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

Summary

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.

Abstract

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.

Introduction

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 pert....

Protocol

Vectors and modules can be obtained from the non-profit repository, Addgene (https://www.addgene.org).

1. Cloning using GreenGate

1. Design primers using the overhangs listed in Table 1, replacing ‘NNNN’ with module type-specific adapter sequences listed below: Forward: 5´AACA GGTCTC A NNNN (n) CA^* + specific sequence 3´, Reverse: 5´AACA GGTCTC A NNNN (n) + reverse complement of specific sequence 3´. Add the underlined.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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