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Pii: s0958-1669(00)00073-2Chemical-inducible systems for regulated expression of plant genes
Jianru Zuo and Nam-Hai Chua
Chemical regulation of transgene expression presents a Chemical-inducible systems for regulated gene expression powerful tool for basic research in plant biology and are extremely useful for basic plant biology research and biotechnological applications. Various chemical-inducible biotechnology applications. For example, the ability to systems based on de-repression, activation and inactivation of activate a specific gene trait in the field by using chemicals the target gene have been described. The utility of inducible might circumvent the problem of saving of transgenic promoters has been successfully demonstrated by the seeds by growers. In response to basic research interests as development of a marker-free transformation system and large- well as commercial needs, many chemical-inducible sys- scale gene profiling. In addition, field applications appear to be tems have been developed in the past decade. Because promising through the use of registered agrochemicals this subject was intensively surveyed 2–3 years ago [2,3•,4] our review will focus primarily on results published in thepast three years. We compare the relative merits of the var- Addresses
ious systems, describe their uses thus far, consider Laboratory of Plant Molecular Biology, Rockefeller University, strategies for future development and discuss their poten- 1230 York Avenue, New York, NY 10021, USA Correspondence: Nam-Hai Chua; e-mail: email@example.com Current Opinion in Biotechnology 2000, 11:146–151
Desired properties of an ideal chemical-
Table 1 lists the desired properties of an ideal chemical- 2000 Elsevier Science Ltd. All rights reserved.
inducible system. Stringent chemical regulation of the Abbreviations
system requires many different properties. To prevent uncontrollable expression, it is important that the chemical inducer should not be a plant metabolite. Chemicals that elicit physiological responses in plants or are otherwise toxic should be avoided for obvious reasons. Taking these properties into consideration, it is clear thatpromoters of plant genes whose expression is triggered bychemicals (e.g. salicylic acid or benzothiadiazole)  are Introduction
probably not suitable. These promoters are likely to have Genetic manipulations by transgenic technology invariably some basal level expression, and because of the various cis involve the introduction of one or more transgenes that can elements embedded in their 5′ upstream region, they are turn on or turn off desired traits in plants. In many cases, con- likely to respond to a number of physiological and envi- stitutive promoters (e.g. the CaMV35S)  are used to ronmental signals, in addition to the chemical in question.
transcribe a gene of interested. A major limitation of consti- For these reasons, several laboratories have been engaged tutive promoters, however, is that they cannot be used to in the development of chemical-inducible systems using investigate genes whose constant over- or under-expression components derived from non-plant sources.
has deleterious effects on the plant. In the more severe cases,expression of a sense or anti-sense transgene in transformed Artificial chemical-inducible systems
cells may be toxic, thereby blocking plant regeneration.
Chemical-inducible expression systems in plants, in gen-eral, are based on de-repression, inactivation, and To a certain extent, the lethality problems can be partially activation of transcription of the target gene (Table 2). All overcome by using tissue-specific promoters. On the other these systems contain two transcription units. Whereas the hand, chemical-inducible systems for regulated gene first unit employs a constitutive (e.g. 35S) promoter to expression offer a more general and flexible solution. In express a chemical-responsive transcription factor, the sec- contrast to constitutive promoters, chemical-inducible sys- ond unit consists of multiple copies of the transcription tems are quiescent in the absence of inducers and factor binding site linked to a minimal plant promoter (a therefore will not inhibit plant regeneration. By the judi- truncated 35S promoter in all reported systems), which is cious application of inducers, it is possible to regulate gene used to express the target gene. The properties of these expression in transgenic plants at a particular developmen- systems are briefly summarized below.
tal stage and for a specific duration. Furthermore, the useof an appropriate promoter to express the chemical-respon- De-repression system
sive transcription factor can further restrict the target The bacterial tetracycline repressor (TetR) binds to the transgene expression to specific organs, tissues, or even tet operator in the absence of tetracycline. Upon associa- tion with tetracycline, TetR is released from its operator, Chemical-inducible systems for regulated expression of plant genes Zuo and Chua 147
Desired protperties of an ideal chemical-inducible system in
A list of chemical-inducible systems in plants.
High specificity with respect to inducers High dynamic range of response with respect to inducerconcentrations Rapid switch-off following inducer withdrawal Inducer not toxic and has no physiologic effects plants (a) Zuo et al., unpublished data.
presumably due to the conversion of the dimeric TetR(DNA-binding form) to the monomeric form. Based on containing tet operator sequences becomes silenced over these observations, Gatz et al. [5,6] developed the first time, presumably as a result of methylation of the tet oper- de-repression system in plants. The target promoter is a ator as originally observed in bacteria cells ([3•] and modified 35S promoter, in which one and two copies of references therein; see also [9••]).
the tet operator were placed upstream and downstreamfrom the TATA-box, respectively. In the absence of tetra- Activation systems
cycline, overexpressed TetR binds to the tet operator, Most inducible expression systems described in plants are thereby preventing target gene expression. Upon binding based on transcriptional activation. The most common tetracycline, TetR is released from the operator, relieving strategy is to constitutively or conditionally express an the repression. The tetracycline de-repression system has inactive chimeric transcription activator, which contains a been successfully used for expressing a number of genes heterologous DNA-binding domain (DBD), an activation in tobacco, tomato and potato but it did not work in domain, a nuclear localization signal (NLS) and, most crit- Arabidopsis, which presumably requires a higher but ically, the regulatory domain of an animal steroid nuclear intolerable repressor concentration for efficient repres- receptor. Regulation of steroid nuclear receptors has been sion ([2,3•] and references therein). Another disadvantage well documented, and the molecular mechanism appears is that fresh tetracycline has to be supplied every other to be highly conserved from insects to mammals. In the day due to the short half-life of the inducer in plants, absence of the hormone ligand, the receptor associates making the system less convenient to use.
with cellular regulatory proteins, including HSP90, andbecomes anchored in the cytosol as a monomer.
Association of a ligand with the hormone-binding domain In the tetracycline-inactivation system, the TetR repressor leads to the release of HSP90 from the receptor. The was converted into an activator (tetracycline transactivator receptor subsequently dimerizes, translocates into the [tTA]) by fusing it to the acidic activation sequence of her- nucleus, and binds to the target DNA. As the hormone pes simplex virus protein 16 (VP16). The target expression inducibility appears to be transferable when the regulatory cassette contains multiple copies of the tet operator domain is fused to a heterologous DBD, and also because sequence. The expression of the target gene is therefore plants do not have an analogous hormonal system, steroid- dependent upon binding of tTA to the tet operator, which based transactivation systems have been used in a number occurs in the absence of tetracycline. Introduction of the of studies . The regulatory domains of the mammalian latter results in the release of the tTA–tetracycline com- glucocorticoid receptor (GR; the GVG system) , estro- plex from the operator, thus turning off target gene gen receptor (ER) ([11•]; J Zuo, Q-W Niu, N-H Chua, transcription . This transcription-inactivation system unpublished data) and an insect ecdysone receptor [12••] has been used in both tobacco and Arabidopsis, and appears have all been shown to give relatively tight control and to be very useful for the study of gene product stability .
