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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Abstract

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

Introduction

Chimeric fluorescent fusion proteins have been regarded as useful tools to study intracellular dynamics and subcellular localization and further understand their physiological functions and working mechanisms1,2,3,4. It is especially beneficial to co-express well-known organelle reporter proteins with the protein in question to better illustrate its spatiotemporal rationale, distribution, and function(s) within the endomembrane system in cells4,5,6,7,8.

A chimeric fluorescent fusion protein can be expressed in plants via transient expression and stable genetic transformation, which have their respective advantages and limitations9,10,11. Transient expression of a protein is a convenient approach that includes biolistic bombardment-, polyethylene glycol (PEG)-, or electroporation-mediated DNA transient expression in protoplasts and Agrobacterium-mediated leaf infiltration in intact plant cells, as shown in Figure 1A,B12,13,14,15,16. However, co-expression of multiple chimeric fluorescent fusion proteins in a single plant cell requires a mixture of several independent expression plasmids. Thus, the drawbacks of employing multiple plasmids for protein co-expression in plants are lower co-expression levels due to the dramatically reduced chance of several plasmids simultaneously entering the same cells when compared to a single plasmid, and the variations of protein expression levels caused by the uncontrollably random amount of each types of plasmid being transferred into the cell17,18. In addition, it is technically challenging to introduce several independent expression plasmids into a single Agrobacterium for protein co-expression9,10,11. Therefore, Agrobacterium-mediated protein transient expression by infiltration of tobacco leaves is only capable of expressing one plasmid at a time, as shown in Figure 1B. In contrast, generation of transgenic plants expressing fluorescent fusion proteins is usually achieved by Agrobacterium that carries a binary transformation vector. However, the binary vector that mediates the gene transfer and insertion into the plant genomes is only capable of expressing a single fluorescent fusion protein (Figure 1B)9,10,12. Generating a transgenic plant which expresses several chimeric fluorescent proteins simultaneously requires multiple rounds of genetic crossing and screening, which can take from months to years depending on the numbers of the genes to be co-expressed.

The employment a single expression vector for co-expression of multiple proteins in plant has been reported by several previous studies19,20,21. However, multiple rounds of enzymatic digestion and DNA ligation of DNA molecules and backbone vectors are usually required for generation of the final plasmid for protein co-expression or over-expression. Here, we have developed a new and robust method for co-expressing multiple chimeric fluorescent proteins in plants. It is a highly efficient and convenient method that achieves multiple protein co-expression in plants for both transient expression and stable transformation in a time-honored fashion. It employs a single vector that contains multiple functionally independent protein expression cassettes for protein co-expression and thereby overcomes the drawbacks of the conventional methods. Moreover, it is a highly versatile system in which DNA manipulations and assembly are achieved by a simple one-step optimized reaction without extra steps of DNA digestion and ligation. The working principle is illustrated in Figure 2. Furthermore, it is fully compatible with current cellular, molecular, and biochemical approaches that are based on chimeric fluorescent fusion proteins.

Protocol

1. Primer Design Strategy and DNA Amplification

  1. Design the primers for molecular cloning of DNA fragments. The primers comprise 20 bp gene specific binding sequences and 20 to 25 bp 5'-end overhang sequences, which are the complementary overlapping sequence of adjacent DNA molecules (see Table 1 for example).
    NOTE: The subsequent assembly of each DNA fragments, linkage of different protein expression cassettes, and integration with the final expression vector all depend on recognition of the adjacent overlapping sequences.
  2. Amplify DNA fragments, including promoter, fluorescent reporter, target gene, and terminator, that are necessary for the construction of the semi-independent protein expression cassettes by standard PCR reactions with their corresponding primers and high fidelity polymerase.
    NOTE: The templates used in this study for DNA amplification are derived from previous studies15,22,23. The annealing temperature and extension time of the PCR reactions are primer and gene dependent.

2. DNA Fragment Assembly and Construction of Protein Expression Cassettes

  1. Examine the quality of the first-round PCR products by DNA electrophoresis and quantify by spectrophotometer. Check whether DNA degradation and contamination have occurred by 1% agarose gel electrophoresis. The OD260/OD280 of the PCR products should be between 1.6 and 1.8.
  2. Mix different DNA fragments (0.05 - 0.1 pmol for each fragment) into a new PCR tube to a final volume of 5 µL.
    NOTE: Mix DNA molecules designed for the same protein expression cassette together in one PCR tube. Avoid mixing DNAs from different expression cassette together, since it will reduce the efficiency of DNA assembly due to the increasing numbers of DNA molecules that need to be linked.
    NOTE: The protocol can be paused here.
  3. Prepare 5x ISO stock buffer: 500 mM Tris-HCl, pH 7.5; 50 mM MgCl2; 1 mM deoxynucleotide (dNTP); 50 mM dithiothreitol; 25% polyethylene glycol (PEG) 8000; and 5 mM nicotinamide adenine dinucleotide (NAD).
  4. Make 1 mL 2x master mixture from 400 µL 5x ISO stock buffer, 7.5 units of T5 exonuclease, 62.5 units of high-fidelity DNA polymerase, 5,000 units of Taq polymerase (see Table of Materials), and sterilized double distilled H2O.
    NOTE: This is adapted from previous studies24,25,26; these volumes should be optimized.
  5. Aliquot 100 µL of 2x master mixture per tube and store at -20 °C.
    Caution: Frequent freezing and thawing of 2x master mixture can cause low DNA assembly efficiency.
  6. Add 15 µL of 2x master mixture to the 5 µL DNA mixture and incubate at 50 °C for 60 min.

3. Construction of the Vector for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

  1. Amplify the entire semi-independent protein expression cassette by a second-round PCR using the product (0.5 - 1.0 µL) from the first-round isothermal assembly reaction as the template and the outermost primers (e.g., 1-FP35S and 1-RNOS for expression cassette 1).
    1. Use 1 unit of high fidelity polymerase in a 50 µL reaction volume followed by 30 cycles (94 °C for 30 s, 55 °C for 30 s, and 68 °C for 2 min), followed by a final extension at 68 °C for 5 min.
  2. Linearize the final protein expression backbone vectors pUC18 and pCAMBIA1300, which are designed for protein transient expression and genetic transformation, respectively, by adding 4 units Sma I into a final 10 µL reaction volume and incubating for 1 - 2 hr at 25 °C. Inactivate the restriction enzyme by incubating at 65 °C for 20 min.
  3. Mix equimolar DNA molecules of protein expression cassettes and linearized final vector into a final reaction volume of 5 µL. Then, perform the second-round DNA recombination by mixing with 15 µL 2x master buffer and incubating at 50 °C for 60 min.
  4. Use the final products of the second-round isothermal recombination reaction to transform competent E. coli cells (e.g., DH5α) according to standard procedures27. Double-check positive colonies by colony PCR and DNA sequencing. Extract the positive plasmids from the E. coli by using a mini plasmid extraction kit and store at -80 °C.
    Caution: For long-term storage, plasmids dissolved in TE buffer or double distilled H2O are more stable than keeping them in E. coli strains.

