Name: inducible, cell type-specific expression in arabidopsis thaliana through lhgr-mediated trans-activation
Uploaded: 2019-04-19T15:00:00
Description: Watch this Scientific Journal Video about Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation at JoVE.com

Work in our laboratories is supported by German Research Foundation (DFG) Grants WO 1660/6-1 (to S.W.) and GR 2104/4-1 (to T.G.) and SFB1101 (to T.G. and J.U.L) and by a European Research Council consolidator grant (PLANTSTEMS 647148) to T.G.&#160; S.W. is supported through the Emmy Noether Fellowship of the DFG through Grant WO 1660/2.

Vectors and modules can be obtained from the non-profit repository, Addgene (https://www.addgene.org).
1. Cloning using GreenGate
<ol>
	<li>Design primers using the overhangs listed in Table 1, replacing &#8216;NNNN&#8217; with module type-specific adapter sequences listed below: Forward: 5&#180;AACA GGTCTC A NNNN (n) CA* + specific sequence 3&#180;, Reverse: 5&#180;AACA GGTCTC A NNNN (n) + reverse complement of specific sequence 3&#180;. Add the underlined...

<ol>
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Here, we describe the steps necessary to generate and apply a versatile and comprehensive toolkit for inducible, cell type specific trans-activation. Crossing lines carrying effector cassettes under control of the pOp promoter with driver lines allows studying the mis-expression effects in the F1 generation, enabling the rapid assessment of genetic perturbation in a wide range of cell types. Alternatively, effector constructs can be used to transform driver lines or, by adapting the cloning strategy, dr...

A major limitation in biology in the postgenomic era is to decipher the context specific role of a given factor or genetic perturbation. Constitutive genetic perturbations such as loss-of-function and gain-of-function approaches often only allow end-point analysis of life-long adaptation processes, obfuscating the distinction between primary and secondary effects. In addition, context specific functions can be masked or diluted by large scale effects in distant tissues or during other phases of development. Moreover, in extreme cases, lethality can preclude any mechanistic insight. Ideally, to circumvent these issues, one could analyze the effect of acute genetic perturbation within a specific context such as a specific cell type in a time-resolved manner. To achieve this, genetic tools for inducible, cell type-specific expression are required1. To provide a resource for the rapid assessment of the spatiotemporal dynamics of a response to a given effector, we have combined the ease of cloning provided by the GreenGate system2 with the proven efficacy of the GR-LhG4 system3,4,5,6. We have generated a set of lines expressing the chimeric transcription factor LhG4 fused to the ligand binding domain of the rat corticoid receptor (GR)7 under the control of well-characterized cell type specific promoters8. In resting conditions, the transcription factor remains outside of the nucleus, as the GR domain is bound by the cytosolic HSP90. With the addition of synthetic ligand dexamethasone (Dex), nuclear translocation is induced and LhG4 will mediate transcription of expression cassettes under control of a cognate synthetic pOp-type promoter, such as an mTurquoise2 reporter included in the driver lines to visualize the expression domain after induction.&#160; Thus, the driver lines technically also contain an effector cassette. The crossing of a set of driver lines with a line carrying an effector cassette with, for example, a gene of interest under control of the same pOp-type promoter thus allows the rapid assessment of the acute or long-term consequences of effector expression in a wide range of cell types.
Here, we provide a protocol describing the procedures necessary to generate the driver and effector lines and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. To illustrate the prowess and specificity of this approach, we utilize the well-known transcription factor VND7 which is capable of driving xylem-like secondary cell wall thickenings ectopically9. Treating F1 from a cross between the pSCR10,11 driver line and the pOp6:VND7 effector line leads to the formation of ectopic xylem-like cells in the starch sheath cells of the Arabidopsis stem.
To facilitate the generation of large DNA assemblies required for constructing driver and effector expression plasmids, we used the fast and efficient GreenGate cloning method2. GreenGate cloning is based on type II S restriction enzymes such as Eco31I or its isoschizomer BsaI2. These enzymes cut downstream of their asymmetric recognition sites producing overhangs with varying base composition. By incorporating Eco31I/BsaI restriction sites in oligonucleotides and allocating specific overhang sequences to DNA elements, modular cloning is achieved, facilitating the generation of large assemblies. In the GreenGate framework, DNA modules fall into categories A-F based on overhang sequence that serve as adapters and are assembled in that order. Therefore, the primers designed to amplify your desired product should be in accordance with the selected module2 (Figure 1A). If there is an internal Eco31I/BsaI site and the sequence is not compatible with the GreenGate entry and destination modules, you can proceed without mutagenizing, but with lower efficiency. Alternatively, use site-directed mutagenesis to remove Eco31I/BsaI restriction sites taking care not to change amino acids in gene products.

<table>
	<tbody>
		<tr>
			<td>Ampicillin</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>K029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C1919</td>
			<td></td>
		</tr>
		<tr>
			<td>Column purification</td>
			<td>Qiagen</td>
			<td>QIAquick PCR Purification Kit (250)</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture chamber for imaging</td>
			<td>Sarstedt AG &#38; Co. KG</td>
			<td>1-well tissue culture chamber, on cover glass II</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4903</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific, UK</td>
			<td>D139-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Eco31I</td>
			<td>Thermo Fisher Scientific</td>
			<td>FD0294</td>
			<td></td>
		</tr>
		<tr>
			<td>injection cannula (0.30 x 12 mm, 30 G x 1/2)</td>
			<td>Sterican, Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>T832.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 CLSM, HCX PL APO lambda blue 63x water immersion objectiv</td>
			<td>Leica, Wetzlar, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MS medium</td>
			<td>Duchefa, Haarlem, Netherlands</td>
			<td>M0221.0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1 CLSM, Apo LWD 25x&#160; 1.1 NA water immersion objective</td>
			<td>Nikon, Minato, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35/10 mm</td>
			<td>Greiner Bio-One GmbH, Germany</td>
			<td>627102</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60/150 mm</td>
			<td>Greiner Bio-One GmbH, Germany</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 120/120/17</td>
			<td>Greiner Bio-One GmbH, Germany</td>
			<td>688102</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant agar</td>
			<td>Duchefa, Haarlem, Netherlands</td>
			<td>P1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid extraction</td>
			<td>Qiagen</td>
			<td>QIAprep Spin Miniprep Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Razorblade</td>
			<td>Classic, Wilkinson Sword GmbH</td>
			<td>7005115E</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes</td>
			<td>Sarstedt AG &#38; Co. KG</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>Kurt Obermeier GmbH &#38; Co. KG, Bad Berleburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>AppliChem GmbH</td>
			<td>3834.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>NanoDrop 2000c</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfadiazine</td>
			<td>Sigma-Aldrich</td>
			<td>S6387</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>AppliChem GmbH</td>
			<td>2228.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Ligase 5 U/&#181;l</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Ligase 30 U/&#181;l</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0013</td>
			<td></td>
		</tr>
	</tbody>
</table>

inducible, cell type-specific expression in arabidopsis thaliana through lhgr-mediated trans-activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Generation of driver and effector lines through GreenGate cloning
The GreenGate cloning system is based on GoldenGate cloning and use the type IIS restriction endonuclease BsaI or its isoschizomer Eco31I. As the enzyme produces overhangs distant from its asymmetric recognition site, the base composition of the overhangs can be freely chosen, which is the basis form the modularity of the system. Each PCR-generated element, for example, a promoter sequence, CDS...

Watch this Scientific Journal Video about Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation at JoVE.com

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

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