Exploration of jasmonate signalling via automated and standardized ...

The Plant Journal (2005) 44, 1065–1076

doi: 10.1111/j.1365-313X.2005.02586.x

TECHNICAL ADVANCE

Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells Valerie De Sutter†, Rudy Vanderhaeghen†, Sofie Tilleman†, Freya Lammertyn, Isabelle Vanhoutte, Mansour Karimi, Dirk Inze´, Alain Goossens and Pierre Hilson* Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Gent, Belgium Received 13 June 2005; revised 18 August 2005; accepted 9 September 2005. * For correspondence (fax þ32 9 331 38 09; e-mail [email protected]). † These authors contributed equally to this work.

Summary Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2þ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed. Keywords: protoplast transient expression assay, automation, Gateway recombinational cloning, Arabidopsis, tobacco BY-2, alkaloid biosynthesis.

Introduction Genome information has grown dramatically in the past few years, but the assignment of functions to genes through experimentation remains an arduous task. In multicellular eukaryotes, even in the best characterized model species, at least one-third of the genes have no known functions; most of the remaining ones are categorized based solely on sequence homology and transcript profiling; and at best one in 10 has been studied in more depth via molecular biology or genetics approaches. Since the advent of plant transformation technologies two decades ago, the ultimate proof of gene function has ª 2005 Blackwell Publishing Ltd

generally been provided by the introduction of transgenes into the chromosomes of stably transformed plants. Although this ‘gold standard’ methodology has been extremely successful, it suffers from several drawbacks. The effects resulting from genetic perturbations may be lethal, pleiotropic or only remotely linked to the molecular mechanisms they directly affect. Conversely, many biological processes are robust and the insertional mutagenesis, silencing or ectopic expression of genes often does not yield any notable phenotype. Despite significant improvements, generation and analysis of large amounts of transgenic 1065

1066 Valerie De Sutter et al. plants are still cumbersome, so that limited numbers of genes can be tested in only a few genetic backgrounds. This latter bottleneck becomes increasingly problematic because many novel gene candidates are identified via the analysis of genome-scale data sets, such as microarray transcript profiles or protein–protein interaction surveys, and only a few can be further characterized via transgenic research. Therefore novel methods need to be developed that rapidly provide additional information about gene function and help to identify the most promising candidates (Hilson, 2006). Although they are not suited for complex organism studies, transient expression assays (TEAs) in plant protoplasts have proven very useful for dissecting a broad range of molecular mechanisms, including signalling and metabolic pathways as well as transcriptional regulatory networks (Asai et al., 2002; Hilson et al., 1990; Hwang and Sheen, 2001; Kovtun et al., 2000; Sugimoto et al., 2003; Tiwari et al., 2003; Zentella et al., 2002; Zimmermann et al., 2004; reviewed by Sheen, 2001). However, TEAs have so far been hampered by the difficulty for a single operator to handle more than a few samples in parallel. Their implementation at a large scale would greatly benefit from standardized, automated protocols. We searched for factors that potentially control the production of alkaloids, including nicotine, in Nicotiana tabacum (tobacco). Some of the key genes coding for the structural enzymes at the core of the corresponding secondary metabolism pathways have been identified, but little information is available yet about their regulation (Nugroho and Verpoorte, 2002). However, herbivory, wounding and phytohormone treatments have been shown to control alkaloid biosynthesis in tobacco. For instance, nicotine production is affected negatively by both auxin and ethylene, whereas it is stimulated by jasmonates in whole plants and in cell suspensions of Nicotiana spp. (Baldwin et al., 1994; Goossens et al., 2003b; Imanishi et al., 1998; Kahl et al., 2000; Shoji et al., 2000a). A previous study, which combined targeted metabolite analysis and cDNA–AFLP transcript profiling of jasmonate-treated tobacco Bright Yellow-2 (BY-2) cells, yielded a large inventory of known and novel genes whose expression varied on elicitation (Goossens et al., 2003b). We pursued systematically the functional analysis of these genes, assuming that a substantial portion might be involved in secondary metabolism of tobacco. Here we describe protocols for Arabidopsis and tobacco BY-2 protoplast TEAs in which polyethylene glycol (PEG)/ Ca2þ transfections and transcriptional readout measurements are performed on robotic platforms. Automation significantly increased throughput and efficiency, and reduced the amount of necessary reagents. Reproducibility was improved markedly with the introduction of multiple replicates and internal standards that combined dual luciferase reporter genes. Our experimental design was imple-

mented in a screen for potential regulators of tobacco alkaloid biosynthetic pathways. As a result, two transcription factors were identified that control the transcription of the gene coding for putrescine N-methyltransferase, involved in the production of nicotine in BY-2 cultured cells. Results Vectors and cloning strategy The ability to clone all necessary DNA segments precisely and easily is crucial in any research programme aimed at the systematic functional analysis of large gene sets. We have adopted the Gateway recombinational cloning system that allows the reliable in vitro transfer of DNA segments flanked with the appropriate att sites, from versatile validated plasmid ‘entry clones’ to a variety of ‘destination vectors’, each designed for a specific assay, resulting in ‘expression clones’. This transfer is performed efficiently, in a single step, in an oriented fashion, and is independent of the segment sequences (Hartley et al., 2000). The destination vectors and the derived expression plasmids introduced into plant cells for the experiments described below are listed in Table 1. These constructs belong to two categories, their purpose being either the transcription of an open reading frame (ORF) under the control of the 35S CaMV promoter (effectors and internal standard); or the quantitative analysis of promoter activity via the measurement of the firefly luciferase enzyme (reporters). Robust cell assays rely on strong, reproducible output signals. Although many recombinant DNA constructs for ORF transcription were already available as Gatewaycompatible binary T-DNA expression plasmids, we investigated the extent to which expression of reporter or effector genes from smaller-backbone plasmids would yield higher transcriptional readout outputs in our automated TEA protocols. For equal DNA amounts, higher signals were always produced by identical expression cassettes in minimal high-copy plasmids compared with large binary T-DNA plasmids. This feature was observed in tobacco BY-2 protoplasts as well as in Arabidopsis cell-culture protoplasts, whether the reporter gene encoded the green fluorescent protein (eGFP) or the firefly luciferase (Figure 1a). Therefore in all experiments described below, ORF expression cassettes were introduced into cells via small plasmids extracted from Escherichia coli cells in which they are produced at high copy numbers. Automation of protoplast PEG transfection Transient expression assay automation was achieved on a TECAN Genesis 200 robotic platform, chosen because it offered functions essential for assay optimization. It was equipped with a mobile pipetting head carrying eight

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 44, 1065–1076

Automated standardized transient expression assays 1067 Table 1 Vectors for transient assays in plant cells Name

Vector type

Cloning site

Composition

Size (kb)

Reference

p2GW7 pK7WG2

Effector, destination Effector, destination

attR1-ccdB-attR2 attR1-ccdB-attR2

5.9 11.1

Karimi et al. (2002) Karimi et al. (2002)

pK2GW7

Effector, destination

attR1-ccdB-attR2

11.1

Karimi et al. (2002)

p2B1fLB27 pK2B1fLB27

Effector, expression Effector, expression

– –

5.9 11.1

This paper This paper

pK7B2FB12

Effector, expression

–

10.2

This paper

p35S::EGFP pm43GWfL7 pB4NsPMT2B3fL7 pB4NtPALaB3fL7 p2B1GUSB27 p2rL7 p2B1rLB27

Effector, expression Reporter, destination Reporter, expression Reporter, expression Internal control Internal standard Internal standard

– attR4-ccdB-attR3 – – – – –

35Spro::GCS::35Ster 35Spro::GCS::35Ster binary vector (Km resistant) 35Spro::GCS::35Ster binary vector (Km resistant) 35Spro::fLUC::35Ster 35Spro::fLUC::35Ster binary vector (Km resistant) 35Spro::eGFP::35Ster binary vector (Km resistant) 35Spro::eGFP::35Ster GCS::fLUC::35Ster PMTpro::fLUC::35Ster PALpro::fLUC::35Ster 35Spro::GUS::35Ster 35Spro::rLUC::35Ster 35Spro::rLUC::35Ster

4.5 6.7 6.9 6.2 6.1 5.2 5.2

Danon et al. (2004) This paper This paper This paper This paper This paper This paper

35S, CaMV 35S; fLUC, firefly luciferase ORF; GCS, Gateway cloning site; rLUC, Renilla luciferase ORF; pro, promoter; ter, terminator. All destination vectors are described with sequence, map and VectorNTI files in the dedicated website http://www.psb.ugent.be/gateway, and can be requested online.

independent channels with dispensible liquid-sensing tips and varying space between channels. Pipetting operations were defined precisely and specifically at each step of the protocol, with parameters controlling lateral tip position in the sample container, tip height (either fixed or determined relative to sensed liquid level), speed of aspiration and dispensing, mixing, timing and delays. A second robotic arm was used to displace sample plates, as well as various containers. The following considerations were important for transposition of the TEA protocol to the robotic platform. (i) Scale and container: two of the main goals of automation were increasing throughput and decreasing reagent amounts. Therefore all volumes were scaled-down and the transfection, washing and lysis steps were all performed in 48-well multititre plates. (ii) Protoplast pelleting: in the manual protocol, protoplasts were concentrated by centrifugation at the bottom of tubes. Because such a step involved complex manipulation of the cell containers, it was bypassed by simply letting the protoplasts sediment at the bottom of the wells in static microtitre plates. (iii) Liquid handling: pipetting operations were significantly more precise when performed by a robot than with a manually operated device. The speed at which solutions were pumped in and out of the tips, as well as the position of the tips in the well, were adapted according to fluid viscosity and to the desired effect. For example, to avoid losing cells by drawing away the soft cell pellet, careful removal of the viscous PEG solution in the last crucial step was performed very slowly (5 ll sec)1) while lowering the pipetting tip progressively as the

supernatant volume decreased, in a manner impossible to perform by hand. In contrast, lysis was favoured by flushing the cell suspension rapidly (150 ll sec)1) in and out of the tip multiple times and simultaneously mixing the samples on a multititre plate shaker. Pipetting of protoplast suspension, DNA and PEG/Ca2þ solutions were carried out with wide-bore liquid-sensing tips, to prevent cell damage by shearing during the successive mixing steps and to position the pipetting device according to the liquid level. (iv) Sterility: in our set-up complete assays lasted 0.05) between columns.

Transformation of tobacco BY-2 cells Stable transformation was performed by co-cultivating 4 ml of a 3-day-old BY-2 ProNsPMT2::GUS/GFP suspension culture (V.D.S. and A.G., unpublished results) with 25–100 ll of a 2-day-old A. tumefaciens culture. Bacteria were grown in liquid LC-medium [1% (w/v) bacto-tryptone, 0.5% (w/v) bacto yeast extract, 0.8% (w/v) NaCl] complemented with appropriate selective antibiotics (100 lg ml)1 rifampicin; 20 lg ml)1 gentamicin sulphate; 300 lg ml)1 streptomycin; 100 lg ml)1 spectinomycin–HCl). Three days after co-cultivation, cells were transferred to selective solid BY-2 medium (10 lg ml)1 G418-disulphate, supplemented with 500 lg ml)1 carbenicillin and 500 lg ml)1 vancomycin to eliminate Agrobacterium). All antibiotics were supplied by Duchefa (Haarlem, The Netherlands). Cells were kept in the dark at 25
C during co-cultivation and selection. After approximately 3 weeks transgenic calluses became apparent, were transferred to fresh selective plates, incubated in the dark at 25
C, and further subcultured when appropriate.

Acknowledgements We wish to thank Wilson Ardilez-Diaz and Caroline Buysschaert for the sequence validation of the expression clones, Joke Allemeersch and Kris Morreel for assistance with statistical analysis, and Martine De Cock for help in preparing the manuscript. The Arabidopsis cultured cell line was kindly provided by Laszlo Bo¨gre. V.D.S. and S.T. are indebted to the Institute for the promotion of Innovation by Science and Technology in Flanders for predoctoral fellowships.

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