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.

1. Primer Design Strategy and DNA Amplification
<ol>
	<li>Design the primers for molecular cloning of DNA fragments. The primers comprise 20 bp gene specific binding sequences and 20 to 25 bp 5&#39;-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.</li>
	<li>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.</li>
</ol>
2. DNA Fragment Assembly and Construction of Protein Expression Cassettes
<ol>
	<li>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.</li>
	<li>Mix different DNA fragments (0.05 - 0.1 pmol for each fragment) into a new PCR tube to a final volume of 5 &#181;L. 
	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.</li>
	<li>Prepare 5x&#160;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).</li>
	<li>Make 1 mL 2x master mixture from 400 &#181;L 5x&#160;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.</li>
	<li>Aliquot 100 &#181;L of 2x master mixture per tube and store at -20 &#176;C. 
	Caution: Frequent freezing and thawing of 2x&#160;master mixture can cause low DNA assembly efficiency.</li>
	<li>Add 15 &#181;L of 2x master mixture to the 5 &#181;L DNA mixture and incubate at 50 &#176;C for 60 min.</li>
</ol>
3. Construction of the Vector for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants
<ol>
	<li>Amplify the entire semi-independent protein expression cassette by a second-round PCR using the product (0.5 - 1.0 &#181;L) 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).
	<ol>
		<li>Use 1 unit of high fidelity polymerase in a 50 &#181;L reaction volume followed by 30 cycles (94 &#176;C for 30 s, 55 &#176;C for 30 s, and 68 &#176;C for 2 min), followed by a final extension at 68 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>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 &#181;L reaction volume and incubating for 1 - 2 hr at 25 &#176;C. Inactivate the restriction enzyme by incubating at 65 &#176;C for 20 min.</li>
	<li>Mix equimolar DNA molecules of protein expression cassettes and linearized final vector into a final reaction volume of 5 &#181;L. Then, perform the second-round DNA recombination by mixing with 15 &#181;L 2x master buffer and incubating at 50 &#176;C for 60 min.</li>
	<li>Use the final products of the second-round isothermal recombination reaction to transform competent E. coli cells (e.g., DH5&#945;) 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 &#176;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.</li>
</ol>
4. Biolistic-bombardment Mediated Transient Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants
<ol>
	<li>Prepare Tobacco BY-2 suspension cells and Arabidopsis juvenile plants for bombardment.
	<ol>
		<li>Culture BY-2 cells in Murashige and Skoog (MS) medium by subculturing twice per week at 25 &#176;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.</li>
		<li>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).</li>
		<li>Transfer the filter paper with the BY-2 cells on it to a new Petri dish (85 mm x 15 mm).</li>
		<li>Surface sterilize Arabidopsis thaliana (Col-0) seeds by vortexing a mixture with 70% (v/v) ethanol containing 0.05% Tween 20 for 10 min.</li>
		<li>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 &#189; MS agar plates.</li>
		<li>Store the plates at 4 &#176;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 &#181;m m-2 s-1 intensity, 22 &#176;C.</li>
		<li>Transfer 7-day old sample plants into a 30 mm diameter circle in the center of a new &#189; MS medium plate to increase the efficiency of bombardment. 
		Caution: Avoid overlapping the plants when transferring and placing them onto the new &#189; MS plate.</li>
		<li>Add several drops of &#189; 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.</li>
	</ol>
	</li>
	<li>Coat gold particles with plasmid DNA.
	<ol>
		<li>Vortex gold microcarrier solution thoroughly, for 3 min. Prepare a new 1.5 mL tube.</li>
		<li>Sequentially add the following solutions into the tube and vortex: 25 &#181;L (1.5 mg) gold particles, vortex 10 s; 10 &#181;L of 25.46 mg/L spermidine, vortex 10 s; 5 &#181;L of 1&#181;g/&#181;L plasmid DNA, vortex 3 min; 25 &#181;L of 277.5 mg/L CaCl2 solution, vortex 1 min. 
		Caution: Keep vortexing during this step.</li>
		<li>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 &#181;L 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.</li>
		<li>Re-suspend the gold particles in 18 &#181;L of absolute ethanol and aliquot 6 &#181;L particles suspension onto the middle of three macrocarriers. Let them air dry.</li>
	</ol>
	</li>
	<li>Transfer DNA into plants via particle bombardment.
	<ol>
		<li>Set the particle delivery system as following: 1100 psi, 28-mm Hg vacuum, 1-cm gap distance, and 9-cm particle flight distance.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C. 