4. Biolistic-bombardment Mediated Transient Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

  1. Prepare Tobacco BY-2 suspension cells and Arabidopsis juvenile plants for bombardment.
    1. Culture BY-2 cells in Murashige and Skoog (MS) medium by subculturing twice per week at 25 °C in a shaker set at 130 rpm. Filter collect 30-mL 3 d cultured BY-2 cells onto a piece of 70-mm autoclaved filter paper via a vacuum pump by setting the vacuum pressure to 40 mbar.
    2. In order to prevent the cells from drying out during the following steps, add several drops of BY-2 cell liquid cultural medium in a Petri dish before placing the filter paper (next step).
    3. Transfer the filter paper with the BY-2 cells on it to a new Petri dish (85 mm x 15 mm).
    4. Surface sterilize Arabidopsis thaliana (Col-0) seeds by vortexing a mixture with 70% (v/v) ethanol containing 0.05% Tween 20 for 10 min.
    5. Spin down the seeds using a bench top centrifuge at max speed for 2 s, remove the supernatant and wash the seeds with 100% ethanol once for 30 s. Pipette out the seeds onto a new sterile filter paper in a sterile hood. Then, air-dry the seeds and spread them onto ½ MS agar plates.
    6. Store the plates at 4 °C for 48 hours prior to transferring them into a plant growth chamber with the following settings: 16 h light /8 h dark with 120 - 150 µm m-2 s-1 intensity, 22 °C.
    7. Transfer 7-day old sample plants into a 30 mm diameter circle in the center of a new ½ MS medium plate to increase the efficiency of bombardment.
      Caution: Avoid overlapping the plants when transferring and placing them onto the new ½ MS plate.
    8. Add several drops of ½ MS liquid medium on the surface of the plants or tissues to preserve moisture and prevent drying out the plants out during the following steps.
  2. Coat gold particles with plasmid DNA.
    1. Vortex gold microcarrier solution thoroughly, for 3 min. Prepare a new 1.5 mL tube.
    2. Sequentially add the following solutions into the tube and vortex: 25 µL (1.5 mg) gold particles, vortex 10 s; 10 µL of 25.46 mg/L spermidine, vortex 10 s; 5 µL of 1µg/µL plasmid DNA, vortex 3 min; 25 µL of 277.5 mg/L CaCl2 solution, vortex 1 min.
      Caution: Keep vortexing during this step.
    3. Spin down the gold microcarriers using a bench top centrifuge at max speed for 5 s and carefully pipette out the supernatant without disturbing the pellet. Wash with 200 µL of absolute ethanol and re-suspend the pellet by vortex for 5 - 10 s. Spin down at max speed for 5 s and remove the ethanol.
    4. Re-suspend the gold particles in 18 µL of absolute ethanol and aliquot 6 µL particles suspension onto the middle of three macrocarriers. Let them air dry.
  3. Transfer DNA into plants via particle bombardment.
    1. Set the particle delivery system as following: 1100 psi, 28-mm Hg vacuum, 1-cm gap distance, and 9-cm particle flight distance.
    2. Bombard the cells/plants on the agar medium plate for three times at three different positions and then keep the cells/plants in dark immediately after the bombardment.
      Caution: Wear safety glasses when operating the particle delivery system because of the association of high-pressure gas and high-speed particles with the system.
  4. Keep bombarded cells/plants in the dark in the plant growth chamber for 6 to 72 hours prior to observation of fluorescent signals. Set the plant growth chamber to 24 h dark and 22 °C.
    Caution: The expression efficiency and fluorescent signal intensity are promoter-, gene-, and plant/tissue-dependent.

5. Generation of Stable Transgenic Arabidopsis Co-expressing Multiple Chimeric Fluorescent Proteins by Agrobacterium-Mediated Transformation.

  1. Thaw the Agrobacterium competent cells (PMP90) on ice and wait for 30 min, then add 2 µL binary vector (100 - 200 ng) (prepared above) into the competent cells. Sit the mixture on ice for 10 min.
  2. Transfer the mixture into a pre-chilled 0.1 cm electroporation cuvette. Insert cuvette into the electroporation system and perform electroporation with following settings: 1.6 kV, 600 ohms, 25 µF.
  3. Add 1 mL of SOC liquid medium into the cuvette immediately after the electroporation, pipette the cells into a new 1.5 mL tube, and incubate at 28 °C in a horizontal orbital shaker at 200 rpm for 120 min.
  4. Centrifuge the cells at 2,348 x g at room temperature for 5 min, discard the majority of the supernatant, gently re-suspend the pelleted cells with a pipette tip, spread them on a LB plate containing 50 mg/L Hygromycin B, and incubate at 28 °C for 2 - 3 days.
  5. Transform Arabidopsis thaliana Col-0 plants with the Agrobacterium, which contains the binary vector pCAMBIA1300 integrated with multiple protein expression cassettes, by the floral dip28 method as previously described to generate stable transgenic plants.
  6. Sterilize the surface of the transgenic Arabidopsis seeds by mixing them with 70% (v/v) ethanol containing 0.05% Tween 20. Vortex for 10 min. Spin down the seeds using a bench top centrifuge at max speed for 2 s, remove the supernatant, and wash the seeds with 100% ethanol once for 30 s.
  7. Pipette out the seeds onto a sterile filter paper in a sterile hood. Thereafter, air dry and spread them onto ½ MS agar plates containing Hygromycin B for screening positive progenies.
  8. Incubate the plates at 4 °C for 2 days. Then, transfer them into the plant growth chamber and culture for 7 days.
  9. Select 7-day old survival juvenile plants by checking for fluorescent signals under the fluorescent microscope, then transfer the plants into soil for further screening of homozygous plants.