high inducibility. The GVG chimeric factor contains the Upon turning off the transcription of a transgene by apply- DBD of the yeast GAL4 transcription factor (G), the acti- ing the inducer, the turnover of the transgene product can vating sequence of VP16 (V), and the regulatory region of be assessed. A negative control by the inducer, however, the rat GR (G). In transgenic tobacco plants, the expres- makes the system less practical to use than a positively sion of a luciferase reporter gene driven by the target controlled system because plants have to be maintained in promoter is stimulated over 100-fold by treatment with the presence of tetracycline in order to turn off transcrip- dexamethasone (dex), a synthetic GR ligand. The system tion. An additional complication is that the promoter has been successfully used to express a number of genes in Plant biotechnology
different studies (see below). Major shortcomings of the In addition to the examples described before, the intact GVG system appear to be dex-dependent toxic effects in Aspergillus nidulans AlcR activator was used to control the some cases, and the induction of defense-related genes expression of target genes in plants using ethanol. In trans- ([13•]; N-H Chua et al., unpublished data). The effects genic tobacco plants AlcR stimulated the expression of a appear to occur in transgenic lines with high expression chloramphenicol acetyltransferase reporter gene upon levels of the GVG transcription factor (T Aoyama, N-H induction by ethanol to a level corresponding to 50% activ- Chua, unpublished data). Although the cause of these ity of the 35S promoter, whereas the background was effects is still under investigation it might result from bind- nearly undetectable . Pending development of non- ing of the GVG transcription factor at high concentrations volatile inducers, this system appears to hold promise for to plant cis-elements with sequence homology to the GAL4 recognition site. One solution to this problem is toselect for experimental lines that express moderate or low The dual-control inducible system
levels of GVG, and use as negative controls empty vector The drawbacks of the de-repression and inactivation sys- transgenic lines expressing the same level of GVG as tems prompted Gatz and co-workers [9••] to develop a dual-control inducible system by combining the advan-tages of these two systems and eliminating most of the An ER-based inducible system has been developed by disadvantages. A chimeric transcription activator TGV was Bruce et al. [11•]. The transactivation domain of the maize made by fusing the TetR DBD (T) to the regulatory region activator C1 was inserted in the activation domain of the of the rat GR (G) and the VP16 transactivating sequence human ER, and this ER–C1 fusion gene was controlled by (V), and the resulting factor is therefore subjected to dual a modified 35S promoter. The target expression promoter regulation by tetracycline and dex. In a dex-dependent contains four copies of ER element (ERE) fused to a min- fashion, TGV activates the expression of a reporter gene imal 35S promoter. In stably transformed maize BMS driven by a synthetic promoter consisting of multiple (Black Mexican Sweet) cell lines, the activity of a luciferase copies of the modified tet operator sequences placed reporter gene ranges from undetectable in uninduced cells upstream of a 35S minimal promoter. This dex-inducible to 14,000 relative light units upon a 48 hour induction with activation is similar to the previously reported GVG system estradiol (see also below). Another ER-based inducible sys- . When dex is removed and tetracycline is applied, the tem, designated the XVE system, was recently developed system is promptly switched off as association of tetracy- by using the DBD of the bacterial repressor LexA (X) and cline renders the chimeric factor incapable of binding the transactivation domain of VP16 (V) (J Zuo et al., unpub- DNA. The redesigned target promoter, modified by elim- lished data). The target promoter consists of eight copies of inating putative CG methylation sites, resolved the LexA-binding sites upstream from a 35S minimal promot- problems caused by methylation of the tet operator over er. The expression of a reporter gene can be readily generations, but the cost is a higher background expres- induced by estradiol 3–5-fold over that of the 35S promoter sion. In addition, because structurally similar activators and without detectable background expression. The GVG-like the same inducer (dex) were used, the TGV system may toxic effects have not been found in the XVE system. This have similar side effects as the GVG system.
system, however, appears to be deregulated in transientlytransformed soybean cells (T Klein, J Zuo, N-H Chua, Present and potential applications
unpublished data), presumably due to the presence of Conditional overexpression studies
An example is the conditional expression of the bacterialavrRpt2 avirulence gene under the control of the GVG sys- All of the systems discussed above employ chemical induc- tem in transgenic Arabidopsis plants carrying the RPS2 ers that are not suitable for field applications because of disease-resistance gene . Induction of the avrRPT2 the toxicity of dex, estradiol and tetracycline to the ecosys- gene by dex led to a hypersensitive cell-death response.
tem. This restriction, however, appears to have been These transgenic plants offer the opportunity to investi- partially overcome by the efforts of Martinez et al. [12••], gate the molecular events surrounding avrRPt2–RPS2 who have developed a non-steroidal agrochemical- gene interaction. Because the latter leads to plant death, inducible system. In this new system, the hybrid activator these transgenic lines can be used to isolate for mutants contains transactivating sequences from GR and VP16, the blocked in the signaling pathway leading from the DBD of GR and the hormone regulatory domain of the avrRPt2–RPS2 gene interaction to cell death, thereby Heliothis virescens ecdysone receptor. In transgenic tobacco identifying components in the pathway.
plants, the activator induced the expression of a reportergene over 400-fold, corresponding to 150% of the activity Other than lethality, expression of transgene in the sense or of a 35S promoter. The system is highly responsive to anti-sense orientation can lead to physiological adaptations of RH5992, a non-steroidal ecdysone agonist that lacks phy- transgenic plants, thus masking the true gene functions. This totoxicity and is currently used as a lepidopteran control problem is most powerfully illustrated by the recent example agent on a range of crops. A main drawback of this system of the TIR1 gene. Estelle and co-workers  found that is the relatively high background expression.
transgenic plants expressing a 35S–TIR1 transgene (thus Chemical-inducible systems for regulated expression of plant genes Zuo and Chua 149
being constitutively expressed) had no apparent phenotype.
transgenic plants without using an antibiotic resistance On the other hand, transgenic plants carrying a GVG–TIR1 marker. The ipt gene of Agrobacterium Ti plasmid is known construct produced more lateral roots in the presence of dex, to cause cytokinin production in transformed cells leading similar to wild-type plants treated with auxin. These results to shoot regeneration. The uncontrolled production of confirm the role of TIR1 in the auxin response pathway. cytokinins, however, causes developmental abnormalities,and the transgenic shoots were unable to produce roots and Co-suppression studies
their flowers were infertile. Recognizing the shoot-regen- Yoshizumi et al.  used the GVG system to express the erating potential of the ipt gene, Kunkel et al. [19••] placed antisense strand of the Arabidopsis CDC2b gene. Inhibition it under the control of the GVG system . Agrobacteria of CDC2b expression upon dex induction resulted in short carrying the GVG–ipt construct were used to inoculate hypocotyls and open cotyledons of the transgenic plants tobacco leaf disc in a medium without auxin and cytokinin.
grown in the dark, and these phenotypes were fairly corre- No shoot regeneration was observed in the absence of dex.
lated to the level of the antisense gene expression. Their By contrast, in the presence of the inducer many shoots results revealed an important aspect of the CDC2b func- regenerated, which were developmentally abnormal, for tion in the light- and hormone-regulated seedling growth example, inhibition of root growth, the loss of apical dom- inance and sterility. These shoots were transferred to amedium without the inducer, and after several weeks nor- Conditional genetic complementation
mal plants developed, most of which were found to be Plant genes that affect development at an early stage (e.g.
transgenic. This work demonstrated the feasibility and embryogenesis) may also play a role in later stages of potential of using a chemical-inducible system to regulate development. Mutations in such genes arrest early devel- expression of genes that promote plant development.
opment, thus precluding investigations on their possiblelate functions. Such a mutant can be transformed with the DNA manipulations
appropriate coding sequence under the control of a chem- Chemical-inducible systems can be used to activate specif- ical-inducible system. In the presence of the inducer, ic recombinases (e.g. cre and flipase) for nuclear DNA transgenic plants will be able to undergo early develop- remodeling in transgenic plants (see  for more compre- ment. Withdrawal of the inducer at a later time will allow hensive discussions). Depending on the configuration of evaluation of the late functions of the gene product.
the recombinase binding sites, the target DNA can beinverted or evicted resulting in activation or inactivation of Identification of downstream target genes
transgenes (S Moller, N-H Chua, unpublished data).
Chemical-inducible systems provide a very important tool Organelle DNA can be similarly manipulated with a recom- for investigating the sequence of events that ensue upon binase appended with the appropriate transit sequences.
transient perturbation of the gene under their control. Basedon theoretical considerations, we can expect two classes of Generation of chimeric plants
downstream target genes: firstly, primary response genes, Specific organs of transgenic plants carrying a chemical- the expression levels of which are effected in the absence of inducible system can be treated with the appropriate new protein synthesis; and secondly, secondary response inducer to activate gene expression only in the treated genes, which require new protein synthesis to change their organ. In this case, however, the induced expression is only transcription rates. Identification of genes from the first class transient. On the other hand, using a chemical-inducible requires that the regulatory gene product itself (transcrip- cre/lox system one can create transgenic plants that are tion factor, kinases, etc.) be directly placed under chemical genetic chimera; this can be done by a confined treatment control. In the case of a transcription factor, fusing a steroid of organs/tissues with an inducer, thereby permanently regulatory domain to it will render the activity of the fusion activating or inactivating the transgene in the treated protein dependent on the appropriate steroid. In the pres- organs/tissues. The resulting genetic chimera may provide ence of the inducer and a protein synthesis inhibitor, only new information on the mechanisms of long distance sig- the primary response genes are activated, which can be identified by methods such as differential display. An exam-ple of this is the identification of a NAM-like gene as an Ablation of specific cells
immediate downstream target of AP3 . More recently, Cell ablation is an important tool for investigating cell Bruce et al. [11•] used an ER–C1 chimeric factor to condi- fate specification, cell lineage and cell–cell interactions tionally overexpress two transcription factors, CRC (a fusion during plant development. Traditionally, this is carried factor between C1 and R) and P, which are believed to be out by laser ablation of a cell or a group of cells in ques- involved in the flavonoid pathway, thereby identifying a tion, followed by tracking the effects of ablation on large number of downstream target genes.
