We thank Yumeng (Helen) Xia (Rutgers University) for manuscript editing. The work on BASL polarity formation is supported by grants from the U.S. National Institute of General Medical Sciences to J.D. (R01GM109080) and Rutgers University.

1. Plasmid Construction
<ol>
	<li>Generate the constructs by cloning technology as previously described1,6.
	<ol>
		<li>First use a high fidelity DNA polymerase with appropriate primers to amplify the coding sequences of BASL, YDA and MPK6 and sub-clone them into entry vectors. Find the detailed protocol in 7.</li>
		<li>To generate the dominant negative versions of MPK6 and YDA (DNmpk68 and DNyda4, respectively), use the entry plasmids of MPK6 and YDA as template and introduce the point mutations by a site-directed mutagenesis method9.</li>
	</ol>
	</li>
	<li>To build the constructs for tobacco transient assays, conduct standard LR (attL x attR recombination) reactions (detailed procedure in 7) to integrate BASL into pH35CG10, DNyda and DNmpk6 into pXNGW and pXCGW vectors11, respectively.
	<ol>
		<li>Confirm the resultant plasmids by enzyme digestion and sequencing. Once successful, transform the binary constructs into the Agrobacterium tumefaciens strain GV310112 and grow them on selection plates at 28 &#176;C for 2 - 3 days.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Agrobacterium Infiltration Solution
<ol>
	<li>Inoculate a few freshly transformed Agrobacterium colonies into 10 ml of Luria-Bertani (LB) medium with appropriate antibiotics (Gentamycin 25 &#956;g/ml, Rifamycin 25 &#956;g/ml,&#160; Spectinomycin 100 &#956;g/ml for pH35CG, pXNGW and pXCGW constructs; the same concentrations of Gentamycin and Rifamycin with Kanamycin 50 &#956;g/ml for P19 (expressing proteins against transgenic silencing)13.</li>
	<li>Grow Agrobacterium cultures in a 28 &#176;C shaker at the speed of 200 rpm for about 16 hr and then measure the OD600 (estimated to reach 1.5 - 1.8) (Figure 2A-B). Spin down the cultures at 2,500 x g for 10 min. Re-suspend and wash the cell pellets with 10 mM of MgCl2 (the infiltration solution).</li>
	<li>Spin down the cultures again and re-suspend the pellets with appropriate amount of the infiltration solution to reach the final OD600 = 0.5 (Figure 2C-D). Before infiltrating plants, leave the cell mixtures at room temperature for 1 - 3 hr. This step helps to maintain cell homeostasis.</li>
</ol>
3. Tobacco Plant and Leaf Injection
<ol>
	<li>Use tobacco plants (Nicotiana benthamiana), grown 4 - 5 week-old in a standard plant growth chamber (23 &#176;C with cycles of 16 hr-light/8 hr-dark)14 for leaf injection. 
	Note: At this stage, tobacco plants typically have 6 leaves (2 large, 2 medium, and 2 small). The two medium-sized leaves are the most optimal for leaf injection (the two large ones are too old, while the two small ones are often too young).</li>
	<li>Before the infiltration day, water the tobacco plants to saturate the soil.&#160;</li>
	<li>On the infiltration day, pre-incubate the plants in the dark for 1 - 3 hr. This step allows stomata in the epidermis to open widely, which greatly facilitates the Agrobacterium when penetrating into the leaf epidermal cells.</li>
	<li>Using color tapes labeled with dates, flag the leaves to be infiltrated. Mark the areas to be infiltrated with a black thick sharpie (optional; the infiltrated areas are visible after being infected.).</li>
	<li>Take equal volumes of the Agrobacterium re-suspension and mix them in a 100 x 15 mm Petri dish by gentle swirling (Figure 2E). Note: In our case, we mixed 1 ml of p19 with 1 ml of CFP-BASL, 1 ml of DNyda-nYFP and 1 ml of DNmpk-cYFP.</li>
	<li>In the process of infiltration, fill a 3 ml needleless syringe with the infiltration mixture (1 - 2 ml) and push into the bottom side (abaxial) of the tobacco leaf (Figure 2F). While infiltrating, it is helpful to use one hand to push and the other hand to gently support the upper side (adaxial, against the pushing force). 
	Note: Once the infiltration mixture is successfully injected, the infected areas will become easily recognizable in the epidermis. However, the infiltration may fail due to harsh injection (tissue damage) or insufficient push (no liquid penetration). For each test, we suggest at least two injections in two independent leaves (from different plants).</li>
	<li>Place the infected plants back into the growth chamber. Generally, protein expressions are detectable after 48 hr following infiltration.</li>
</ol>
4. Confocal Imaging&#160;
<ol>