	Caution: The expression efficiency and fluorescent signal intensity are promoter-, gene-, and plant/tissue-dependent.</li>
</ol>
5. Generation of Stable Transgenic Arabidopsis Co-expressing Multiple Chimeric Fluorescent Proteins by Agrobacterium-Mediated Transformation.
<ol>
	<li>Thaw the Agrobacterium competent cells (PMP90) on ice and wait for 30 min, then add 2 &#181;L binary vector (100 - 200 ng) (prepared above) into the competent cells. Sit the mixture on ice for 10 min.</li>
	<li>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 &#181;F.</li>
	<li>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 &#176;C in a horizontal orbital shaker at 200 rpm for 120 min.</li>
	<li>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 &#176;C for 2 - 3 days.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Pipette out the seeds onto a sterile filter paper in a sterile hood. Thereafter, air dry and spread them onto &#189; MS agar plates containing Hygromycin B for screening positive progenies.</li>
	<li>Incubate the plates at 4 &#176;C for 2 days. Then, transfer them into the plant growth chamber and culture for 7 days.</li>
	<li>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.</li>
</ol>
6. Pharmaceutical treatments
<ol>
	<li>For pharmaceutical treatments, dilute each drug in liquid MS medium to its appropriate working concentrations before incubation with cells or plants.
	<ol>
		<li>Wortmannin treatment: prepare 1 mM stock solutions of wortmannin by dissolving wortmannin powder in DMSO and store the stocks at -20 &#176;C. Transfer plant cells or juvenile plants into the liquid MS medium containing 16.5 &#181;M wortmammin and incubate for 30 - 45 min before imaging.</li>
		<li>Brefeldin A (BFA) treatment: prepare 1 mM stock solutions of BFA by dissolving BFA powder in DMSO and store the stocks at -20 &#176;C. Transfer plant cells or juvenile plants into the liquid MS medium containing 10 &#181;g/mL BFA for 30 - 45 min before imaging.</li>
	</ol>
	</li>
</ol>
7. Confocal Microscope Imaging and Protein Subcellular Co-localization Analysis
<ol>
	<li>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&#160;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.</li>
	<li>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&#39;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.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-adapted+green+fluorescent+protein+is+a+versatile+vital+reporter+for+gene+expression,+protein+localization+and+mitosis+in+the+filamentous+fungus,+Aspergillus+nidulans.">Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.</a> Mol Microbiol. 27 (1), 121-130 (1998).</li><li>Bruening, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+gene+silencing+regularized.">Plant gene silencing regularized.</a> Proc Natl Acad Sci U S A. 95 (23), 13349-13351 (1998).</li><li>Wassenegger, M., Pelissier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+RNA-mediated+gene+silencing+in+higher+plants.">A model for RNA-mediated gene silencing in higher plants.</a> Plant Mol Biol. 37 (2), 349-362 (1998).</li><li>Dehio, C., Schell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+plant+genetic+loci+involved+in+a+posttranscriptional+mechanism+for+meiotically+reversible+transgene+silencing.">Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing.</a> Proc Natl Acad Sci U S A. 91 (12), 5538-5542 (1994).</li><li>Zhong, G., Zhu, Q., Li, Y., Liu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Once+for+all:+A+novel+robust+system+for+co-expression+of+multiple+chimeric+fluorescent+fusion+proteins+in+plants.">Once for all: A novel robust system for co-expression of multiple chimeric fluorescent fusion proteins in plants.</a> Front Plant Sci. 8, 1071 (2017).</li><li>Li, X. T., Xie, Y. Y., Zhu, Q. L., Liu, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+editing+in+genes+and+cis-regulatory+regions+improves+qualitative+and+quantitative+traits+in+crops.">Targeted genome editing in genes and cis-regulatory regions improves qualitative and quantitative traits in crops.</a> Molecular Plant. 10 (11), 1368-1370 (2017).</li><li>Zhu, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+&amp;#34;purple+endosperm+rice''+by+engineering+anthocyanin+biosynthesis+in+the+endosperm+with+a+high-efficiency+transgene+stacking+system.">Development of &amp;#34;purple endosperm rice'' by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system.</a> Molecular Plant. 10 (7), 918-929 (2017).</li><li>Tang, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+formation,+evolution,+and+functionalization+of+new+cytoplasmic+male+sterility+genes+in+the+plant+mitochondrial+genomes.">Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes.</a> Cell Research. 27 (1), 130-146 (2017).</li></ol>