6. Pharmaceutical treatments

  1. For pharmaceutical treatments, dilute each drug in liquid MS medium to its appropriate working concentrations before incubation with cells or plants.
    1. Wortmannin treatment: prepare 1 mM stock solutions of wortmannin by dissolving wortmannin powder in DMSO and store the stocks at -20 °C. Transfer plant cells or juvenile plants into the liquid MS medium containing 16.5 µM wortmammin and incubate for 30 - 45 min before imaging.
    2. Brefeldin A (BFA) treatment: prepare 1 mM stock solutions of BFA by dissolving BFA powder in DMSO and store the stocks at -20 °C. Transfer plant cells or juvenile plants into the liquid MS medium containing 10 µg/mL BFA for 30 - 45 min before imaging.

7. Confocal Microscope Imaging and Protein Subcellular Co-localization Analysis

  1. Transfer the juvenile plants or suspension cells onto a conventional glass slide and gently put a cover slide on the top for imaging by standard confocal laser scanning microscopy. Use the following settings: 63X water objective (N.A 1.4), 0 background, 700 gain, 0.168 mm pixel size, and photomultiplier tube detector. Excite GFP-tagged proteins at 485 nm and detect fluorescence at 525 nm. For RFP-tagged proteins, excite at 514 nm and detect at 575 nm.
  2. Calculate the co-localization ratio of fluorescent signals using Image J software (https://imagej.nih.gov/ij/) with the Pearson-Spearman correlation (PSC) co-localization plug-in as previously described8. Pearson correlation coefficients or Spearman's rank correlation coefficients are shown in the results (Figure 4). The produced r values will be from -1 to 1. 0 demonstrates no detectable correlation of two signals, whereas +1 and -1 show full positive and negative correlation, respectively, of two signals.

Results

We have developed a robust and highly efficient method for the co-expression of multiple chimeric fluorescent fusion proteins in plants. It breaks through the barriers of the conventional approaches use multiple separated plasmids for protein co-expression, as shown in Figure 1A,B, via either transient expression or stable genetic transformation. In this new method, we generate a single expression vector that is composed of multiple ...

Discussion

Here we have demonstrated a novel method to robustly co-express chimeric fluorescent fusion proteins in plants. It can be used for both transient expression and genetic transformation and is compatible with current fluorescent protein-based bio-imaging, molecular, and biochemical applications and technologies9,10,13. In addition, it overcomes the difficulties of the conventional methods that use several individual expression pla...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank the members of the Wang laboratory for helpful discussions and comments. This work is supported by the National Natural Science Foundation of China (NSFC, grant no. 31570001) and the Natural Science Foundation of Guangdong Province and Guangzhou City (grant no. 2016A030313401 and 201707010024) to H.W.

Materials

NameCompanyCatalog NumberComments
KOD-FX PolymeraseTOYOBOKFX-101
Sma INEBR0141L/S/V
Tris-HClBBIA600194-0500
MgCl2BBIA601336-0500
dNTPNEB#N0447V
DTTBBIC4H10O2S2
PEG 8000BBIA100159-0500
NADBBIA600641-0001
T5 exonucleaseEpicentreT5E4111K
Phusion High-Fidelity DNA polymeraseNEBM0530S
Taq DNA polymeraseNEBB9022S
Murashige and Skoog Basal Salt Mixture(MS)SigmaM5524
EthanolBBIA500737-0500
Tween 20BBIA600560-0500
AgarBBIA505255-0250
SpermidineBBIA614270-0001
Gold microcarrier particlesBio-Rad165-22631.0 µm
CaCl2BBICD0050-500
MacrocarriersBio-Rad165-2335
Rupture diskBio-Rad165-2329
Stopping screenBio-Rad165-2336
TryptoneOXOIDLP0042
Yeast ExtractOXOIDLP0021
NaClBBIA610476-0001
KClBBIA610440-0500
GlucoseBBIA600219-0001
Hygromycin BGenviewAH169-1G
WortmanninSigmaF9128
Brefeldin ASigmaSML0975-5MG
Dimethylsulphoxide (DMSO)BBIA600163-0500
T100 Thermal CyclerBio-Rad1861096
Growth chamberPanasonicMLR-352H-PC
PSD-1000/He particle delivery systemBio-Rad165-2257
Gene PulserBio-Rad1652660
CuvetteBio-Rad1652083
Benchtop centrifugeEppendorf5427000097
Confocal microscopeZeissLSM 7 DUO (780&7Live)
NanoDrop 2000/2000c SpectrophotometersThermo Fisher ScientificND-2000
EPS-300 Power SupplyTanonEPS 300
Fluorescent microscopeMshotMF30
AgroseBBIA600234
AmpicillinBBIA100339
Ethylene Diamine Tetraacetie AcidBBIB300599

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