; Y. Xia et al., unpubl.).
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