The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>KOD-FX Polymerase</td><td>TOYOBO</td><td>KFX-101</td><td></td></tr><tr><td>Sma I</td><td>NEB</td><td>R0141L/S/V</td><td></td></tr><tr><td>Tris-HCl</td><td>BBI</td><td>A600194-0500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>BBI</td><td>A601336-0500</td><td></td></tr><tr><td>dNTP</td><td>NEB</td><td>#N0447V</td><td></td></tr><tr><td>DTT</td><td>BBI</td><td>C4H10O2S2</td><td></td></tr><tr><td>PEG 8000</td><td>BBI</td><td>A100159-0500</td><td></td></tr><tr><td>NAD</td><td>BBI</td><td>A600641-0001</td><td></td></tr><tr><td>T5 exonuclease</td><td>Epicentre</td><td>T5E4111K</td><td></td></tr><tr><td>Phusion High-Fidelity DNA polymerase</td><td>NEB</td><td>M0530S</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>NEB</td><td>B9022S</td><td></td></tr><tr><td>Murashige and Skoog Basal Salt Mixture(MS)</td><td>Sigma</td><td>M5524</td><td></td></tr><tr><td>Ethanol</td><td>BBI</td><td>A500737-0500</td><td></td></tr><tr><td>Tween 20</td><td>BBI</td><td>A600560-0500</td><td></td></tr><tr><td>Agar</td><td>BBI</td><td>A505255-0250</td><td></td></tr><tr><td>Spermidine</td><td>BBI</td><td>A614270-0001</td><td></td></tr><tr><td>Gold microcarrier particles</td><td>Bio-Rad</td><td>165-2263</td><td>1.0 &amp;micro;m</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>BBI</td><td>CD0050-500</td><td></td></tr><tr><td>Macrocarriers</td><td>Bio-Rad</td><td>165-2335</td><td></td></tr><tr><td>Rupture disk</td><td>Bio-Rad</td><td>165-2329</td><td></td></tr><tr><td>Stopping screen</td><td>Bio-Rad</td><td>165-2336</td><td></td></tr><tr><td>Tryptone</td><td>OXOID</td><td>LP0042</td><td></td></tr><tr><td>Yeast Extract</td><td>OXOID</td><td>LP0021</td><td></td></tr><tr><td>NaCl</td><td>BBI</td><td>A610476-0001</td><td></td></tr><tr><td>KCl</td><td>BBI</td><td>A610440-0500</td><td></td></tr><tr><td>Glucose</td><td>BBI</td><td>A600219-0001</td><td></td></tr><tr><td>Hygromycin B</td><td>Genview</td><td>AH169-1G</td><td></td></tr><tr><td>Wortmannin</td><td>Sigma</td><td>F9128</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>SML0975-5MG</td><td></td></tr><tr><td>Dimethylsulphoxide (DMSO)</td><td>BBI</td><td>A600163-0500</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>Growth chamber</td><td>Panasonic</td><td>MLR-352H-PC</td><td></td></tr><tr><td>PSD-1000/He particle delivery system</td><td>Bio-Rad</td><td>165-2257</td><td></td></tr><tr><td>Gene Pulser</td><td>Bio-Rad</td><td>1652660</td><td></td></tr><tr><td>Cuvette</td><td>Bio-Rad</td><td>1652083</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>5427000097</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>LSM 7 DUO (780&amp;amp;7Live)</td></tr><tr><td>NanoDrop 2000/2000c Spectrophotometers</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>EPS-300 Power Supply</td><td>Tanon</td><td>EPS 300</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Mshot</td><td>MF30</td><td></td></tr><tr><td>Agrose</td><td>BBI</td><td>A600234</td><td></td></tr><tr><td>Ampicillin</td><td>BBI</td><td>A100339</td><td></td></tr><tr><td>Ethylene Diamine Tetraacetie Acid</td><td>BBI</td><td>B300599</td><td></td></tr></tbody></table>

co-expression of multiple chimeric fluorescent fusion proteins in an efficient way in plants

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.

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 ...

Watch this Scientific Journal Video about Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants at JoVE.com

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

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.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

co-expression-multiple-chimeric-fluorescent-fusion-proteins-an

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

9.6K Views.  South China Agricultural University. 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.

Video: Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Environment

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetics

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Immunology and Infection

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Biology

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

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Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection