Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Summary

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.

Abstract

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 "polarization") 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.

Introduction

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 m....

Protocol

1. Plasmid Construction

1. Generate the constructs by cloning technology as previously described^1,6.
   1. 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.
   2. 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 introduce the point mutations by a site-directed mutagenesis method⁹.
2. To build the constructs for tobacco transient assays....

Results

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 (

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phusion DNA polymerase	New England Biolabs	M0530S
pENTR/D/TOPO	Life Technologies	K2400-20
QuickChange II XL Site-directed Mutagenesis Kit	Agilent Technology	200521
LB broth	AMRESCO	J106-2KG
Bacto Agar	AMRESCO	J673-1KG
Gentamycin	Sigma-Aldrich	G3632-1G
Rifampicin	Sigma-Aldrich	R3501-1G
Kanamycin	Sigma-Aldrich	K4000-25G
Spectinomycin	Sigma-Aldrich	S4014-5G
MgCl2	Sigma-Aldrich	M8266-100G
25 x 75 mm Slide	VWR	16004-382
24 x 50 mm Cover glass	VWR	48393-241
Laser scanning confocal microscope	Leica	TCS SP5 II	LAS AF Lite software
40X objective lens	Leica	HCX Plan APO	HCX Plan APO, NA 1.30
Centrifuge	Thermo Scientific	SORVALL 6+
Hole puncher	Staples
50 ml falcon tubes	Genesee Scientific	21-108
No. 5 Forceps	Canemco & Marivac	205EQA-Spec