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 recognit...

<ol>
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H., Hillmer, S., Robinson, D. G., Jiang, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuolar+sorting+receptor+(VSR)+proteins+reach+the+plasma+membrane+in+germinating+pollen+tubes.">Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes.</a> Mol Plant. 4 (5), 845-853 (2011).</li><li>Chen, W. X., 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=Rapid+in+vitro+splicing+of+coding+sequences+from+genomic+DNA+by+isothermal+recombination+reaction-based+PCR.">Rapid in vitro splicing of coding sequences from genomic DNA by isothermal recombination reaction-based PCR.</a> Biotechnology &amp; Biotechnological Equipment. 30 (5), 864-868 (2016).</li><li>Zhu, Q. L., Yang, Z. F., Zhang, Q. Y., Chen, L. T., Liu, Y. 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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>MgCl2</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 &#181;m</td>
		</tr>
		<tr>
			<td>CaCl2</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&#38;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 ...

This method can help answer key questions in the plant molecular and cell biology field such as, how can we proteins in the cell. The main advantage of this technique is the high efficiency of co-expressing cellular fusion proteins in one expression vector. Demonstrating the procedure will be Guitao Zhong, a graduate student from our lab.To begin, design the primers for molecular cloning of DNA fragments as described in the text protocol. Amplify DNA fragments 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. These include the promoter, fluorescent reporter, target gene, and terminator.Examine the quality of the first round PCR products by looking for DNA degradation and contamination with DNA electrophoresis using a 1%agarose gel. Quantify the PCR products with a spectrophotometer. The ratio between the readings at 260 and 280 nanometers of the PCR products should be between 1.6 and 1.8.Mix the DNA fragments designed for the same protein expression cassette together in one PCR tube to a final volume of five microliters. About mixing DNS from different expression cassette data. Since equal reduces the efficiency of DNA assembly, due to increasing numbers of DNA molecules that need to be linked.Now, add 15 microliters of 2x master mixture to the 5 microliter DNA mixture and incubate at 50 degrees celsius for 60 minutes. Amplify the entire semi-independent protein expression cassette by a second round of PCR. Use 0.5 to one microliters of the product from the first round isothermal assembly reaction as the template in the outermost primers.Use one unit of high-fidelity polymerase in a 50 microliter reaction volume for 30 cycles, followed by a final extension at 68 degrees celsius for five minutes. Next, linearize the final protein expression backbone vector POC 18, and vector CAMBIA 1300, by adding four units of small one into a final 10 microliter reaction volume. These vectors are designed for protein transient expression and genetic transformation.Incubate the restriction digest for one to two hours at 25 degrees celsius. Following digestion, inactivate the restriction enzyme by incubating at 65 degrees celsius for 20 minutes. Next, mix equimolar DNA molecules of protein expression cassettes and linearized final vector into a final reaction volume of five microliters.Finally, perform the second round of DNA recombination by mixing the reaction with 15 microliters of 2x master buffer, and incubating at 50 degrees celsius for 60 minutes. To prepare tobacco BY-2 suspension cells for bombardment, filter-collect 30 milliliters of 3-day cultured BY-2 cells onto a piece of 70 milliliter autoclaved filter paper via a vacuum pump, by setting the vacuum pressure to 40 millibar. Meanwhile, add several drops of BY-2 cell liquid cultural medium into a petri dish.Transfer the filter paper with the BY-2 cells on it to the petri dish. To prepare arabidopsis juvenile plants for bombardment, prepare seven-day-old sample plants as detailed in the text protocol. Then, transfer them into a rectangle in the center of a new half-strength MS medium plate to increase the efficiency of bombardment.Take care to avoid overlapping the plants when transferring and placing them onto the plate. Add several drops of half-strength MS liquid medium on the surface of the plants or tissues to preserve moisture and prevent drying the plants out during the remaining steps. To coat gold particles with plasmid DNA, first vortex the gold microcarrier solution thoroughly for three minutes.Now, add 25 microliters of gold particles to a new 1.5 milliliter tube, and vortex for 10 seconds. Add 10 microliters of 25.46 milligrams per liter spermidine and vortex for 10 seconds. Next, add five microliters of one microgram per microliter plasmid DNA, and vortex for three minutes.Finally, add 25 microliters of 277.5 milligrams per liter calcium chloride solution, and vortex for one minute. Spin down the gold microcarriers using a bench-top centrifuge at maximum speed for five seconds. Following centrifugation, carefully pipe it out the supernatent without disturbing the pellet.Next, wash the pellet with 200 microliters of absolute ethanol, and re-suspend the pellet by vortexing for five to 10 seconds. Once gain, spin down the gold microcarriers at maximum speed for five seconds, and remove the ethanol. Re-suspend the gold particles in 18 microliters of absolute ethanol.Then, aliquot six microliters of particle suspension onto the middle of three microcarriers and let them air dry. To transfer the DNA into the cells and plants via particle bombardment, first set the particle delivery system as detailed in the text protocol. Then, bombard the cells or plants on the agromedium plate for three times at three different positions.Keep the bombarded cells and plants in the dark in the plant growth chamber for six to 72 hours prior to observation of fluorescent signals. Set the plant growth chamber to 24 hours dark and 22 degrees celsius. 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.Using the settings listed in the text protocol, excite GFP tagged proteins at 485 nanometers, and detect fluorescence at 525 nanometers. For RFP tagged proteins, excite at 585 nanometers and detect at 608 nanometers. Finally, calculate the collocalization ratio of fluorescent signals as described in the text protocol.Co-expression of arabidopsis VSR-2 and arabidopsis SCAMP4 in tobacco BY-2 cells was achieved via particle bombardment and show correct localizations. RFP arabidopsis VSR-2 displays a punctate pattern, which was distinct from the plasma membrane localization of arabidopsis SCAMP4-GFP, with some cytosolic punctate dots. Moreover, arabidopsis transgenic plants that co-express arabidopsis SCAMP4-GFP and RFP arabidopsis VSR-2 were generated via agrobacteria-mediated transformation.The subcellular localizations of co-expressing the two chimeric proteins in root and root hair cells is shown. Consistent with pervious studies, treatment of transgenic arabidopsis caused RFP arabidopsis VSR-2 labeled prevacualar compartments, forming a small ring-like structure. Whereas treatment with prefoldin A induced arabidopsis SCAMP GFP labeled transgold G-network aggregation.As a negative control, little autofluorescent signal is observed in tobacco BY-2 cells and arabidopsis root and root hair cells by applying the same settings of image collection. This method can provide insight into the subcellular localization of the proteins. It can also be applied to the study of protein in direction.After its development, this technique paved the way for researchers in the field of plant cell biology. To conveniently export the subcellular localizations and spacial interaction of proteins in the living plant cell. Following this procedure, other methods like PET mediated transformation and genetic crossing can be performed in order to answer additional questions like, how to co-express several fusion proteins in plants.Don't forget that working with bombardment can be extremely hazardous and precautions such as eye protectors should always be taken while performing this procedure.

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9.5K visualizações.  South China Agricultural University. This method can help answer key questions in the plant molecular and cell biology field such as, how can we proteins in the cell. The main advantage of this technique is the high efficiency of co-expressing cellular fusion proteins in one expression vector. Demonstrating the procedure will be Guitao Zhong, a graduate student from our lab.To begin, design the primers for molecular cloning of DNA fragments as described in the text protocol. Amplify DNA fragments that are necessary for th...