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 introdu...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-mediated+site-directed+mutagenesis.">PCR-mediated site-directed mutagenesis.</a> Cold Spring Harbor protocols. , 738-742 (2013).</li><li>Kubo, M., 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=Transcription+switches+for+protoxylem+and+metaxylem+vessel+formation.">Transcription switches for protoxylem and metaxylem vessel formation.</a> Genes Dev. 19, 1855-1860 (2005).</li><li>Yuan, 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=Allosteric+regulation+of+transport+activity+by+heterotrimerization+of+Arabidopsis+ammonium+transporter+complexes+in+vivo.">Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo.</a> Plant Cell. 25, 974-984 (2013).</li><li>Weigel, D., Glazebrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+agrobacterium+using+the+freeze-thaw+method.">Transformation of agrobacterium using the freeze-thaw method.</a> CSH protocols. 2006 (7), (2006).</li><li>Voinnet, O., Rivas, S., Mestre, P., Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+transient+expression+system+in+plants+based+on+suppression+of+gene+silencing+by+the+p19+protein+of+tomato+bushy+stunt+virus.">An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.</a> Plant J. 33, 949-956 (2003).</li><li>Rivero, 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=Handling+Arabidopsis+plants:+growth,+preservation+of+seeds,+transformation,+and+genetic+crosses.">Handling Arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses.</a> Methods in molecular biology. 1062, 3-25 (2014).</li><li>Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+polarity+signaling+in+Arabidopsis.">Cell polarity signaling in Arabidopsis.</a> Annu Rev Cell Dev Biol. 24, 551-575 (2008).</li><li>Zhang, J., Nodzynski, T., Pencik, A., Rolcik, J., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIN+phosphorylation+is+sufficient+to+mediate+PIN+polarity+and+direct+auxin+transport.">PIN phosphorylation is sufficient to mediate PIN polarity and direct auxin transport.</a> Proc Natl Acad Sci U S A. 107, 918-922 (2010).</li><li>Furutani, M., 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=Polar-localized+NPH3-like+proteins+regulate+polarity+and+endocytosis+of+PIN-FORMED+auxin+efflux+carriers.">Polar-localized NPH3-like proteins regulate polarity and endocytosis of PIN-FORMED auxin efflux carriers.</a> Development. 138, 2069-2078 (2011).</li><li>Takano, J., 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=Polar+localization+and+degradation+of+Arabidopsis+boron+transporters+through+distinct+trafficking+pathways.">Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways.</a> Proc Natl Acad Sci U S A. 107, 5220-5225 (2010).</li><li>Truernit, E., Bauby, H., Belcram, K., Barthelemy, J., Palauqui, J. 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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+stomatal+meristemoids+reveals+new+component+of+asymmetric+cell+division+and+commonalities+among+stem+cell+populations+in+Arabidopsis.">Molecular profiling of stomatal meristemoids reveals new component of asymmetric cell division and commonalities among stem cell populations in Arabidopsis.</a> Plant Cell. 23, 3260-3275 (2011).</li><li>Mouchel, C. F., Osmont, K. S., Hardtke, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRX+mediates+feedback+between+brassinosteroid+levels+and+auxin+signalling+in+root+growth.">BRX mediates feedback between brassinosteroid levels and auxin signalling in root growth.</a> Nature. 443, 458-461 (2006).</li></ol>

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 event...

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>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>25x75mm Slide</td>
			<td>VWR</td>
			<td>16004-382</td>
			<td></td>
		</tr>
		<tr>
			<td>24x50 mm Cover glass</td>
			<td>VWR</td>
			<td>48393-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica&#160;</td>
			<td>TCS SP5 II</td>
			<td>&#160;LAS AF Lite software</td>
		</tr>
		<tr>
			<td>40 x objective lens</td>
			<td>Leica</td>
			<td>HCX&#160;Plan APO&#160;</td>
			<td>HCX&#160;Plan APO&#160;, 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 &#38; 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 (<st...

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 ...

The overall goal of this procedure is to visualize dynamic spatial distribution of protein

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

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9.7K vistas.  Rutgers, The State University of New Jersey. The overall goal of this procedure is to visualize dynamic spatial distribution of protein