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 ...

The key goal in developmental biology is to understand the context-specific role of a gene. And this can be difficult to achieve by conventional knockout mutants or by constitutive overexpression lines. We used the modular green gate cloning system to generate a resource for inducible cyto-specific expression in the three main meristems of the model plant Arabidopsis thaliana.Using the same modular cloning strategy, this method can be applied to other plant species that are immutable to transformation. Visual demonstration of this procedure is valuable because analyzing the effect of trans-activation in the meristems of the stem and the short apex requires dissection before imaging, which can be quite challenging. To begin, sterilize the seeds as outlined in the text protocol.Next, prepare half-strength Murashige and Skoog medium at pH 5.8 and add one percent sucrose and 0.9%agar. After autoclaving, add Dex dissolved in DMSO to the induction plates at a final concentration between 10 and 30 micromolar. Add an equal amount of DMSO to the control plates.Put the seeds for root imaging on plates and stratify them for 48 hours in darkness and at four degrees Celsius. Put the plates in a vertical position in a plant incubator and grow them for five days. Five days after germination, use confocal laser scanning microscopy to image the seedlings.To begin, prepare and grow the seeds as outlined in the text protocol. Six to seven days after germination, transfer each seedling to soil in a separate pot. If inducing the plants by watering, use a 25 micromolar DEX solution in water, prepared from a stock of 25 millimolar DEX dissolved in ethanol.Water every two to three days until the desired time of induction. If inducing by dipping, prepare a one-liter beaker with 750 milliliters of water containing 0.02%Silwet L77 and DEX at 25 micromolar final concentration or an equivalent amount of DMSO for the DEX and mock treatment. Dip a single plant into the induction or mock solution for 30 seconds.Repeat every two to three days, until the desired time of imaging. To begin, prepare and grow the seeds as outlined in the text protocol. Six to seven days after germination, transfer each seedling to soil in a separate pot.When the stem is around one centimeter long, spray the influorescent SAM with between 10 and 50 micromolar DEX solution in water. For induction and imaging at later stages of development, induce SAMs of longer stems or side shoots. 24 to 48 hours after induction, dissect the SAMs and proceed to imaging.First, transfer the seedlings from the plate to a 10 microgram per millileter solution of propidium iodide and counterstain them for five minutes. Place the roots into a microscope imaging chamber and image them using a confocal laser scanning microscope with a 63x water immersion objective. To visualize propidium iodide fluorescence, use an excitation wavelength of 488 nanometers and collect emission between 590 and 660 nanometers.For mTurquoise2 fluorescence, use 458 nanometer excitation and collect emission between 460 and 615 nanometers using sequential scanning. First, fix a stem with a finger on the opposite side of the desired section. Using a razor blade, cut a segment of approximately three centimeters and perform several fine cuts.Rinse the razor blade in a Petri dish containing tap water and collect the stem sections. Either stain the sections or directly mount them onto microscope slides. For staining, prepare one milliliter of a 250 microgram per milliliter solution of propidium iodide in a reaction tube.Remove the tap water from the Petri dish with a pipette and replace it with the propidium iodide solution and stain for five minutes. Then, remove the propidium iodide solution and rinse with water. Using either fine forceps or a fine paintbrush, transfer the stained sections to microscope slides.Use a confocal laser scanning microscope with a 25x dipping water immersion lens to image the samples. Use a 561 nanometer laser light to excite propidium iodide fluorescence and collect emission from 570 to 620 nanometers. Use a 405 nanometer laser light to excite the mTurquoise2 fluorophore effector encoded by the driver line constructs and collect emission from 425 to 475 nanometers.First, use forceps to cut the stem two centimeters below the shoot tip. Hold the stem in one hand and use fine forceps to remove the flower buds and large primordia. To remove young primordia, fix the SAM in an upright position in a Petri dish containing three percent agarose.Next, use a binocular and forceps to remove any young primordia close to the SAM. Transfer the dissected SAM to a tube containing a 250 microgram per milliliter solution of propidium iodide. Let the sample stain for five to 10 minutes while making sure the sample stays fully submerged during the staining procedure.Place the stained SAM into a small Petri dish containing three percent agarose medium. Cover the SAM with double distilled water. Use a confocal laser scanning microscope equipped with a 25x dipping water immersion lens to image the dissected SAMs.Use a 561 nanometer laser light to excite propidium iodide fluorescence and collect emissions from 570 to 620 nanometers. Use a 405 nanometer laser light to excite the mTurquoise2 fluorophore and collect emission from 425 to 475 nanometers. Record image stacks spanning 50 micrometers in the Z direction with a step size of 0.5 micrometers.Induction with DEX leads to cell-type specific mTurquoise2 expression in the root endodermis the phloem precursors and cambium, and the stem cells in the shoot apical meristem. As a test case for transactivation, an effector line encoding the secondary cell wall master transcription factor VND7 fused to the V16 activation domain, is generated. After five days of single treatment with either 15 microliters of DEX or DMSO, stem sections are prepared for the visualization of the ectopic lignification in the start sheath.The start sheath cells in induced samples show a strong signal for the propidium iodide channel and some cells show the typical reticulate thickening of the cell wall in xylem cells. After watching this video, you should have a clear understanding of how to induce gene expression in different tissues and how to prepare your samples for imaging. Following this procedure, the established driver lines can be used with a wide variety of effector constructs generated by other users according to their research interests.This shortcut should help plant researchers to rapidly assess the effect of cell-type specific expression or knockdown of a gene of interest in a time reserved manner. This cell-type specific induction system requires the use of the corticosteroid dexamethasone. Direct contact should be avoided, so it is important to wear gloves and a face mask during treatment.

Research

JoVE Journal

Genetics

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...