Vectors and modules can be obtained from the non-profit repository, Addgene (https://www.addgene.org).
1. Cloning using GreenGate
2. Generation of Arabidopsis transgenic plants
3. Induction of trans-activation in Arabidopsis driver lines
4. Imaging of reporter expression in Arabidopsis driver lines.
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, or terminator, is first inserted into a designated entry vector with matching overhangs produced by restriction digest to generate a module. After subcloning, a number of matching modules, usually six, are used for the GreenGate ligation reaction resulting in the assembly of the construct in a binary plant destination vector.
For driver lines, modules containing the DNA sequences of tissue-specific promoter (pTS), the GR-LHG4 transcription factor, the pOp6 promoter, and the mTurquoise2 reporter fused to an N-terminal signal peptide and a C-terminal ER retention signal were fused including terminators and various adapter modules and a module for transgenic selection as described previously 2,8 (Figure 2). Effector lines were constructed with pOp6 promoter, and an effector cassette, for example consisting of a gene of interest and terminator as well as a module encoding a resistance gene for transgenic plant selection (Figure 2).
Induction of driver lines and visualization of reporter fluorescence
Induction with Dex leads to cell type specific mTurquoise2 expression in the root endodermis (pSCR>>SP-mTurquoise2-HDEL, Figure 3A), phloem precursors and cambium (pSMXL5>>SP-mTurquoise2-HDEL 17, Figure 3B), and the stem cells in the shoot apical meristem (pCLV3>>SP-mTurquoise2-HDEL 18, Figure 3C).
Trans-activation of VND7 in starch sheath cells of the cambium
As a test case for trans-activation, we generated an effector line encoding the secondary cell wall master transcription factor VND7 fused to the VP16 activation domain, which has been shown to induce secondary cell wall formation cell autonomously when misexpressed 8,9,19,20.
After 5 days of a single treatment with either 15 µM of Dex or DMSO for the induced or the mock plants respectively, stem sections were prepared for the visualization of ectopic lignification in the starch sheath. Propidium Iodide shows in the stem strong affinity to lignified tissue. The starch sheath cells in induced samples by Dex, but not in the mock showed a strong signal for the PI channel and some cells show the typical reticulate thickening of the cell wall in xylem cells (Figure 4).
Figure 1. The GreenGate cloning principle. A) Type IIS restriction endonucleases, like BsaI/Eco31I, recognize non-palindromic sequences (red) and cut asymmetrically in a defined distance independently of the sequence (blue). Eco31I recognizes ‘GGTCTC’, cuts from the second nucleotide downstream of the recognition site and creates a four base 5’ overhang. B) The GreenGate cloning system is based on a modular system with six different entry vectors pGGA000-pGGF000. These vectors contain an Ampicillin resistance cassette (AmpR) and a ccdB cassette flanked by the specific adaptors for each entry vector (e.g pGGA000 entry vector) and the ‘GGTCTC’ Eco31I recognition sites. Eco31I digestion of pGGA000 releases ccdB and creates the pGGA000-specific four nucleotide overhangs (dark blue). Insert1 is amplified by primers harboring ‘GGTCTC’ and pGGA000 specific adaptors and after digestion ligated into pGGA000. The same procedure is followed with the other modules. C) The final GreenGate reaction combines the Eco31I digestion of the destination vector pGGZ001 and the six entry vectors pGGA000-pGGF000 and the simultaneous ligation of all modules into the destination vector. Please click here to view a larger version of this figure.
Figure 2. Overview of the Dex-inducible LhGR/pOp system with driver and effector lines. In driver lines, tissue-specific promoters (pTS) control the expression of the synthetic transcription factor LhG4, which is translationally fused to the ligand binding domain of rat glucocorticoid receptor (GR) and thereby prevents, in the absence of Dex, the nuclear translocation. The effector line harbors a transcriptional cassette driven by a pOp element and a TATA box with a minimal 35S promoter. Crossed with a driver line and upon Dex induction, GR-LhG4 binds to the pOp-type elements in the reporter cassette and to those in the effector module, inducing the transcription of mTurquoise2 and the effector. Please click here to view a larger version of this figure.
