Introduction

Plant protoplasts without cell walls offer a versatile cell-based experimental system. Macromolecules such as DNA, RNA and proteins can be delivered into protoplasts using various methods, e.g., PEG–calcium fusion, electroporation and microinjection. Signal transduction and metabolic pathways as well as transcription and translation machineries can be transiently manipulated to investigate cell-autonomous regulation and responses. The method is especially powerful in functional genomics analysis when combined with rich genetic resources, such as insertion libraries, available in the model plant Arabidopsis thaliana. We describe here a protocol for a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) and provide troubleshooting advice and detailed information on useful reporter constructs. Applications, advantages and limitations of the TEAMP assay are also discussed. More general information about plant protoplasts can be found in a review1 and a live performance of the TEAMP procedure can be seen at the Sheen lab website (http://genetics.mgh.harvard.edu/sheenweb/protocols_reg.html).

Overview

The isolation of plant protoplasts was first reported more than 40 years ago2. Protoplasts had been used to observe cellular processes and activities such as cell wall synthesis, cell division, embryogenesis, differentiation during regeneration, photosynthesis activity, calcium signaling and regulation, and the modulation of ion channels by light, stress and hormone responses in various plant species1. Protoplasts were also isolated from special cell types to investigate cell type–specific responses. For example, gibberellin and abscisic acid actions on α-amylase synthesis and blue light–induced H+-ATPase activity have been studied with protoplasts isolated from barley aleurone layer3 and Vicia faba guard cells4, respectively.

After the development and significant improvement of various DNA transfection methods (e.g., PEG, electroporation and microinjection), diverse cellular functions of genes of interest have been studied using protoplasts generated from several plant species1. Since Arabidopsis became the central model system in plant biology with the availability of its whole genome sequence, we and several other laboratories have established the TEAMP system as a cell-based functional genomics tool1,5.

The TEAMP protocol is based on mesophyll protoplasts freshly isolated from leaves of Arabidopsis wild-type or mutant plants growing on soil (see Fig. 1a). After cell walls are removed using fungal cellulase and macerozyme, protoplasts are released, washed and collected (see Fig. 1b,c). Remarkably, these protoplasts maintain many of the same physiological responses and cellular activities as intact plants, despite the enzymatic treatment step1. PEG–calcium-mediated transfection is used to deliver DNA into protoplasts, followed by 2–24 h incubation to allow gene expression (see Fig. 1d). A sterile technique is not necessary owing to the short incubation time. To allow constitutive expression of transfected genes, the cauliflower mosaic virus 35S promoter derivative with the maize C4PPDK basal promoter and the 5′-untranslated region (5′-UTR) enhancer is used6 (see Fig. 2a). To control the level of gene expression, a chemically inducible gene expression system has also been developed (see Advantages and limitations).

Figure 1: Arabidopsis protoplast isolation and transient gene expression.
figure 1

(a) A healthy 4-week-old Arabidopsis plant suitable for protoplast isolation. Asterisks indicate the optimal true leaves (fifth, sixth and seventh) to be used as a mesophyll protoplast source. (b) Mesophyll protoplasts ( × 400). (c) Isolated protoplasts retain the characteristics of the genetic backgrounds from which they were obtained; protoplasts isolated from wild-type Col-0 (top), ethylene-insensitive etr1-1 mutant (middle) and the constitutive ethylene response ctr1-1 mutant (bottom) show different cellular morphology and characteristics under light microscopy ( × 600). (d) Protoplasts expressing the nuclear-targeted GFP driven by the constitutive 35S enhancer fused to the maize C4PPDK basal promoter are shown under fluorescence microscopy ( × 400). More than 90% of protoplasts were transfected successfully with the GFP reporter gene construct.

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Figure 2: Commonly used plasmid constructs in the transient expression in Arabidopsis mesophyll protoplast (TEAMP) system.
figure 2

(a) Schematic representation of a regulatory gene construct under the control of the constitutive 355 enhancer fused to the maize (C4PPDK) basal promoter. GOI, gene of interest; NOS, nopaline synthase terminator. (b) Schematic representation of a luciferase (LUC) reporter gene construct driven by the promoter of interest (POI) and terminated by NOS. (c) Schematic representation of the dexamethasone (DEX)-inducible constructs. The top picture illustrates the artificial transcription activator (ATA), constructed with a bacterial DNA-binding domain (LexA-DB), a viral transcription activation domain (VP16) and a mammalian (rat) glucocorticoid receptor (GR). The ATA is driven under the constitutive 35S enhancer with the maize C4PPDK basal promoter. Upon DEX treatment, the ATA (LexA-VP16-GR) is released from the cytoplasm HSP90 complex and moves into the nucleus to induce target gene transcription (represented by the black arrow). In the nucleus (bottom picture), the ATA controls the transcription of a DNA construct that is driven by eight copies of the bacterial LexA operator (8xLexA-OP) and the minimum 35S promoter. In the example presented, the LUC reporter gene is included in the 8xLexA-OP-driven construct. (d) Time course analysis of the LUC reporter activation in the absence and presence of 10 μM soluble DEX. Values are means of ratio in triplicates. The β-glucuronidase (GUS) activity expressed from the co-transfected UBQ10-GUS-NOS serves as an internal control. LUC expression is induced upon DEX treatment and shows linear expression kinetics.

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Luciferase7 (LUC) and β-glucuronidase8 (GUS) are routinely used as reporters for the quantitative measurement of gene expression in TEAMP assays (Fig. 2b, Table 1). A set of LUC reporters controlled by diverse promoters that can respond to specific signals (Table 1) have been donated by the Sheen lab to the public stock center, the Arabidopsis Biological Resource Center (ABRC). These reporters can serve as positive controls to help set up the TEAMP assay in laboratories new to the system. However, the application of TEAMP is not limited to reporter assays. Physiological and cell-autonomous responses can be observed and scored using various analytical methods to investigate protein activity, localization, trafficking, interaction and stability.

Table 1 List of useful reporter constructs for transient expression in Arabidopsis mesophyll protoplast assays.
Full size table

Applications

Physiological and cell-autonomous responses and assays in protoplasts have been widely used to identify key regulators and elucidate molecular mechanisms underlying intracellular signal transduction pathways involved in plant physiology, immunity, growth and development. The application of TEAMP system has been significantly broadened in recent years and will be further expanded by integration with Arabidopsis genomics and genetic resources for high-throughput explorations.

Using TEAMP assays, molecular mechanisms underlying plant hormone signaling pathways have been elucidated. In auxin signaling, regulatory functions and responses of AUX/IAA proteins9, RAC GTPase10 and auxin response factors11 have been characterized. The protein stability control of EIN3, a key nuclear transcription factor in ethylene signaling, has been shown to be the main regulatory event involved in an unexpected cross-talk between glucose and ethylene signaling12. Significantly, the phospho-relay via the Arabidopsis two-component system has been established as the main cytokinin regulatory circuitry13, which has been verified by numerous genetic and phenotypic analyses of corresponding Arabidopsis mutants14. Both abiotic and biotic stress signaling can also be studied in the TEAMP system. For example, the functions of the evolutionarily conserved mitogen-activated protein kinase (MAPK) cascades have been identified and characterized in Arabidopsis mesophyll protoplasts for oxidative stress signaling5 and innate immune responses15. Subsequently, the virulence functions of type III effectors, AvrPto and AvrPtoB from the pathogenic bacterium Pseudomonas syringae, have been shown to be potent suppressors of the MAPK-dependent plant innate immunity16.

More recently, protein–protein interactions in vivo between the glucose sensor hexokinase 1 (HXK1) and its partners have been demonstrated for HXK1-dependent nuclear glucose signaling17. Through application of the yeast two-hybrid concept, protein–protein interaction has also been examined in Arabidopsis mesophyll protoplasts to establish a heterodimerization map of specific groups of bZIP transcription factors18.

Single-cell imaging of diverse fluorescence marker proteins, e.g., GFP and its variants, fused to functional proteins have also been extensively utilized to examine protein localizations, protein domain functions in protein targeting and protein transport machinery functions in vesicle trafficking. For instance, subcellular localization of various proteins has been determined using the GFP tag11,12,17,18,19. Novel roles of the transmembrane domain and C-terminal 7 amino acids of Arabidopsis small outer envelope membrane protein 7 have been identified for chloroplast targeting20. Interesting regulatory roles of dynamin-like ADL6 (ref. 21), small GTP binding protein Rha1 (ref. 22) and actin filaments23 have been studied in vacuolar trafficking. The protoplast isolation method and fluorescence-assisted cell sorting technique have been further applied to reveal genome-wide cell type–specific transcript profiles in Arabidopsis roots24.

Advantages and limitations

Uniform and abundant mesophyll protoplasts isolated from mature Arabidopsis leaves can respond to diverse signals in a physiological manner similar to the responses observed in leaves of whole plants. Importantly, application of the TEAMP system is not limited to processes and activities that occur in leaves. Proteins that are normally expressed in other cell types and tissues can be ectopically expressed and examined in mesophyll protoplasts to learn their molecular and cellular functions. Analysis using mesophyll protoplasts often provides information of greater functional and physiological relevance to plants than data obtained using heterologous cell systems, e.g., bacteria, yeast, insect and mammalian cells. As most of the quantitative and physiological responses can be observed and measured within 2–10 h after DNA transfection, the experiments do not require sterile techniques and complex culture medium, which represents a tremendous saving in time and costs. There is no other biological system for gene expression analysis (including bacterial, yeast and mammalian cell culture) that can be handled with the same minimal laboratory requirement. The requirement for a relatively small number of protoplasts is another advantage of the TEAMP system owing to its high efficiency and sensitivity. In some other protoplast protocols, 10–1,000 times more protoplasts (105–107) are recommended for each DNA transfection experiment25.

On the other hand, the conserved functions of molecular domains and regulatory mechanisms among bacteria, yeasts, animals and plants allow us to utilize molecular switches, originally identified in other organisms, to control gene and protein expression levels in plant mesophyll protoplasts. For instance, the mammalian steroid nuclear receptor has been used to provide a ligand inducible gene expression system in mesophyll protoplasts12. As depicted in Figure 2c, an artificial transcription activator (ATA) is constructed with a bacterial DNA-binding domain (LexA-DB), a viral transcription activation domain (VP16) and a mammalian (rat) glucocorticoid receptor (GR). The ATA is driven under the constitutive 35S enhancer fused to the maize C4PPDK basal promoter6. Upon dexamethasone (DEX) treatment, the ATA (LexA-VP16-GR) is released from the cytoplasm HSP90 complex and moves into the nucleus to induce target gene transcription26. In the nucleus, the ATA controls the transcription of a DNA construct that is driven by eight copies of the bacterial LexA operator (8xLexA-OP) and the minimum 35S promoter (see Fig. 2c). In the example presented (in which the LUC reporter gene is included in the 8xLexA-OP-driven construct; see Fig. 2d), LUC expression is induced upon DEX treatment and shows linear expression kinetics. The expression level of LUC is also controlled in a ligand dosage–dependent manner, indicating that the expression level of desired genes and proteins can be manipulated using this inducible system.