development of other cells (reviewed in [21,22]). Thistechnique, requiring an expensive and highly specialized Marker-free transformation
facility, however, can be replaced by fusing a cell-specif- A novel use of chemical-inducible systems has been ic promoter to a toxic gene (e.g. dipthera toxin) and the reported by Kunkel et al. [19••], who were able to select cells are killed at the onset of the specific promoter Plant biotechnology
activity. The incorporation of a chemical-inducible sys- merits are difficult to assess at this time. In general, VP16 tem will allow one to investigate cell fate and lineage at appears to work well in a number of species.
The regulatory domain not only confers tight control and Conclusions and future challenges
high inducibility to a system, but also provides the diversity The development of chemical-inducible systems for tight of inducers that can be used. A regulatory domain with a high control of plant gene expression is a challenging task and is binding affinity to its cognate inducer is preferable, as only the subject of considerable current activities. Because the low concentrations of inducer would be required for activa- components for such systems are usually derived from tion. For example, the hormone-binding domain of the ER non-plant sources, progress in this area depends to a large binds estradiol with a very high affinity of 0.05 nM  as extent on discoveries of chemical-responsive transcription compared to ~10 nM for binding of dex to the GR . In factors in other organisms. Based on the published exam- theory, the regulatory domains of many mammalian nuclear ples, some general rules have evolved that would be receptors (e.g. thyroid hormone, androgen, vitamin D3, and helpful to the future development of new inducible sys- peroxisome-proliferator-activated receptors) can be incorpo- tems. For expression of the target gene, the 35S minimal rated into chemical-inducible systems for plants. Several of promoter was used in all cases, which ranges in length from them may not be suitable, however, because they bind to –60 to –31 at the distal end of the promoter. A longer min- chemicals present in plants (e.g. peroxisome-proliferator- imal promoter (e.g. up to –60) [12••] will enhance the activated receptors bind to linelenic and linoleic acids, and overall promoter strength but it will also lead to a higher androgen receptors may bind to intermediates of the brassi- basal expression level. On the other hand, a shorter mini- nosteroid biosynthetic pathway) . In some cases, an mal promoter (e.g. –31 to +1)  has the opposite effect.
inducible system that works in one species may not function In most cases (e.g. the GVG, XVE and TGV systems), in others. It is therefore prudent to first test the activity of truncations to around –46 to –48 appear to be optimal for candidate regulatory domains in plant cells before using low background activity and high inducibility.
them as a component of the chimeric transcription factor. The transactivator tTA binds to tet constitutively but this As mentioned before, most of the systems reported thus far interaction is disrupted by tetracycline or its derivatives.
are unsuitable for field applications because of the chemi- By mutagenesis, tTA can be converted into the so-called cal nature of the inducers. Further work should focus on reverse tTA (rtTA), which now requires the association of systems suitable for applications with transgenic crop tetracycline for DNA binding . Baron et al.  devel- plants, with particular emphasis on agricultural chemicals oped a new system that co-expresses tTA and rtTA in (e.g. insecticides and safeners; the latter chemicals are HeLa cells; the DNA-binding domain of the latter was also used in agriculture to render crops tolerate to herbicides) modified in order to recognize a variant tet operator that have already been registered for field usage. An addi- sequence upon association of doxycycline. This new sys- tional interest would be to develop multiple-inducible tem allows one to reversibly control the expression of two systems to independently regulate several target genes.
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