Figure 3. Induced driver lines in root, stem, and SAM. A) Dex-induced driver line pSCR>>SP-mTurquoise2-HDEL (germinated in 50 µM Dex) in the root and mock treatment. The SCARECROW promoter (pSCR) mediates expression in the endodermis, cortex/endodermis initial (CEI) and quiescent center (QC). Cells are counter-stained with propidium iodide (PI). Scale bars = 20 µm. B) Dex-induced driver line pSMXL5>>SP-mTurquoise2-HDEL (dipped in a 50 µM Dex solution and visualized after 3 days) in in the stem and mock treatment. SMXL5 promoter (pSMXL5) mediates expression in the cambium stem cell domain and phloem precursors. Cells are counter-stained with propidium iodide (PI). Scale bars = 100 µm. C) Dex-induced driver line pCLV3>>SP-mTurquoise2-HDEL (10 µM, 48 h) in the SAM. The CLAVATA3 promoter (pCLV3) mediates expression in the stem cell domain. Pictures in the bottom show XZ and YZ cross sections, Dex-induced and mock treated, respectively. Cells are counter-stained with propidium iodide (PI). Scale bars = 20 µm. Please click here to view a larger version of this figure.
Figure 4. Ectopic lignification in the starch sheath of the stem. A) Ectopic lignification is seen five days after Dex induction of the driver line pSCR>>VND7-VP16 in the stem. The SCARECROW promoter (pSCR) mediates expression in the starch sheath cells in the stem. Left image shows merge of PI channel and bright field, right image only shows PI channel. B) The mock control shows no signal in the starch sheath cells. Cells are counter-stained with propidium iodide (PI). Scale bars = 100 µm. PI fluorescence is false-colored in green while chloroplast auto fluorescence is red in all images. White arrowheads point to starch sheath cells. Left image shows merge of PI channel and bright field, right image only shows PI channel. Please click here to view a larger version of this figure.
Module | 5'overhang | typically used for | 3' overhang |
A | ACCT | promoter | AACA |
B | AACA | N-terminal tag | GGCT |
C | GGCT | Coding sequence | TCAG |
D | TCAG | C-terminal tag | CTGC |
E | CTGC | terminator | ACTA |
F | ACTA | resistance cassette | GTAT |
Table 1: Overhangs used for primer design
Ampicillin | Carl Roth GmbH + Co. KG | K029.1 | |
ATP | Sigma-Aldrich | A9187 | |
Chloramphenicol | Sigma-Aldrich | C1919 | |
Column purification | Qiagen | QIAquick PCR Purification Kit (250) | |
Culture chamber for imaging | Sarstedt AG & Co. KG | 1-well tissue culture chamber, on cover glass II | |
Dexamethasone | Sigma-Aldrich | D4903 | |
DMSO | Fisher Scientific, UK | D139-1 | |
Eco31I | Thermo Fisher Scientific | FD0294 | |
injection cannula (0.30 x 12 mm, 30 G x 1/2) | Sterican, Braun | ||
Kanamycin | Carl Roth GmbH + Co. KG | T832.2 | |
Leica TCS SP5 CLSM, HCX PL APO lambda blue 63x water immersion objectiv | Leica, Wetzlar, Germany | ||
MS medium | Duchefa, Haarlem, Netherlands | M0221.0050 | |
Nikon A1 CLSM, Apo LWD 25x 1.1 NA water immersion objective | Nikon, Minato, Tokyo, Japan | ||
Petri dish 35/10 mm | Greiner Bio-One GmbH, Germany | 627102 | |
Petri dish 60/150 mm | Greiner Bio-One GmbH, Germany | 628102 | |
Petri dish 120/120/17 | Greiner Bio-One GmbH, Germany | 688102 | |
Plant agar | Duchefa, Haarlem, Netherlands | P1001 | |
Plasmid extraction | Qiagen | QIAprep Spin Miniprep Kit | |
Propidium iodide (PI) | Sigma-Aldrich | P4170 | |
Razorblade | Classic, Wilkinson Sword GmbH | 7005115E | |
Reaction tubes | Sarstedt AG & Co. KG | 72.690.001 | |
Silwet L-77 | Kurt Obermeier GmbH & Co. KG, Bad Berleburg, Germany | ||
Spectinomycin | AppliChem GmbH | 3834.001 | |
Spectrophotometer | Thermo Fisher Scientific | NanoDrop 2000c | |
Sucrose | Carl Roth GmbH + Co. KG | 4621.1 | |
Sulfadiazine | Sigma-Aldrich | S6387 | |
Tetracycline | AppliChem GmbH | 2228.0025 | |
T4 Ligase 5 U/µl | Thermo Fisher Scientific | EL0011 | |
T4 Ligase 30 U/µl | Thermo Fisher Scientific | EL0013 |
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.
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.
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.