	<li>At 48 hr post infiltration, use a hole puncher to excise a leaf disk from the injected area (normally 3 - 4 disks/area can be produced) (Figure 2G). Use tweezers to gently transfer the leaf disks to a slide (the abaxial side upward) and mount them with water drops. Avoid pressing too hard on the cover slip because squeezed/damaged cells will not show well under the confocal microscope (Figure 2H).</li>
	<li>Before confocal imaging, scan the mounted samples on an epi-fluorescent compound microscope to check the expression levels. Note: Based on our experience, the cells that show intermediate expression levels are best represented under the confocal microscope.</li>
	<li>Start with the 40X (NA 1.3) lens on a confocal microscope. Adjust the slide to the desired position so that the cells expressing medium levels of florescent protein remain at the center. The excitation/emission spectrums for CFP and YFP are 458 nm / 480 - 500 nm and 514 nm / 520 - 540 nm, respectively.</li>
	<li>Activate the &#34;Live&#34; button to preview the images then adjust laser intensity and smart gain for the most balanced fluorescence intensity/noise ratio. To improve imaging quality, increase scanning pixels and/or frame/line average. Finally, adjust the focusing knob to reach the median focal plane and click &#34;Capture&#34; to take the image. 
	Note: The CFP and YFP images can be captured sequentially or simultaneously by checking on or off the option of &#34;sequential&#34; scanning mode, respectively.</li>
	<li>When Z-projection is desired for the in-depth view of the cells, select &#34;xyz&#34; imaging mode, define the depth of the scanning range (10 - 15 &#181;m) and collect the images from the top to the bottom of the target cell. Compile the obtained images with the Leica software by right clicking on the image file to select &#34;rename&#34; and export images as .tiff files.</li>
	<li>Image 30 - 40 cells for quantification analysis.</li>
</ol>
5. Image Processing and Quantification Analysis
<ol>
	<li>Use Fiji software (http://fiji.sc/Fiji) to process images and conduct quantification.
	<ol>
		<li>Plot the fluorescence intensity graphs.
		<ol>
			<li>To better visualize whether the CFP/YFP expression is uniformly distributed, use Fiji to demonstrate the signal strength of CFP and YFP along the cell periphery. To achieve this, first launch Fiji then select &#34;File&#34; and &#34;Open&#34; to start the image. Click &#34;line&#34; on the tool menu and select &#34;Segmented line&#34; by right clicking. Decide the region of interest and draw a line along the cell periphery to trace the CFP or the YFP signal (Figure 3A-C). 
			Note: Fiji measures the absolute fluorescence intensity value along the segmented lines.</li>
			<li>Select the drop-down menu &#34;Analyze&#34; and &#34;Plot Profile&#34; to generate a graph with position and intensity values that represent the levels of CFP/YFP along the segmented line. If desired, duplicate the intensity values (by selecting the menu &#34;Copy&#34;) into EXCEL or other graphic software to generate comparable intensity graphs.</li>
		</ol>
		</li>
		<li>Quantify polarity degree.
		<ol>
			<li>Collect approximately 20 representative cells from 3 replicate experiments to evaluate the polarity degree of CFP and YFP in tobacco cells. Calculate the polarity degree based on the ratio of the high fluorescence intensity (defined as H) over the low fluorescence intensity (defined as L). Note: The quantification method is explained in Figure 3.
			<ol>
				<li>To measure the H and L values, draw a segmented line along the interested region at the cell periphery (described above), click &#34;Analyze&#34; from the drop-down menu, and then select &#34;Measure&#34;. When a &#34;Results&#34; window pops up, use &#34;Mean&#34; values to perform quantification.</li>
				<li>From the cells displaying relatively uniform expression of CFP and YFP, e.g., CFP-BASL (Figure 3A) or the YFP complemented from co-expression of DNyda-nYFP and DNmpk6-cYFP (Figure 3B), obtain the H and L values by measuring two randomly selected peripheral segments with equal length.</li>
				<li>From the cells exhibiting obvious polar accumulation of fluorescent proteins, in this case, the co-expression of CFP-BASL, DNyda-nYFP and DNmpk6-cYFP (Figure 3C), collect the H values from the peripheral regions with enriched fluorescence signals and the Ls from the regions with low/no fluorescence accumulation.</li>
				<li>Pool the resultant ratios of H/L from 20 cells together and test for normal distribution. Calculate the p values by the Student&#39;s t test (for normal distribution) or the Kolmogorov-Smirnov (K-S) test (for non-normal distribution).</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

