Chemically inducible promoters in transgenic plants

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Chemically inducible promoters in transgenic plants Christiane Gatz Chemicals such as tetracycline, dexamethasone, copper, salicylic acid and herbicide safeners have been explored as effector molecules to regulate the expression of transgenes in higher plants. The tetracycline-inducible promoter is, at present, the most advanced system and has already been used to study the function of a family of transcription factors in vivo. Dexamethasone inducible gene expression has been employed for cell-fate mapping in Arabidopsis thaliana. In addition, salicylic acid, which induces genes involved in systemic acquired resistance against pathogens, has been used to elicit protection against insect feeding in transgenic plants.

Address Institut fur Genetik, Universit~it Bielefeld, 33501 Bielefeld, Germany; e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:168-172 © Current Biology Ltd ISSN 0958-1669 Abbreviations CaMV cauliflower mosaic virus GR glucocorticoid receptor GST glutathione S-transferase IPTG isopropyl-I~-D-thiogalactopyranoside PR pat hogenesis-related rtTA reverse tTA tTA tetracycline-controlled transactivator VP16 virus protein 16

Introduction Systems that allow the regulation of gene expression by chemical stimuli offer great advantages both for studying gene function in vivo and for biotechnological applications. T h e properties of such systems are tailored to meet the requirements of their applications. For research experiments in the laboratory, tight control and high specificity of the inducing agent are important. For field experiments, the chemical should be cheap, easy to apply and environmentally compatible. Depending on the gene product, compromises can be made in terms of specificity and 'leakiness' of expression. This review focuses on chemically inducible promoters that can be used for either of the above purposes.

Chemically inducible promoters as tools to study gene function in vivo One of the most efficient ways of studying gene function in vivo is the analysis of phenotypes that arise when the level of a given gene product is altered. This can be achieved either by isolating mutants or by constructing transgenic plants. Expression of antisense RNAs or dominant-negative proteins leads to a reduction in the

amount of a functional gene product. Enhanced expression of a gene product may provide valuable information from gain-of-function experiments, and expression of a gene product with altered molecular properties allows new insights into cellular control mechanisms. T h e ability to control the activity of a gene in a reversible and temporally defined manner renders the above-mentioned concept of studying genc function in vivo more efficient. Use of such a system is a necessity if expression of the transgene interferes with the regeneration process. Moreover, a regulatable promoter not only offers the opportunity to study the function of a gene product at different stages of development, but also enables the correlation of the phenotype with the kinetics of induction, thereby allowing differentiation between primary and secondary effects of transgene expression. Position effects and somaclonal variation, which can also give rise to an altered phenotype, can be excluded when the phenotype is visible only after induction of the transgenc. Ideally, a chemically inducible promoter should have a low background activity in the absence of the inducer and demonstrate high expression in the presence of the inducer. This is, however, not an absolute requirement. For some experiments, low background activity and only moderate expression of the transgene is required; for other experiments, low background activity can be tolerated, but high expression in the induced state is a prerequisite. In any case, the inducer should affect expression only of the transgene. This last requirement renders endogenous promoters unsuitable and favours the use of well characterized regulatory elements from organisms distant in evolution, such as yeast, Escherichia coli, Drosophila or mammalian cells, that respond to chemical signals that are usually not encountered by higher plants. On this basis, two different concepts to gene control can be realized, namely, promoter-repressing systems and promoter-activating systems.

Promoter-repressing systems

T h e repression principle is based on the sterical interference of a repressor protein with proteins important for transcription. It is a common mechanism in bacteria, but occurs much less frequently in higher eukaryotes, where protein-protein interactions mediate stimulation or inhibitory effects on the transcription machinery. Two bacterial repressor/operator systems (Lac and "let) have been employed to control the activity of RNA polymerase II promoters, according to the prokaryotic paradigm. The first paper reporting the successful use of a bacterial repressor protein to control the activity of a modified

Chemically inducible promoters in transgenic plants Gatz 169

cauliflower mosaic virus (CaMV) 35S promoter in transient assays appeared in 1988 [1]. T h e Tnl0-encoded Tet repressor was used in this study, the DNA-binding activity of which is abolished by very low amounts of the antibiotic tetracycline [2]. As tetracycline readily enters eukaryotic cells without the need for a specific uptake system, it is a suitable chemical inducer for laboratory experiments. Systematic analysis of the effect of repressor-operator complexes in different positions within the CaMV 35S promoter [3,4] led to the design of a tightly repressible CaMV 35S promoter that contains one tet operator directly upstream of the TATA-box and two tet operators downstream of the TATA-box [5]. Repression depends on high intracellular repressor concentrations [6] as the repressor has to compete with at least 40 proteins that assemble around the TATA-box to form a competent transcription initiation complex. In tobacco, the expression of the tetracycline-inducible promoter can be modulated several hundred fold [5]. Induction at the whole plant level is most efficient in plants growing in hydroponic culture with tetracycline added to the nutrient solution [7]. Regulation is stringent enough to completely suppress the rolB phenotype, which is chlorosis and inhibition of growth ([8°]; Fig. 1). T h e system has also been used to express a dominant-negative mutant of the TGA-family of transcription factors in transgenic tobacco plants [9°]. In addition, it has been shown to work in potato (R H6fgen, L Willmitzer, personal communication) and tomato (A Thompson, B Thomas, personal communication), but thus far, has not been successfully established in Arabidopsis thaliana. At present, it seems that sufficiently high repressor concentrations cannot be tolerated in Arabidopsis, a phenomenon that has also been reported for mammalian cells [10]. Transgenic tomato plants that overexpress the bacterial Tet repressor have been shown to grow more slowly under optimal growth conditions than controls. This effect can be reversed with tetracycline, indicating that the repressor is harmful only in its DNA-binding conformation (A Thompson, B Thomas, personal communication). Early efforts to use the Lac repressor/operator system to establish isopropyl-13-1)-thiogalactopyranoside (IPTG) inducible transcription gave promising results [11]. Lac operators were inserted into the vicinity of the TATA box of the promoter for chlorophyll a/b binding protein, CAB. Though the constructs were stably integrated into the genome of tobacco plants, induction was not done at the whole plant level, but only in protoplasts.

Promoter-activating systems

A different approach for the construction of a chemically inducible system for higher plants is to use transcriptional activators from other higher eukaryotes. T h e mammalian glucocorticoid receptor (GR), which activates eukaryotic transcription only in the presence of steroids like dex-

Figure 1

Isogenic cuttings of transgenic tobacco plants encoding the rolB gene from Agrobacterium rhizogenes under the control of the tetracycline-inducible promoter. Only the left plant was treated with tetracycline. In the absence of tetracycline (right plant), the rol B phenotype is completely suppressed, leading to 'normal' growth. The picture was taken 5 weeks after the onset of induction.

amethasone, has been used previously to establish a regulatory system in Schizosaccharomyces pombe [12]. In transiently transformed tobacco cells, transcription of a target promoter containing GR-binding sites upstream of a TATA-box was shown to be strictly dependent on the presence of dexamethasone [13]. In stably transformed Arabiclopsis plants, however, this arrangement of regulatory elements does not work [14"']. However, if the ligand-binding domain of the receptor is fused to maize transcription factor R, a steroid-inducible receptor, R-GR, is generated [14°°]. T h e regulation is based on the retention of the R - G R fusion protein in the cytoplasm resulting from the interaction of the steroid-binding domain with heat-shock protein Hsp70. Lloyd et al. [14 °°] have transformed the R - G R fusion protein into an Arabidopsis mutant (ttg) that can be complemented with maize transcription factor R. R - G R allowed steroid-inducible complementation of the phenotype and was used for epidermal cell fate mapping experiments. Thus, fusing the steroid-binding domain to transcriptional activators that would recognize a binding site absent in plant promoters might be a sensible way to construct a steroid inducible expression system. Another eukaryotic ligand-dependent activator is ACE1, a copper-dependent transcriptional activator from yeast. Mett etal. [15] have shown that ACE1 regulates transcription of a suitable target promoter in a copper-dependent manner in transgenic plants. As copper is likely to affect other processes in plants, the specificity of this inducer remains to be established. A third strategy is based on the construction of fusion proteins between transcriptional transactivation domains and bacterial repressor proteins such as the Lac repressor

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or the Tet repressor. T h e favourable thermodynamic properties of the interaction between the Tet repressor/operator and tetracycline prompted Gossen et al. [16] to construct a tetracycline-controlled transactivator (tTA) by fusing the virus protein 16 (VP16) activation domain to the carboxyl terminus of TetR. From a target promoter containing seven tet operators upstream of a minimal promoter (i.e. sequences between -53 and +75 of the human cytomegalovirus promoter), tTA is able to regulate gene expression over a range of five orders of magnitudes in stably transformed H e L a cells. T h e same principle has been demonstrated to work in transgenic tobac~:o plants [17"], thus establishing a promoter system that can be shut off in the presence of tetracycline. T h e possibility of switching-off transcription from one specific promoter should allow analysis of mRNA or encoded protein decay rates for individual genes. Thus, the tTA-dependent promoter might provide a good alternative to the use of general inhibitors such as actinomycin D and cycloheximide. As the tTA-based system has not been optimized as thoroughly as the tetracycline-inducible promoter, its potential has not yet been fulfilled. Expression levels reach 30% of the levels reached by the inducible system, and, more seriously, expression levels drop as transgenics grow older. T h e tTA system has also been successfully transferred into Arabidopsis (M Roever, U Treicheh, C Gatz, J Schiemann, R Hehl, unpublished data) and the moss Physcomytrella patens [18], where silencing of gene expression does not seem to occur. As Gossen etal. [10] and our group [17 °] discuss in more detail, stringent control of transcription in higher eukaryotic systems is achieved by promoter activation, rather than by repression. Nonetheless, regulatory control over a range of three orders of magnitude has been achieved in trypanosomes based on the repression principle, again using the bacterial Tet repressor [19]. T h e tTA system is definitely intriguing because of its low background activity in the presence of tetracycline. However, one of the disadvantages of this system is that plants have to be permanently cultivated on tetracycline to keep the transgene silent, which is time-consuming given the instability of tetracycline in the light. In order to induce expression of the transgene, plants must be transferred to tetracycline-free medium. Inactivation of tetracycline is slower than uptake of tetracycline, thus rendering the induction kinetics less favourable. A promising alternative to the above system is a mutant "let repressor that shows a 'reverse phenotype'. This reverse repressor binds DNA only in the presence of tetracycline. By fusing this repressor derivative to the VP16 activation domain to form a reverse tTA (rtTA), Gossen et al. [20] have been able to develop a tetracycline-inducible promoter system that is based on the activation system. Preliminary experiments indicate, however, that rtTA does not work in transgenic tobacco

or transgenic Arabidopsis plants (C Gatz, HM Rupp, T Schmtilling, unpublished data).

Endogenous chemically inducible promoters Several chemically inducible promoters from various plant species are currently available. Chemicals such as hormones, nitrate, carbohydrates, and elicitors of plant defence responses can be used to stimulate gene expression. T h e corresponding promoters are not suitable for studying gene function in vivo, however, because of the pleiotropic effects they exert. For applied purposes, substances that alter growth and development (e.g. hormones) can be excluded as potential inducers. On the other hand, signals such as salicylic acid derivatives, elicitors or safeners (see below) are potentially useful. If such a chemical were used to induce a transgene in the field, the simultaneous induction of plant defence genes might even be beneficial for crop protection. Salicylic acid is involved in the coordinate induction of genes involved in the onset of systemic acquired resistance, an inducible defence system that combats pathogen infection. Induction of gene expression has been analyzed using chimaeric genes comprising a reporter gene fused with the promoters of the genes for pathogenesis-related (PR) proteins PR-la, PR-2, PR-3, PR-5 and a glycine-rich protein [21]. T h e best studied promoter is the PR-la promoter from tobacco. Plants treated with salicylic acid exhibit an impressive increase of PR-la mRNA or 13-glucuronidase mRNA (i.e. in transgenic plants encoding the 13-glucuronidase gene under the control of the PR-la promoter) I22,23]. When 13-glucuronidase enzyme activities were measured, background levels were between 0.1 pmol min -I (mgprotein) -1 and 100pmolmin-l(mgprotein) -1 which is similar to background levels obtained using the tetracycline-inducible promoter mentioned above. Even so, salicylic acid treatment led to only a 10-fold increase in 13-glucuronidase activity, which does not correspond to the massive induction at the RNA level. T h e PR-la promoter has been used to induce Baci//us thunngiensis 5-endotoxin expression in transgenic plants [24]. As a matter of fact, insect feeding damage was inhibited in plants that had been pre-treated with a salicylic acid derivative [P1], but not in untreated isogenic lines. From this experiment, it can be concluded that the window between the uninduced and the induced state is suitable for applied purposes. In laboratory experiments, if salicylic acid or derivatives are used as inducers, it should be taken into account that PR genes, chitinases, glucanases, catalases, stress proteins, cyclophilines, glutathione S-transferases (GSTs), alternative oxidases and manganese superoxide dismutase are all induced as well [21]. Using a 273 bp promoter fragment of a defence gene from potato (prop1-1) to drive the barnase gene from Baci//us amylo/iquefaciens, Strittmatter eta/. [25] have been able to

Chemically inducible promoters in transgenic plants Gatz

induce necrotic lesions after infection of transgenic plants with Phytophthora infestans. Tissue destruction could also be observed after the plants were treated with ethylene. T h e promoter fragment is not responsive to abiotic stimuli and it is not active in any non-infected tissue except the root tip. Barnase activity resulting from basal expression was inactivated by co-expression of the specific barnase inhibitor, barstar. Safeners are commonly used to prevent plant injury from pre-emergent herbicides, such as chloroacetanilides, and are thus substances routinely released into the environment. T h e y act primarily by elevating herbicide metabolism via increased G S T activity. T h e promoters of different G S T genes show different levels of background expression and responsiveness to different stimuli [26,27]. Transcripts corresponding to GST-27 from maize were visible only after application of herbicides [27]. A range of hormonal, environmental and physiological stimuli failed to elevate GST-27 levels, demonstrating that the inducibility of this promoter is relatively specific. Nevertheless, expression was constitutive in roots and an increase of expression was observed in the late stages of leaf senescence. In contrast, the soybean GH2/4 gene, which also encodes a GST, is activated by a wide range of chemical agents (heavy metals, active and inactive auxines, salicylic acid etc.) as well as by elevated temperatures [26]; thus, its activity might be accidentally increased under field conditions.

Concluding remarks T h e value of any system for controlling gene activity is ultimately judged by its applicability. For studying gene function in vivo, the 'ideal' system has not yet been determined. T h e tetracycline-dependent expression systems are the most advanced at this time, and they have already been used for research purposes [8",9"]. T h e tetracycline-inducible system is too 'leaky' for experiments involving genes with a higher lethality than the rolB gene, however. A second major drawback of this system is that it does not work in Arabidopsis. T h e tetracycline 'inactivatable' system (i.e. tTA based expression) suffers from unfavourablc kinetics with regard to the induction process. Nonetheless, further improvements to systems utilizing Tet regulatory molecules can be envisioned, and these are presently under study in our laboratory.

References and recommended reading

2.

Hillen W, Berens C: Mechanisms underlying expression of TnlO encoded tetracycline resistance. Annu Rev Microbio/1994, 48:345-369.

3.

Frohberg C, Heins L, Gatz C: Characterization of the interaction of plant transcription factors using a bacterial repressor protein. Proc Nat/Acad Sci USA 1991, 88:10470-104?4.

4.

Heins L, Frohberg C, Gatz C: The TnlO encoded "let repressor blocks early but not late steps of assembly of the RNA polymerase II inititaUon complex in vivo. Mol Gen Genet 1992, 232:328-331.

5.

Gatz C, Frohberg C, Wendenburg R: Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. P/ant J 1992, 2:397-404.

6.

Gatz C, Kaiser A, Wendenburg R: Regulation of a modified CaMV 35S promoter by the Tnl0-encoded-Tet repressor in transgenic tobacco. Mo/Gen Genet 1991, 227:229-237

7.

Gatz C: Novel inducible/repressible gene expression systems. Methods Cell Bio11995, 50:411-424.

8. •

R6der FT, Schm~Jlling T, Gatz C: Efficiency of the tetracycline dependent gene expression system-complete suppression and efficient induction of the RolB phenotype in transgenic plants. Mo/Gen Genet 1994, 243:32-38. The rolB gene is studied as an example of a transgene that, when highly expressed, interferes with the regeneration process. The tetracycline-inducible promoter is shown to exhibit sufficiently tight control to suppress growth inhibition and necrosis; the application of tetracycline induces a severe phenotype. 9. •

Rieping M, Fritz M, Prat S, Gatz C: A dominant negative mutant of PG13 suppresses transcription from a cauliflower mosaic virus 35S truncated promoter in transgenic tobacco plants. Plant Cell 1994, 6:1087-1098. Expression of a dominant-negative mutant of transcription factor PG13, which leads to reduced amounts of DNA-binding complex ASF-1 in transgenic plants, is placed under the control of the tetracycline-inducible promoter. Use of the tetracycline regulatory system helps to show that the activity of the truncated CaMV 355 promoter is dependent on the presence of ASF-1. Low expression of the truncated CaMV 35S promoter does not result from position effects in these plants. In the presence of tetracycline, expression is low because the dominant-negative PG 13 derivative is induced. In the absence of tetracycline, expression is high because the dominant-negative PG13 mutant is not expressed. 10.

Gossen M, Bonin AL, Bujard H: Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci 1993, 18:471-475.

11.

Wilde RJ, Shufflebottom E, Cooke S, Jasinska I, Merryweather A, Beri R, Brammar WJ, Bevan MW, Schuch W: Control of gene expression in tobacco cells using a bacterial operatorrepressor system. EMBO J 1992, 11:1251-1259.

12.

Picard D, Schena M, Yamamoto R: An inducible expression vector for both fission and budding yeast. Gene 1990, 257-261.

13.

Schena M, Lloyd AM, Davis RW: A steroid-inducible gene expression system for plant cells. Proc Nat/Acad Sci USA 1991, 88:10421-10425.

14. ••

Lloyd AM, Schena M, Walbot V, Davis RW: Epidermal cell fate determination in Arabidopsis: patterns defined by a steroidinducible regulator. Science 1994, 266:436-439. A fusion protein consisting of maize transcriptional activator R and the ligandbinding domain of GR is expressed in the Arabidopsis mutant ttg. Thus, an Arabidopsis plant is created containing a steroid hormone depend•n1 conditional allele of R. The response of the chimaeric protein to pulses ot hormone is used to define the pattern and timing of trichorne formation on the developing leaf epidermis. 15.

Mett VL, Lochhead LP, Reynolds PHS: Copper controllable gene expression system for whole plants. Proc Nat/Acad Sci USA 1993, 90:4567-4571.

of special interest of outstanding interest

16.

Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Nat/Acad Sci USA 1992, 89:5547-5551.

Gatz C, Quail PH: Tnl0-encoded tet repressor can regulate an operator-containing plant promoter. Proc Nat/Acad Sci USA 1988.85:1394-1397,

17. •

Weinmann P, Gossen M, Hillen W, Bujard H, Gatz C: A chimeric transactivator allows tetracycline-responsive gene expression in whole olants. P/ant J 1094. 5:559-569.

Papers of particular interest, published within the annual period of review, have been highlighted as: • •*

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This work describes the function of the tTA system in transgenic plants. The advantages and disadvantages of the two tetracycline-dependent expression systems are discussed. 18.

19.

24.

Zeidler M, Gatz C, Hartmann E, Hughes J: Tetracycline regulated reporter gene expression in the moss Physcomytrella patens. P/ant Mo/ Bio/1996, in press.

Williams S, Frieddch L, Dincher S, Carozzi N, Kessmann H, Ward E, Ryals J: Chemical regulation of Bacillus thuringiensis 5-endotoxin expression in transgenic plants. Biotechno/ogy 1992, 10:540-543.

25.

Wirtz E, Clayton C: Inducible 9ene expression in trypanosomes mediated by a prokaryoUc repressor. Science 1995, 268:1179-1183.

Strittmatter G, Janssens J, Opsomer C, Botterman J: Inhibition of fungal disease development in plants by engineering controlled cell death. Biotechno/ogy 1995, 13:1085-1089.

26.

Ulmasov 1", Ohmiya A, Hagen G, Guilfoyle T: The soybean GH2/4 gene that encodes a gluthatlone S-transferase has a promoter that is activated by a wide range of chemical agents. P/ant Physiol 1995, 108:919-927.

27.

Jepson I, Lay VJ, Holt DC, Bright SWJ, Greenland AJ: Cloning and characterization of maize herbicide safener-induced cDNAs encoding subunits of gluthatione S-transferase isoforms I, II and IV. P/ant Mo/Bio/1994, 26:1855-1866.

20.

Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracycline in mammalian cells. Science 1995, 268:1766-1769.

21.

Klessig DF, Malamy J: The salicylic acid signal in plants. P/ant Mol Biol 1994, 126:1439-1458.

22.

Van de Rhee MD, Van Kan JAL, Gonz&lez-Jaen MT, Bol JF: Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection. P/ant Cel/1990, 2:357-366.

23.

Uknes S, Dincher S, Friedrich L, Negrotto D, Williams S, Thompson-Taylor T, Potter S, Ward E, Ryals J: Regulation of pathogenesis-related protein-la gene expression in tobacco. P/ant Ge//1993, 5:159-169.

Patents • of special interest oo of outstanding interest PI.

Ryals J, Harms C, Duesing J, Sperisen C, Meins F, Payne G: Chemically regulatable DNA sequence and genes and uses thereof. 1990 EPO332104A2.