Mesophyll protoplasts can be isolated from fresh leaves of wild-type and most Arabidopsis mutant plants and used immediately for experiments without the time-consuming step of cell culture establishment. Wild-type mesophyll protoplasts maintain most leaf features and responses, which are often lost in undifferentiated suspension culture cells. The mutant protoplasts possess the cellular nature that is characteristic of the original mutant plants. For example, mesophyll protoplasts isolated from the ethylene-insensitive etr1-1 and the constitutive ethylene-response ctr1-1 mutants show cellular morphology and characteristics distinct from those in the wild type (see Fig. 1c). There are many successful examples of using the TEAMP assay with various mutant plants, including the analysis of ARF7 functions in auxin signaling11, the antagonistic interaction between HXK1-dependent glucose and ethylene signaling12, the receptor kinase FLS2 function in flg22 signaling15 and roles of various plant hormone signaling pathways in programmed cell death induced by the fungal toxin fumonisin B1 (ref. 27). Conceptually, the use of protoplasts generated from mutant plants in TEAMP assays to screen for new regulators is similar to performing mutant suppressor or enhancer screens, which are commonly applied in the genetic analysis of specific signaling pathways. In general, it is necessary empirically to optimize the protoplast isolation and DNA transfection conditions for different Arabidopsis accessions and mutants. Once conditions are optimized, the use of genetically diverse mesophyll protoplasts makes the TEAMP system even more powerful in functional genomics research.

Unlike plant and animal cell culture lines, mesophyll protoplasts do not divide in the simple incubation buffer described in this protocol. Thus, this TEAMP protocol is not suitable for the study of cell cycle regulation. To use protoplasts to study cell division and dedifferentiation or other long-term plant responses, specialized plant growth conditions and sterile culture media will be required. Such protoplast regeneration protocols have been developed over the past two decades as well28,29,30.

For successful experiments, a high level of protoplast transfection efficiency (greater than 50%) is very important to obtain reliable and reproducible data using the TEAMP system. We recommend the use of an improved GFP vital marker donated to the ABRC to visually monitor protoplast transfection efficiency19 (see Fig. 1d). Careful practice, keen observation and accumulated experience contribute to the acquisition of meaningful results. All experiments need to be repeated multiple times to ensure consistency and to avoid pitfalls and false positive results. Even though the protocol appears to be simple and straightforward, the importance of practice and empirical optimization of each experiment for the individual researcher's purpose cannot be over-emphasized.

Materials

Reagents

  • 0.2 M 4-morpholineethanesulfonic acid (MES, pH 5.7; Sigma, cat. no. M8250), sterilize using a 0.45-μm filter

  • 0.8 M mannitol (Sigma, cat. no.M4125), sterilize using a 0.45-μm filter

  • 1 M CaCl2 (Sigma, cat. no. C7902), sterilize using a 0.45-μm filter

  • 2 M KCl (Sigma, cat. no. P3911), sterilize using a 0.45-μm filter

  • 2 M MgCl2 (Sigma, cat. no. M9272), sterilize using a 0.45-μm filter

  • β-Mercaptoethanol (Sigma, cat. no. M6250)

  • 10% (wt/vol) BSA (Sigma, cat. no. A-6793), sterilize using a 0.45-μm filter

  • Cellulase R10 (Yakult Pharmaceutical Ind. Co., Ltd., Japan)

  • Macerozyme R10 (Yakult Pharmaceutical Ind. Co., Ltd., Japan)

    Critical

    We have obtained the best results with this enzyme provider.

  • PEG4000 (Fluka, cat. no. 81240)

    Critical

    We found that the PEG source is very critical to achieve high transfection efficiency.

  • 1 M Tris–phosphate (pH 7.8), sterilize using a 0.45-μm filter

  • 100 mM trans-1,2-diaminocyclo-hexane-N,N,N′,N′-tetraacetic acid (DACTAA; Sigma, cat. no. D-1383)

  • 50% (vol/vol) glycerol (Fisher, cat. no. 15892), sterilize using a 0.45-μm filter

  • 20% (vol/vol) Triton X-100 (Sigma, cat. no. T-8787)

  • 1 M DTT (Sigma, cat. no. D-9779)

  • LUC assay system (Promega, cat. no. E1501)

  • 1 M Tris–HCl (pH 8.0) (US Biological, cat. no. T8650), sterilize using a 0.45-μm filter

  • 0.1 M 4-methylumbelliferyl glucuronide (MUG; Gold BioTechnology, Inc., cat. no. MUG-1G)

  • 0.2 M Na2CO3 (Sigma, cat. no. S7795)

  • 1 M methylumbelliferone (MU; Fluka, cat. no. 69580)

  • Metro-Mix 360 (Sun Gro Horticulture, Inc.)

  • Jiffy7 (Jiffy Products Ltd., Canada)

  • Arabidopsis accessions: Col-0 and Ler (ABRC)

  • Enzyme solution (see REAGENT SETUP)

  • Washing and incubation (WI) solution (see REAGENT SETUP)

  • W5 solution (see REAGENT SETUP)

  • MMG solution (see REAGENT SETUP)

  • PEG–calcium transfection solution (see REAGENT SETUP)

  • Protoplast lysis buffer (see REAGENT SETUP)

  • MUG substrate mix for GUS assay (see REAGENT SETUP)

  • Ampicillin

Equipment

  • Fluorescence microscope(Leica DM5000; Leica)

  • Luminometer (Monolite3010; Pharmigen)

  • Fluorometer (TK100; Hoefer)

  • Bench-top centrifuge (IEC Centra CL2; International Equipment Company)

  • 0.45-μm syringe sterilization filter (Whatman, cat. no. 6870-2504)

  • Generic razor blade (single-edged blade; VWR Scientific, cat. no. 55411-055)

  • Petri dish (100 × 25 mm2 for 10 ml enzyme solution; VWR Scientific, cat. no. 25389-000)

  • Nylon mesh (75 μm, laboratory sifters, Carolina Biological Supplies, cat. no. 65-2222N)

  • Improved Neubauer 0.1-mm-deep Reichert hemacytometer (Hausser Scientific, cat. no. 1483)

  • 30-ml round-bottomed tube (Sarstedt, cat. no. 55.517)

  • 2-ml round-bottomed natural microcentrifuge tube (SealRite; USA Scientific, cat. no. 1620-2700)

  • 6-well culture dish (Falcon, cat. no. 3046)

Reagent setup

  • Enzyme solution Prepare 20 mM MES (pH 5.7) containing 1.5% (wt/vol) cellulase R10, 0.4% (wt/vol) macerozyme R10, 0.4 M mannitol and 20 mM KCl. Warm the solution at 55 °C for 10 min to inactivate DNAse and proteases and enhance enzyme solubility. Cool it to room temperature (25 °C) and add 10 mM CaCl2, 1–5 mM β-mercaptoethanol (optional) and 0.1% BSA.

    Critical

    Addition of 1–5 mM β-mercaptoethanol is optional, and its use should be determined according to the experimental purpose.

    Critical

    Before the enzyme powder is added, the MES solution is preheated at 70 °C for 3–5 min. The final enzyme solution should be clear light brown. Filter the final enzyme solution through a 0.45-μm syringe filter device into a Petri dish (100 × 25 mm2 for 10 ml enzyme solution).

    Critical

    The enzyme solution should be prepared fresh.

  • WI solution Prepare 4 mM MES (pH 5.7) containing 0.5 M mannitol and 20 mM KCl. The prepared WI solution can be stored at room temperature (22–25 °C).

  • W5 solution Prepare 2 mM MES (pH 5.7) containing 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl. The prepared W5 solution can be stored at room temperature.

  • MMG solution Prepare 4 mM MES (pH 5.7) containing 0.4 M mannitol and 15 mM MgCl2. The prepared MMG solution can be stored at room temperature.

  • PEG–calcium transfection solution Prepare 20–40% (wt/vol) PEG4000 in ddH2O containing 0.2 M mannitol and 100 mM CaCl2.

    Critical

    Prepare PEG solution at least 1 h before transfection to completely dissolve PEG. The PEG solution can be stored at room temperature and used within 5 d. However, freshly prepared PEG solution gives relatively better protoplast transfection efficiency. Do not autoclave PEG solution.

  • Protoplast lysis buffer Prepare 25 mM Tris–phosphate (pH 7.8) containing 1 mM DTT, 2 mM DACTAA, 10% (vol/vol) glycerol and 1% (vol/vol) Triton X-100. The lysis buffer should be prepared fresh.

  • MUG substrate mix for GUS assay Prepare 10 mM Tris–HCl (pH 8) containing 1 mM MUG and 2 mM MgCl2. The prepared GUS assay substrate can be stored at −20 °C.

Procedure

Plant growth

Timing 3–4 weeks

  1. 1

    Grow Arabidopsis plants on either Metro-Mix 360 or Jiffy7 soil in a greenhouse or an environment-controlled chamber with a relatively short photoperiod (10–13 h light at 23 °C/11–14 h dark at 20 °C) under low light (50–75 μE m−2 s−1) and 40–65% relative humidity.

    Critical Step

    Col-0 and Ler have been extensively tested in our lab. In general, Arabidopsis plants are very sensitive to all kinds of environmental changes (e.g., drought, flooding, extreme temperature and constant mechanical perturbation). Try to maintain a constant environment as much as possible.

Protoplast isolation

Timing 4–5 h

  1. 2

    Choose well-expanded leaves from 3–4-week-old plants (usually true leaf numbers five to seven) before flowering (see Fig. 1a).

    Critical Step

    The selection of healthy leaves at the proper developmental stage is a very important factor in protoplast experiments. Protoplasts prepared from leaves recovered from stress conditions (e.g., drought, flooding, extreme temperature and constant mechanical perturbation) may look similar to those from healthy leaves. However, we have often experienced low transfection efficiency with the protoplasts from stressed leaves. High stress–induced cellular status can also be a problem in the study of stress, defense and hormonal signaling pathways.

  2. 3

    Cut 0.5–1-mm leaf strips from the middle part of a leaf using a fresh sharp razor blade without tissue crushing at the cutting site. A good preparation yields approximately 107 protoplasts per gram fresh weight (approximately 100–150 leaves digested in 40–60 ml of enzyme solution). For routine experiments, 10–20 leaves digested in 5–10 ml enzyme solution will give 0.5–1 × 106 protoplasts, enough for more than 25–100 samples (1–2 × 104 protoplasts per sample).

    Critical Step

    Change the blade after cutting four to five leaves. We routinely cut leaves on a piece of clean white paper (8" × 11") on top of the solid and clean laboratory bench, which provides for good support and easy inspection of wounded/crushed tissue (juicy and dark green stain).

  3. 4

    Transfer leaf strips quickly and gently into the prepared enzyme solution (10–20 leaves in 5–10 ml) by dipping both sides of the strips (completely submerged) using a pair of flat-tip forceps.

    Critical Step

    Immediate dipping and submerging of leaf strips is very critical for protoplast yield. When leaf strips are dried out on the paper during cutting, the enzyme solution cannot penetrate and protoplast yield is decreased significantly.

  4. 5

    Vacuum infiltrate leaf strips for 30 min in the dark using a desiccator.

  5. 6

    Continue the digestion, without shaking, in the dark for at least 3 h at room temperature. The enzyme solution should turn green after a gentle swirling motion, which indicates the release of protoplasts.

    Critical Step

    Digestion time depends on the experimental goals, desirable responses and materials used, and it needs to be optimized empirically. After 3 h digestion, most protoplasts are released from leaf strips in case of Col-0. However, the protoplast isolation efficiency differs significantly for different ecotypes and genotypes. The digesting time has to be optimized for each ecotype and genotype. Prolonged incubation of leaves (16–18 h) in the dark is stressful and might eliminate physiological responses of leaf cells. However, the stress might be important for the dedifferentiation and regeneration processes recommended in other protoplast protocols.

  6. 7

    Check for the release of protoplasts in the solution under the microscope; the size of Arabidopsis mesophyll protoplasts is approximately 30–50 μm.

    Critical Step

    It is not necessary to release all the protoplasts from leaf strips. Be gentle with protoplasts, but you can handle them with regular pipettes and pipette tips.

  7. 8

    Dilute the enzyme/protoplast solution with an equal volume of W5 solution before filtration to remove undigested leaf tissues.

  8. 9

    Wash a clean 75-μm nylon mesh with water to remove ethanol (the mesh is normally kept in 95% ethanol) then remove excess water before protoplast filtration. Filter the enzyme solution containing protoplasts after wetting the 75-μm nylon mesh with W5 solution.

  9. 10

    Centrifuge the flow-through at 100g to pellet the protoplasts in a 30-ml round-bottomed tube for 1–2 min. Remove as much supernatant as possible and re-suspend the protoplast pellet by gentle swirling.

    Critical Step

    A higher speed (200g) of centrifugation may help to increase protoplast recovery.

    Troubleshooting

  10. 11

    Re-suspend protoplasts at 2 × 105 ml−1 in W5 solution after counting cells under the microscope (× 100) using a hemacytometer. Rest the protoplasts by keeping on ice for 30 min.

    Critical Step

    If cold response is desired, keep protoplasts at room temperature. Although the protoplasts can be kept on ice for at least 24 h, freshly prepared protoplasts should be used for the study of gene expression regulation, signal transduction and protein trafficking, processing and localization.

  11. 12

    Protoplasts should begin to settle at the bottom of the tube by gravity after 15 min. Remove the W5 solution as much as possible without touching the protoplast pellet. Re-suspend protoplasts at 2 × 105 ml−1 in MMG solution kept at room temperature.

DNA-PEG–calcium transfection

Timing Less than 1 h for 20 samples

  1. 13

    Add 10 μl DNA (10–20 μg of plasmid DNA of 5–10 kb in size) to a 2-ml microfuge tube.

    Critical Step

    To study the role of a candidate regulator, three plasmids expressing a regulatory effector, a specific reporter and a transfection control reporter are used at the ratio of 5:4:1. However, it is highly recommended to optimize the plasmid ratio depending on each reporter's sensitivity. For example, a highly sensitive reporter needs only half the amount. Whenever the plasmid amount is changed, the same total plasmid DNA amount is compensated with a control plasmid that does not express any effector or reporter. The plasmid DNA has to be purified appropriately to support high transfection efficiency. Additional information on the preparation of plasmid DNA using the economical CsCl gradient is provided on the Sheen lab website (http://genetics.mgh.harvard.edu/sheenweb/protocols.html).

  2. 14

    Add 100 μl protoplasts (2 × 104 protoplasts) and mix gently.

  3. 15

    Add 110 μl of PEG solution, and then mix completely by gently tapping the tube (handle six to ten samples for each transfection).

  4. 16

    Incubate the transfection mixture at room temperature for up to 15 min (5 min is sufficient).

    Critical Step

    Transfection time should be optimized empirically.

  5. 17

    Dilute the transfection mixture with 400–440 μl W5 solution at room temperature and mix well by gently rocking or inverting the tube to stop the transfection process.

  6. 18

    Centrifuge at 100g for 2 min at room temperature using a bench-top centrifuge and remove supernatant.

  7. 19

    Re-suspend protoplasts gently with 1 ml WI in each well of a 6-well tissue culture plate.

    Critical Step

    We also use 12-well (0.5 ml WI) or 24-well (0.25 ml WI) plates for larger-scale experiments (50–100 samples). The culture plate is normally coated with 5% (vol/vol) sterile calf serum for a short time (1–2 s) to prevent sticking of the protoplasts to the plastic surface. The depth of the WI solution is approximately 0.1 mm to prevent hypoxia stress during protoplast incubation.

    Critical Step

    The use of carrier DNA is unnecessary for this type of DNA delivery.

    Critical Step

    High transfection efficiency can be achieved using 10–20% PEG final concentration. The optimal PEG concentration should be determined empirically for each experimental purpose. Visual reporters such as GFP can be used to determine optimal DNA transfection conditions (Fig. 1d, Table 1). If protoplasts are derived from healthy leaf materials, most protoplasts should remain intact throughout the isolation, transfection, culture and harvesting procedures.

    Critical Step

    Protoplast transfection can be scaled up or down depending on the experimental needs. Approximately 103–4 protoplasts are sufficient for reporter enzyme assays, 104–5 protoplasts are required for protein labeling and immunoprecipitation or western blot analysis and 106 protoplasts for RNA extraction and microarray analysis. If the protoplast transfection efficiency is low (less than 20%), the quality of plasmid DNA or the ratio of plasmid DNA and protoplast number should be systematically examined to find the optimal condition.

    Troubleshooting

Protoplast culture and harvest

Timing 2–24 h

  1. 20

    Incubate protoplasts at room temperature (20–25 °C) for the desired period of time.

    Critical Step

    A stimulus can be applied during the incubation according to the experimental purposes. For example, the synthetic flg22 peptide derived from bacteria can be added for 10 min or 1 h for the analysis of protein kinase activity or early transcription responses, respectively. For the inducible gene expression system (Fig. 2c,d), DEX can be added to the protoplasts 15–30 min after DNA transfection.

    Critical Step

    In general, the protoplast incubation time is 2–6 h for RNA analysis and 2–16 h for enzyme activity analysis and protein labeling. The TEAMP system is most suitable for the study of early events in signal transduction pathways, gene regulation and cell death.

    Critical Step

    The protoplast incubation temperature has to be optimized according to experimental needs.

    Critical Step

    It is not necessary to perform experiments under sterile conditions in general. Ampicillin (50 μg ml−1) can be added during the protoplast incubation after DNA transfection if bacterial growth is a concern.

  2. 21

    Re-suspend and harvest protoplasts by centrifugation at 100g for 2 min.

  3. 22

    Remove the supernatant and freeze samples on dry ice.

    Pause point

    Samples can be stored at −80 °C until further analysis.

Reporter assays

Timing 1–2 h

  1. 23

    Several assays can be carried out using option A (LUC assay), B (GUS assay) or C (GFP assay).

    1. A

      LUC assay

      1. i

        Add 100 μl of protoplast lysis buffer to the frozen protoplasts and mix vigorously by vortexing for 2 s to rupture the protoplasts.

      2. ii

        After 5 min incubation on ice, centrifuge at 1,000g for 2 min.

      3. iii

        Use 5–20 μl of the lysate and 100 μl LUC mix (Promega) to measure LUC activity with a luminometer.

    2. B

      GUS assay

      1. i

        Add 10 μl of the protoplast lysate from Step 23A(ii) into 100 μl of MUG substrate mix and incubate for 30–180 min at 37 °C.

      2. ii

        Add 0.9 ml 0.2 M Na2CO3 to stop the reaction.

      3. iii

        Measure the fluorescence of MU using a fluorometer.

        Critical Step

        Further dilution with 0.2 M Na2CO3 for GUS assay should be determined empirically.

    3. C

      GFP assay

      1. i

        View live cellular image of GFP or GFP fusion proteins (using protoplasts obtained in Step 22) under fluorescence microscopy ( × 400–600) without any additional sample preparation steps.

Troubleshooting

Troubleshooting advice can be found in Table 2.

Table 2 Troubleshooting table.
Full size table

Timing

Step 1: 3–4 weeks

Steps 2–4: approximately 1 h (depending on numbers of leaves being collected)

Steps 5–12: approximately 3–4 h (depending on Arabidopsis accession and mutants)

Steps 13–19: approximately 1 h (depending on numbers of samples being transfected)

Steps 20–22: approximately 2–24 h

Step 23: approximately 1–2 h

Anticipated results

Protoplast transfection efficiency is approximately 60–90% with this protocol in general (Fig. 1d). It is necessary to have higher than 50% transfection efficiency for a reliable analysis. A typical use of the TEAMP assay is to determine the regulatory function of an effector using the readout of a specific reporter in cell-autonomous responses. The effect of regulatory candidates that are transfected with a specific reporter (Table 1) can be quantified within 2–12 h after transfection of mesophyll protoplasts. The specific reporter readout value should be normalized with that of a co-transfected control reporter driven by a constitutive promoter, e.g., UBQ10-GUS-NOS8.