General Use and Possible Modifications of the Tobacco Transient Expression System for Signal Transduction: Overexpression of fluorescent protein (FP)-tagged proteins in the tobacco epidermis has been successfully used for fast examination of protein subcellular localization in plant cells. However, studying dynamic signaling distribution of protein complex in planta is a challenging topic due to complicated subcellular contexts, transient protein-protein interaction and signal transduction events. To address this challenge, we took advantage of the BiFC system to examine MAPK signaling events. By co-expressing YDA-nYFP and MPK6-cYFP, the recovered YFP signal indicated that the activation of the MAPK signaling relay might occur at the plasma membrane.
It would be ideal to co-express fluorescently tagged YDA and MPK6 in Arabidopsis and to examine the dynamic MAPK signaling in vivo. However, generating transgenic plants is time-consuming, particularly when more than one constructs is desired. Protein transient expression in tobacco cells can be a quick preliminary test for the in vivo expression in Arabidopsis or other plants. The other advantage that the tobacco transient expression system offers, but cannot be easily achieved by generating transgenic plants, is that up to 4 or 5 fusion proteins can be co-expressed simultaneously and examined by confocal microscopy when the combination is achievable.
Using tobacco epidermal cells to investigate MAPK signaling in plant cells can be further implemented to other signaling pathways and under different environmental conditions. For example, would the interaction of YDA-MAPK change its intensity or location when the expressing cell is treated with local H2O2 application (a MAPK signaling stimulus)? Such assays can be quickly designed and performed to address specific questions. Multiple members exist in each tier of the MAPK cassettes (3 - 4 tiers in general), but how signaling specificity is achieved in plants or other systems remain largely unknown. By making different combinations of MAPKKKs with MAPKKs, or MAPKKs with MAPKs, the tobacco transient expression system allows a relatively large-scale analysis of not only protein-protein interaction, but also the spatial organization of the signaling flow in plant cells.
Customized Co-expression and BiFC Assay for Investigation of Protein Polarization: One long-standing fundamental question in cell biology is how universal signaling pathways, e.g., MAPKs, achieve their specificity at certain developmental stage or under particular environmental conditions. Co-expressing the polarity protein BASL with the BiFC components of YDA and MPK6 in tobacco cells has allowed us to demonstrate that the YDA-MAPK interaction and signaling is spatially re-distributed by the presence of BASL, a specific polarity proteins during stomatal ACD. Therefore, the YDA-MAPK signaling specificity can be achieved by interaction with BASL to promote cell polarity and asymmetric division.
Although it is not a universal phenomenon, quite a few proteins have been found to be unevenly distributed in plant cells, e.g., small GTPase ROPs15, auxin effluxer PIN proteins16 and their regulators17, boron transporters18 and some unknown proteins (OCTOPUS19, POLAR20, and BRX21). This protocol was not intended to search the genetic or physical partners in a particular protein polarization pathway, but will likely be useful for testing whether the interacting proteins with ROPs, PINs or others may form a polarity complex in plant cells. However, when trying to simulate a polarization event using tobacco cells, one should keep in mind that complications may occur in the assays. For example, the Arabidopsis polarization event may not be recapitulated in tobacco cells, in particular when more components are required than what can be accommodated in the assay. Some unknown molecules derived from the tobacco background, not necessarily the proteins under examination, may be involved in inducing a polarization event. Therefore, it is essential to set up strict control experiments in tobacco and to validate the data in Arabidopsis or other plants.
Key Elements to Succeed in the Experiments: Firstly, greenish and healthy tobacco plants should be used. Since quantification analyses need to pool data from various cells, it is crucial to use thriving tobacco plants with consistently healthy cells. Secondly, the cells expressing medium protein levels should be used for confocal imaging. Low expression levels may not provide sufficient proteins for the assay and saturated expression levels would mask the polarization/translocation effect.

