HaloTag : a new versatile reporter gene system in plant cells

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plant cells. The ligand-specific labelling of HaloTag protein was not hampered by the plant cell wall. Key words: cLSM, HaloTagTM, particle bombardment, proto-.
Journal of Experimental Botany, Vol. 57, No. 12, pp. 2985–2992, 2006 doi:10.1093/jxb/erl065 Advance Access publication 26 July, 2006

RESEARCH PAPER

HaloTagTM: a new versatile reporter gene system in plant cells Christina Lang, Jutta Schulze, Ralf-R. Mendel and Robert Ha¨nsch* Institut fu¨r Pflanzenbiologie, Technische Universita¨t Braunschweig, Humboldtstraße 1, D-38106 Braunschweig, Germany Received 20 March 2006; Accepted 23 May 2006

Abstract HaloTagTM Interchangeable Labeling Technology (HaloTag) was originally developed for mammalian cell analysis. In this report, the use of HaloTag is demonstrated in plant cells for the first time. This system allows different fluorescent colours to be used to visualize the localization of the non-fluorescent HaloTag protein within living cells. A vector was constructed which expresses the HaloTag protein under the control of the 35S promoter of cauliflower mosaic virus. The functionality of the HaloTag construct was tested in transient assays by (i) transforming tobacco protoplasts and (ii) using biolistic transformation of intact leaf cells of tobacco and poplar plants. Two to fourteen days after transformation, the plant material was incubated with ligands specific for labelling the HaloTag protein, and fluorescence was visualized by confocal laser scanning microscopy. The results demonstrate that HaloTag technology is a flexible system which generates efficient fluorescence in different types of plant cells. The ligand-specific labelling of HaloTag protein was not hampered by the plant cell wall. Key words: cLSM, HaloTagTM, particle bombardment, protoplasts, reporter gene expression.

Introduction Labelling of proteins is an important tool in the study of their functions and dynamics in living cells (Giuliano and Taylor, 1998). The introduction of the fluorescent protein GFP (green fluorescent protein) and its derivatives has been a great breakthrough in cell biology. GFP is widely used as

an extremely powerful vital marker in a large number of organisms (Chalfie et al., 1994; Sheen et al., 1995; Zimmer, 2002), and labelling of proteins by genetic fusion has extended our understanding of protein function in the last decade. Fluorescent proteins as reporter genes have the primary advantage that their in vivo assay requires neither long sample preparation nor the uptake of exogenous substrates, as compared with alternatives such as luciferase or b-glucuronidase (Haseloff and Amos, 1995; for a review see Hanson and Ko¨hler, 2001). Moreover, these proteins can be expressed and monitored within intact tissues, cells, or cell organelles without any destruction of the material. To this end, more than 40 variants of the original jellyfish GFP have been constructed which fall into seven main classes of excitation and emission spectra (Palm and Wlodower, 1999). These different colours can be used to study protein localization and interaction especially in colocalization experiments with double or multi-labelling of cells. Here, the different GFP colours have to be fused to the proteins of interest prior to the transformation experiment. Yet, experience shows that some of the proteins have to be labelled with more than one of the GFP variants to give a clear and distinguishable image under the confocal laser scanning microscope (cLSM). The new HaloTagTM Interchangeable Labeling Technology, developed for mammalian cells (Promega, Mannheim, Germany), introduces a new flexibility to fluorescence microscopy. Instead of a gene for a fluorescent protein, a cDNA encoding a nonfluorescent HaloTag protein or protein fusion is introduced into cells either by transient transformation or generation of stably transformed cell lines. By contrast to commonly used fluorescent proteins, HaloTag proteins are expressed as monomers. These cells are briefly incubated with an appropriate HaloTag ligand which readily crosses the cell

* To whom correspondence should be addressed. E-mail: [email protected] Abbreviations: CaMV, cauliflower mosaic virus; cLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; PEG, polyethylene glycol. ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]

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membrane. The HaloTag ligand harbours a reactive linker that covalently binds to the HaloTag protein and a flexible reporter group that can be a fluorophore. Unbound ligand is washed out and fluorescence can be detected in living or fixed cells (Los et al., 2005). Since the HaloTag protein is of prokaryotic origin, endogenous activities are not detectable in mammalian cells. The covalent bond is highly specific and essentially irreversible, yielding a complex that is stable even under denaturing conditions (Technical Manual; Promega, 2005). Like other fluorescent proteins, HaloTag protein can be fused in-frame with any protein or peptide sequence of interest in N- and C-terminal orientation without disturbing their function, so that physiological processes including cellular and subcellular activities, protein trafficking, and protein interactions can be studied in vivo and noninvasively over longer time periods (Los et al., 2005). As shown for animal systems, cells expressing the HaloTag protein and labelled with the HaloTag TMR (tetramethyl rhodamine which is red with an excitation peak of 555 nm and a fluorescence emission peak of 585 nm) ligand, the diAcFAM (diacetyl derivative of fluorescein which is green: 494Ex/526Em) ligand, or the coumarin (blue: 353Ex/ 434Em) ligand are brightly fluorescent. By contrast, cells that do not express the HaloTag protein show no detectable fluorescence under the same labelling and imaging conditions. The HaloTag ligands have no toxicity or morphological side-effects in the cell lines tested (Los et al., 2005). Besides fluorescence labelling, HaloTag technology gives other additional functionalities with a ligand containing biotin which is suitable for use as an affinity tag to capture the protein of interest (Los et al., 2005). In this paper, the use of the HaloTag technology was evaluated for localization experiments in plant cells. Expression of the HaloTag protein was visualized in tobacco and poplar cells followed by staining with the HaloTag TMR and diAcFAM ligands. HaloTag technology was found to be a flexible system generating efficient fluorescence in different cell types of plants.

Materials and methods Plasmid construction and purification All restriction endonuclease and ligase reactions were performed using the buffer conditions recommended by their respective manufacturers using standard techniques (Sambrook et al., 1989). The cDNA of HaloTag was amplified from pHT2-vector (GenBank Accession number AY773970, kindly provided by Promega) using Taq-DNA polymerase (Peqlab, Erlangen, Germany), with the following primer set: forward primer 59-tcg gat ccA TGg gat cag aaa tcg gta c-39 reverse primer 59-tag cat gct aTT Agc cgg cca gcc cgg-39. The PCR product was cloned into pGEMTeasy (Promega) and sequenced. After cutting with BamHI and SphI, the resulting fragment (902 bp) was transferred into the pFF19-vector (Timmermans et al.,

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1990) to create pFF19-HT. This vector harbours the cauliflower mosaic virus (CaMV)-35S promoter with double enhancer and the poly(A) sequence from CaMV-35S. Plasmid-DNA was prepared with Qiagen Plasmid Midi-Kit (Qiagen, Hilden, Germany). Co-transformation of either pFF-GFP-PTS1 (Nowak et al., 2004) or pGreen0229:MPmRFP (Hellens et al., 2000), kindly provided by Dr S Chapman (SCRI, Norwich, UK), was used to distinguish between transformed and non-transformed cells. Plant material Experiments were performed with in vitro-grown cultures of Nicotiana plumbaginifolia and Populus tremula3Populus alba (No. 7171-B4; Institut de la Recherche Agronomique, INRA, France) grown on modified MS-based medium (Murashige and Skoog, 1962; Lloyd and McCown, 1980). The soil-grown plants of N. tabacum and N. benthamiana were cultivated in controlled environment chambers (Hereaus-Vo¨tsch, HPS 1500, Balingen, Germany) with a 14 h light (300 lE mÿ2 sÿ1)/10 h dark period and relative humidity of 80%. Isolation and transformation of protoplasts In vitro shoot cultures of Nicotiana plumbaginifolia were used for protoplast isolation. Plants were maintained on MS medium (Murashige and Skoog, 1962) without growth regulators at 23 8C and 25 lE mÿ2 sÿ1 under a 16 h light/8 h dark regime. Mesophyll protoplasts were isolated from 2–3-month-old plants after overnight digestion of leaves in 0.6% w/v Onozuka Cellulase R10 (Duchefa, Haarlem, The Netherlands) and 0.2% w/v Macerocyme (Duchefa) dissolved in T0medium (Crepy et al., 1982) but omitting Tween. For transformation, 13106 protoplasts were resuspended per 1 ml MaMg solution (Negrutiu et al., 1987). Aliquots of 500 ll were incubated at 45 8C for 5 min, cooled on ice for 30 s followed by addition of 20 ll plasmid DNA (1 lg llÿ1) and 520 ll polyethylene glycol (PEG) solution containing 0.1 M Ca(NO3)2, 0.4 M mannitol, and 30% PEG 4000 (Merck, Darmstadt, Germany). After 20 min incubation at room temperature, the PEG solution was removed with washing solution W5 (Negrutiu et al., 1987) by centrifugation at 80 g for 5 min. Protoplasts were cultured at a density of 13105 mlÿ1 in liquid T0 medium supplemented with 5 mM glutamine in the dark at 25 8C using 6-cm-diameter Petri dishes. Transformation of leaves using biolistics Fully developed leaves of tobacco as well as poplar plants grown in soil or in vitro were harvested; leaf discs (3 cm in diameter) were cut with a metal punch and placed upside down on water-soaked filter paper in Petri dishes. Coating of gold particles with plasmid DNA was carried out as described earlier (Koprek et al., 1996). The transformation was performed with the Particle Delivery System PDS-1000 (Bio-Rad, Munich, Germany) using pressures of 350 and 700 psi and a distance of 45 and 75 mm between the macrocarrier and the target tissue. Plant material was incubated in low light conditions at 25 8C. First analyses were done 24 h post-bombardment. Staining of plant material with Calcoflour White, TMR and diAcFAM ligands After checking the transformation efficiency using GFP or monomeric red fluorescent protein (mRFP), the plant material was stained as follows: transformed protoplasts or protoplast-derived cultures 2– 14 d after transformation were collected by centrifugation at 80 g for 5 min. The pellet was resuspended in 200 ll of W5 and 200 ll of 2-fold concentrated ligand solution dissolved in W5 was added with a final concentration of 0.2, 1.0, and 5.0 lM. After incubation for 15–60 min in the dark, the staining solution was carefully removed by washing twice with 10 ml W5, and protoplasts were resuspended in 200 ll W5. The presence of a new cell wall was

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determined using the fluorescent brightener Calcofluor White (Sigma, Deisenhofen, Germany) as described by Nagata and Takebe (1970). One volume of 0.1% w/v Calcofluor White dissolved in W5 was added to the samples and analyses were carried out with cLSM. Selected leaf areas or stripped lower epidermis of transformed leaves were incubated for 30 min in 1.0 lM ligand solution diluted in water. Careful washing for at least 4 h up to overnight in water was necessary to reduce unspecific background. Microscopic detection Reporter gene expression was visualized with the confocal laser scanning microscope cLSM-510META (Carl Zeiss, Go¨ttingen, Germany) using a sample of transformed protoplasts, selected leaf areas, or the stripped lower epidermis of leaves. The specimens were examined either using the Plan-Neofluar 103/0.3 or the C-Apochromat 403/1.2 water-immersion objectives. For confocal microscopy, the argon laser (488 nm line for GFP, diAcFAM, and chlorophyll autofluorescence) and the helium laser (543 nm) for mRFP and TMR were used. The emitted light was detected as follows: with the bandpass filter GFP (505–530 nm), diAcFAM (505–550 nm), TMR (560– 615 nm), and mRFP (560–615 nm), and with the META-channel the chlorophyll autofluorescence. Additionally, a UV-laser (364 nm) was used to detect Calcofluor White; primary beam splitting mirrors UV/488/543/633 nm, emitted light was detected with a band pass filter 385–470 nm using the Multitracking mode. If necessary, the bright field of samples was taken with the transmitted light photomultiplier, and the lambda mode was used to examine the spectral signature of the fluorochromes.

Results and discussion

Fig. 1. HaloTag transformation vector pFF19-HT: pBluescript-based high copy plasmid carrying the cDNA of HaloTag protein (HT) under the control of the CaMV-35S promoter with double enhancer and the poly(A) sequence from CaMV-35S.

transformation marker pFF-GFP-PTS1 facilitates transfer of GFP directly into peroxisomes. The other transformation marker pGreen0229:MPmRFP is described as a plasmodesmata marker (S Chapman, personal communication). Thus in both cases, an accumulation of the fluorochromes GFP and mRFP was expected in distinct cell compartments, whereas the HaloTag protein should be distributed over the cytoplasm and the nucleus because of the small size of the protein (33 kDa).

Vector construction and transformation

The cDNA of HaloTagTM protein was transferred from the pHT2-vector (Promega) into a plant-specific expression system via PCR-based cloning. Intermediate cloning steps were performed in the pGEMTeasy-vector (Promega). Expression of the protein was driven by the strong CaMV-35S promoter with double enhancer in the pFF vector (Timmermans et al., 1990), a pUC-based high copy plasmid for direct DNA-transfer techniques. The final pFFHT plasmid is shown in Fig. 1. Gene transfer experiments were started using PEGmediated protoplast transformation. Protoplasts, as plant cells surrounded only by a plasmamembrane, were chosen to investigate the possible negative effect of the plant cell wall for staining. Cell walls are known to adsorb the fluorochromes and could therefore disturb or inhibit the colouring procedure. Subsequently, biolistics was used for transformation of intact plant tissues. In both approaches, protoplast transformation and biolistics, a second plasmid was co-transformed in order to distinguish between transformed and non-transformed plant cells. For this purpose, the constructs pFF-GFP-PTS1 (Nowak et al., 2004) or pGreen0229:MPmRFP were used. The targeting signal PTS1 is a C-terminal sequence (SKL, SNL, or other variants; Gould et al., 1989; Mullen et al., 1997) known as signal targeting exclusively for peroxisomes (for a review, see Sparkes and Baker, 2002). Thus the

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Detection of transiently expressed fluorescent proteins in protoplasts

Protoplasts have the advantage being comparable to animal cells due to the removal of the plant cell wall. Two days after PEG-mediated transformation of N. plumbaginifolia protoplasts, the transformation efficiency was checked using GFP and mRFP fluorescence, respectively. Incubation with TMR or diAcFAM ligand was performed for 15, 30, or 60 min in a final concentration of 0.2, 1.0, and 5.0 lM ligand solution. After two washing cycles the cells were immediately subjected to fluorescence microscopy. The protoplasts showed a clear GFP, mRFP, TMR, and diAcFAM fluorescence (Fig. 2). It was found that staining for 30 min with 1.0 and 5.0 lM ligand solution gave brilliant signals, whereas 0.2 lM was not sufficient to colour the material. Two protoplasts stained with 1.0 lM TMR are shown in Fig. 2a–e, one of them (the lower one) has a clear GFP-fluorescence. As pointed out before, this GFP is nearly exclusively localized in peroxisomes mediated by the PTS1 sequence (Nowak et al., 2004). Only this protoplast had a strong TMR fluorescence (in the fourth channel of the cLSM) which is exclusively localized in the cytoplasm because the HaloTag protein possesses no targeting signal. As a next step, the lambda mode of the cLSM510META (Zeiss) was used to prove the specificity of TMR

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Fig. 2. Images of tobacco cells with transient expression of HaloTag protein and GFP/mRFP. Protoplasts and protoplast-derived cells are visualized in the transmitted light channel (a, f, l), GFP (b, g, m), chlorophyll (c, h, n), TMR (d, i, o), and Calcofluor White fluorescence (j, p) and in the merged picture (e, k, q). Only the lower protoplast in (a–e) which shows GFP fluorescence in the peroxisomes 2 d after transformation (b) also has TMR fluorescence in

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HaloTagTM: a new plant reporter gene

fluorescence. The META-detector of the cLSM-510, a combination of an optical grating in line with a 32channel photomultiplier array, can acquire simultaneously several emission signals with complete separation of their wavelengths. This allowed a reference spectrum (emission fingerprint) of the colours showing an emission maximum at 585 nm for TMR (Fig. 2r) and 522 nm for diAcFAM to be taken (Fig. 2s). No unspecific labelling was detectable with the TMR ligand, either from unknown cell compounds or by cell walls. Only for diAcFAM was an unspecific labelling of the vacuole and of a few chloroplasts found in some cases. The diAcFAM ligand is based on a non-fluorescent diacetyl derivative of fluorescein which is converted to a brightly fluorescent fluorophore upon cleavage of the diacetyl groups by cellular esterases, known also from determination of cellular viability assays (Amano et al., 2003). Fluorescein itself is no longer membrane-permeable which could be the reason for the unspecific staining found. No cross-talk was observed between the channels: TMR and diAcFAM ligands, red autofluorescence of chloroplasts, mRFP, and the GFP of peroxisomes were clearly separated from each other. The following experiments were designed to find out if the newly developing cell wall of transformed protoplasts can interfere with the colouring process. Therefore protoplast-derived cultures (4 and 14-d-old) were incubated with the TMR and diAcFAM-ligand solutions and the cell wall stained in parallel with Calcofluor White. Figure 2j and p illustrates cell wall staining. The two protoplasts in Fig. 2f–k started to develop a cell wall and therefore look more oval-shaped. Figure 2l–q shows the first division of a protoplast-derived cell with a compact cell wall. In both cases the transformed cells, which express GFP transiently in the peroxisomes, also have an intensive TMR fluorescence in the cytoplasm. These results demonstrate that the staining of transiently expressed HaloTag protein in intact plant cells is not hampered by the plant cell wall. Detection of transiently expressed fluorescent proteins in intact plant cells Biolistic transformation was used to transform intact plant cells. Leaf material of two tobacco species (Nicotiana tabacum cv. Gatersleben and N. benthamiana) and from poplar (Populus tremula3Populus alba) was tested for possible application in herbaceous plants and trees, shooting routinely into the upper epidermis. Two days after bombardment, the leaf discs were inspected and GFP/ mRFP-expressing areas were cut out for subsequent staining. Based on the results of the protoplast experiments,

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only the 1.0 lM ligand solution and staining for 30 min were used, and the focus was mainly on the destaining procedure. Immediately after staining and destaining, a high background of TMR and diAcFAM fluorescence was found (data not shown), and even 4 h destaining in water did not completely eliminate this background fluorescence. Therefore, longer destaining was essential. Overnight incubation in water gave the pictures shown in Fig. 3. A GFP- or mRFP-expressing cell gave a strong TMR or diAcFAM fluorescence, respectively. There was nearly no background in the surrounding cells as shown in the low magnification picture for diAcFAM (Fig. 3j) and TMR (Fig. 3k). The epidermal cell (Fig. 3a–e for N. tabacum and Fig. 3l, m for N. benthamiana) and the guard cell of N. tabacum (Fig. 3f– i) showed an intensive TMR fluorescence exclusively in the cytoplasm. Since the size exclusion limit of the nucleus is in the range of 40–60 kDa (Shiota et al., 1999), and the protein used has a molecular weight of 33 kDa, staining of the nucleus was also detected (Fig. 3e, i, j). In Fig. 3f–i, only the guard cell with the GFP fluorescence (=transformation marker; Fig. 3f) also had TMR fluorescence (Fig. 3h); the second cell of the stomatal apparatus was not transformed during bombardment and hence served as a negative control. The reference spectra of the colours detected with the META detector of the cLSM-510 showed an emission maximum for TMR and diAcFAM in the range found for the protoplasts (Fig. 2t, u). There were no differences when using intact leaf areas or stripped lower epidermis of leaves during the observation on the cLSM, except that the transmitted light channel gave a more brilliant picture in the case of the mono-cell layer of the epidermis. In addition to tobacco, poplar was also tested as the model for woody plants (Taylor, 2002) and leaf material from in vitro and hydroponically grown plants was transformed. The results are summarized in Fig. 3n and o. In some cases, there were problems with destaining of the hydroponically grown leaf material because of the big masses of hairs that created obstacles either for the penetration of gold particles into epidermal cells or for destaining. Benefits of the HaloTag protein for plant investigations

The HaloTagTM-technology as a new detection system for rapid, site-specific labelling of proteins was first established in mammalian cells. The technology is based on the formation of a covalent bond between the HaloTag protein, which is transiently or stably expressed in cells, and a synthetic ligand which is used in a staining procedure prior to visualization. These ligands carry a variety of

the cytoplasm (d); the upper protoplast serves as a negative control. Four days after transformation (f–k), the protoplasts developed a new cell wall (j); here also only the cell with GFP fluorescence (g) has a clear TMR signal (i). Both daughter cells of a dividing protoplast-derived cell have GFP fluorescence (m), the thick cell wall (p) does not disturb the staining with TMR (o). In (r–u), the spectral signatures taken by the META channel of the different fluorochromes are presented in protoplasts of N. plumbaginifolia (r, s) and epidermal cells of N. tabacum (t, u). Scale bars=10 lm.

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Fig. 3. Images of plant cells with transient expression of HaloTag protein after biolistic transformation. Epidermal (a–e, j) and guard cells (f–i) of N. tabacum are visualized in the transmitted light channel (a), GFP (b, f), chlorophyll (c, g), and TMR fluorescence (d, h) and, in the merged picture, with TMR (e, i), and diAcFAM fluorescence (j). Nicotiana benthamiana guard cells expressing TMR and GFP fluorescence are shown in a low magnification overview (k), a section with only the TMR signal (l), and in the merged picture (m). TMR fluorescence in Populus tremula3Populus alba is presented in (n) and in the merged picture with GFP and chlorophyll fluorescence (o). Scale bars=20 lm.

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HaloTagTM: a new plant reporter gene

functionalities, including fluorescent labels, affinity tags, and attachments to a solid phase (Los et al., 2005). The efficiency of transformation and expression of the HaloTag protein followed by staining ligands in plant cells was studied. A high transformation and labelling efficiency under the conditions used (strong promoter and protoplast transformation or biolistics) were found. Depending on the experiments, 2–30% of the protoplasts or in the biolistics 30–150 cells per bombarded leaf area showed transient foreign gene expression. In all of the cells observed, the co-transformation rate of the two plasmids used was in the 100% range, which means that, in the case where GFP fluorescence was found, the Halo-Tag could also be detected and vice versa. The HaloTag-system is comparable to the well-established GFP system (Haseloff and Amos, 1995). It allows fusion of any protein of interest to its N- or C-terminus. The molecular weight of the protein (33 kDa) does not significantly diverge from the size of GFP (27 kDa). As with any fusion protein, fusing with HaloTag protein may affect the functionality of the protein. However, the incorporation of an appropriate peptide linker between the protein of interest and the HaloTag protein may help to minimize this effect (Los et al., 2005). Since HaloTag proteins are expressed as monomers and exhibit a compact protein structure, steric hindrance of genetic fusions is strongly reduced. In terms of flexibility, the HaloTag technology offers additional benefits in comparison to the use of fluorescent proteins. Because of the open architecture of the technology which allows the use of different ligand colours, one cloning procedure is sufficient. Thus, timeconsuming re-cloning work is reduced, which is important for co-labelling experiments where two or more fluorescent proteins need to be tested to overcome background issues. To this end, Promega offers three different fluorescently labelled ligands and two ligands containing biotin; additional ligands are under development (Truc N Bui, personal communication). When analysed in mammalian cell lines, the HaloTag TMR, coumarin, diAcFAM, and biotin ligands are cell permeable and show no toxicity or morphological side-effects at recommended labelling conditions in the cell lines tested (Los et al., 2005). These results are consistent with what was found using living plant cells. Moreover, comparable to mammalian systems, plant material can be fixed in 3% formaldehyde solution and mounted in Mowiol 4.88 (Hoechst Farbwerke, Frankfurt, Germany) (data not shown). In addition to high flexibility, the use of HaloTagTM Interchangeable Labeling Technology will help to reduce toxicity issues due to overexpression of GFPs, as discussed by Haseloff et al. (1997). These authors pointed out that the accumulation of fluorescent proteins led to the generation of free radicals, especially H2O2, in cells due to the formation of the chromophore by cyclization, dehydration, and aerial oxidation which has not been reported in the HaloTag-system. It was found that a slow decrease in the

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number and intensity of the stained cells, as known for transient gene expression, could be monitored in the ‘long term’ transient protoplast system for 14 d. But no dramatic drop was seen as would have been expected for a toxic substrate. For live cell imaging studies that require longterm microscopic observation, toxicity issues need to be considered to establish proper experimental outcomes. Acknowledgements This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ha3107/3-1). We are grateful to students Michael Schaller, Jana Tiefenau, and Yasemine So¨mer for excellent technical work during their practical course in our laboratory, and to Promega (Dr Stephan Brockmann and Dr Truc N Bui) for providing the pHT2-Vectorsystem, the colouring solution, and helpful discussion.

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