Proteins interact within an intricate hierarchical network and form complexes that play a pivotal role in nearly all biological processes occurring in an unpredictable cellular environment. However, from plant development to growth responses, there has been a lack of convenient tools that can not only quickly but also efficiently identify and monitor these dynamic signaling events at the subcellular level.
The transient protein expression system in tobacco leaf epidermis has appealing advantages for visualizing fluorescent proteins in living cells. This system provides semi-in vivo conditions that allow post-translational protein modification and quick examination of protein localization. By examining the complemented YFP signal, the bimolecular fluorescent complementation (BiFC) assay informs the possibility of protein-protein interaction in plant cells. Compared to other methods, e.g., yeast-two hybrid (Y2H) and co-immunoprecipitation (Co-IP), BiFC also provides a powerful means of visualizing compartments where protein-protein interactions may occur at the subcellular level.
Because asymmetric cell division (ACD) maintains stem cell population while generating new cell types for tissue/organ formation, it is an indispensable mechanism for promoting eukaryotic multicellularity. Arabidopsis stomatal development has been used as a model system for studying ACD in plants. The precursor cell, meristemoid mother cell, divides asymmetrically to produce two different daughter cells, a meristemoid (undergoes stem cell-like divisions before terminating into a pair of guard cells) and a stomatal lineage ground cell (SLGC) (may divide and differentiate into a pavement cell), respectively (Figure 1). In stomatal ACD, the novel protein Breaking of Asymmetry in the Stomatal Lineage (BASL) is polarized premitotically to drive division asymmetries, which include physical asymmetry and cell fate asymmetry1. A MAPK cascade composed of the MAPKKK YODA and the MAPKs, MPK3 and 6 is central for stomatal division patterning and fate adoption2,3, 4,5.
Recently, Zhang et al. linked the polarity protein BASL to the YDA-MAPK signaling pathway in Arabidopsis stomatal ACD 6. The canonical YODA-MAPK pathway, through MPK3/6, phosphorylates BASL and activates its polarization. Phosphorylated BASL functions as a scaffold and recruits YODA (YDA) and MPK3/6 to form a protein complex and concentrate the signaling at the cell cortex6. Polarization of the MAPK components and the positive feedback loop between BASL and the YDA-MAPK pathway represents a novel mechanism for protein polarization in plant cells. Locally enriched MAPK signaling is hypothesized to be closely linked to cell fate differentiation in stomatal ACD6 (Figure 1). One of the key experimental data that supported this model came from the tobacco assays that demonstrated the spatial redistribution of MAPKs induced by the expression of BASL6.
In general, it is not easy to monitor where MAPK signaling occurs because MAPK molecules were often found everywhere inside of a cell. In this study, we utilized the split-YFP system to visualize the interaction between the upstream kinase and downstream ones to suggest where the signaling relay occurs. We further extended the use of the BiFC system by co-expressing a third protein (CFP-tagged) with the split YFP pair (suggestive of protein-protein interaction) to visualize whether and how the complemented YFP could be spatially modulated by the co-expressed CFP protein. By doing so, we demonstrated that co-expression of CFP-BASL induced spatial reorganization of interactions between YDA and MPK6, from even distribution to polarized patterning at the cell cortex of the tobacco epidermal cells. This system therefore has the potential to be developed for monitoring dynamic signaling events in plant cells under conditions when cells are challenged by the internal or external stimuli (e.g., co-expression of other proteins, chemical application, pathogen attack or environmental changes, etc.).

<table><tbody><tr><td>Phusion DNA polymerase</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>pENTR/D/TOPO</td><td>Life Technologies</td><td>K2400-20</td><td></td></tr><tr><td>QuickChange II XL Site-directed Mutagenesis Kit</td><td>Agilent Technology</td><td>200521</td><td></td></tr><tr><td>LB broth</td><td>AMRESCO</td><td>J106-2KG</td><td></td></tr><tr><td>Bacto Agar</td><td>AMRESCO</td><td>J673-1KG</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G3632-1G</td><td></td></tr><tr><td>Rifampicin</td><td>Sigma-Aldrich</td><td>R3501-1G</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>K4000-25G</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>S4014-5G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266-100G</td><td></td></tr><tr><td>25 x 75 mm Slide</td><td>VWR</td><td>16004-382</td><td></td></tr><tr><td>24 x 50 mm Cover glass</td><td>VWR</td><td>48393-241</td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Leica&amp;nbsp;</td><td>TCS SP5 II</td><td>LAS AF Lite software</td></tr><tr><td>40X objective lens</td><td>Leica</td><td>HCX&amp;nbsp;Plan APO&amp;nbsp;</td><td>HCX&amp;nbsp;Plan APO, NA 1.30</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>SORVALL 6+</td><td></td></tr><tr><td>Hole puncher</td><td>Staples</td><td></td><td></td></tr><tr><td>50 ml falcon tubes</td><td>Genesee Scientific</td><td>21-108</td><td></td></tr><tr><td>No. 5 Forceps</td><td>Canemco &amp;amp; Marivac</td><td>205EQA-Spec</td><td></td></tr></tbody></table>

imaging spatial reorganization of a mapk signaling pathway using the tobacco transient expression system

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

To prevent cell death induced by the hyperactive MAPK signaling, the kinase inactive versions of YDA (DNyda) and MPK6 (DNmpk6) were used in the co-expression assay in tobacco cells. Neither the interaction between DNyda and DNmpk6 revealed by BiFC nor CFP-BASL itself generated uneven distribution pattern (Figure 3A-B). However, when CFP-BASL was introduced into the DNyda-DNmpk6 interaction pair, both CFP and YFP signals were redistributed to a highly polarized manner (Figure 3C). This redistribution suggests that BASL interacts with YDA and MPK6 to spatially re-organize the MAPK signaling pathway in plant cells. The phosphorylation status of BASL has differential impacts on the polarity degree of the MAPK signaling: a phospho-mimicking version of BASL generated very strong polarity, whilst a phospho-deficient version showed weaker activity6. These data supported our working model of a positive feedback regulation between BASL and the YDA-MAPK pathway in generating cell polarity6.
<img alt="Figure 1" src="/files/ftp_upload/53790/53790fig1.jpg" /> 
Figure 1. Diagram of the BASL-YDA-MPK6 Polarity Complex During Stomatal Asymmetric Cell Division (ACD) in Arabidopsis. In the leaf epidermis, the stomatal linage initiates from the protodermal cells, which expand to become ACD precursor cells, the Meristemoid Mother Cells (MMCs). A MMC undergoes an ACD to create one Meristemoid and one SLGC (stomatal lineage ground cell), which may divide and eventually differentiate into stomatal guard cells and pavement cells, respectively. At the cell cortex of the ACD precursor cells (MMCs), BASL recruits two components of a MAPK pathway, the MAPKKK YDA and the MAPK MPK6, to form a polarity complex and regulate stomatal ACD. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53790/53790fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53790/53790fig2.jpg" /> 
Figure 2. Diagram of the Injection Procedure in Tobacco Leaf Epidermis. (A) Transform the plasmids harboring CFP-BASL, DNyda-nYFP, DNmpk6-cYFP and P19 into Agrobacterium tumefaciens GV3101 and grow them on respective selection plates (from top to bottom in A). (B) Pick up colonies from (A) to inoculate liquid culture for overnight growth. (C) Harvest cells by spinning and decant the supernatant. After briefly pipetting and washing the pellets, spin down the cells again. (D) Re-suspend and dilute the cell pellets with the infiltration solution (see the protocol) to achieve the final OD600 = 0.5. (E) Gently mix the re-suspended cells in a Petri dish. (F) Use a needleless syringe to push the infiltration mixture into the abaxial side of the tobacco leaves. (G) About 48 hr after infiltration, the leaves are ready for confocal imaging. Excise leaf disks containing infected cells with a hole puncher around the infiltration region. Dashed circles indicate excised leave disks. (H) Use water to mount the leaf disks onto a standard slide for confocal imaging. The box diagrams in the shaded area describe the proteins injected into tobacco leaves for polarization assay. Protein sizes are not in scale. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53790/53790fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53790/53790fig3.jpg" /> 
Figure 3. Display and Quantification of Polarity Formation. (A-C) Confocal images to show CFP and YFP florescence in tobacco epidermal cells. Cyan indicates the expression of CFP. Yellow indicates the expression of YFP. (A) Overexpression of CFP-BASL alone. (B) Co-expression of DNyda-nYFP and DNmpk6-cYFP. The YFP expression is complemented when two proteins interact. (C) Co-expression of CFP-BASL, DNyda-nYFP and DNmpk6-cYFP. Uneven distribution (polarity) is evident for both CFP and YFP. Scale bar = 50 &#181;m in (A-C). (D-F) Graphically plotting the CFP/YFP fluorescence intensity along the dashed lines (Magenta) in (A-C). Magenta triangles mark the start and end point of the regions used for intensity plotting. Quantifications of the polarity degree in tobacco cells are presented underneath. The values of florescence intensity (H for high and L for low) are measured and collected by Fiji from the regions traced with red dashed lines in (A-C). For each sample, the values from 20 cells are averaged for significance test. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53790/53790fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System at JoVE.com

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

imaging-spatial-reorganization-mapk-signaling-pathway-using-tobacco

Universidad San Sebastian

Research

JoVE Journal

Biology

9.7K Views.  Rutgers, The State University of New Jersey. At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells. 

Video: Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

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

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Cancer Research

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

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 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 ...

Chemistry

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

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 ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Neuroscience

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

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A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

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

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

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

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging