Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Summary

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Abstract

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Introduction

Plasmodesmata (Pd) function as conduits for intercellular transport of key regulators of plant development and morphogenesis, ranging from transcription factors to mRNA and small RNA molecules. Furthermore, this macromolecular transport capacity of Pd is utilized by most plant viruses for their intercellular spread during infection; to move through Pd, plant viruses have evolved specialized proteins, termed movement proteins (MPs), that specifically target to Pd^1,2,3,4,5,6....

Protocol

1. Plant Material

1. Choice of plant species
2. Use the plant species native to the protein of interest, i.e., one which encodes this protein for endogenous proteins or which represents the natural host for the pathogen for viral proteins. In addition, the selected plant species must be amenable to the chosen method of transient genetic transformation.
   NOTE: The studies routinely employ Nicotiana benthamiana, which represents a good host for TMV and is transformed efficiently by the Agrobacterium-mediated genetic transformation technique, i.e., agroinfiltration (see Step 3). N. benthamiana plants also are easi....

Results

The representative data, which faithfully illustrate the results expected from the described protocols and identify the TMV MP PLS, are adapted from Yuan et al.²¹. Figure 1A first summarizes major constructs expressing the full-length TMV MP (1-268), TMV MP PLS (comprising the first 50 amino acid residues of the protein, 1-50), and its alanine scanning V4A derivatives fused to CFP (generated as described in Steps 2.2, 5.2 and 6) whereas

Discussion

This protocol has four core constituents: the concept of identifying a sequence that is both necessary and sufficient for targeting to Pd, systematic division of the protein of interest into fragments that are progressively reduced in length, fusing the tested fragments to an autofluorescent protein that serves both as tag and as macromolecular cargo, and functional assay for Pd targeting in living plant tissues following transient expression of the tested fusion proteins. Note that Agrobacterium-mediated transient expre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Confocal laser scanning microscope (CLSM)	Zeiss	LSM5	Any CLSM with similar capabilities is appropriate
Zen software for confocal microscope imaging	Zeiss	2009 version	The software should be compatible with the CLSM used
Quickchange II site-directed mutagenesis kit	Agilent	200523
Acetosyringone	Sigma-Aldrich	D134406
MES	Sigma-Aldrich	69892
Syringes without needles	BD	309659
MgCl2	FisherScientific	M33-500
Spectinomycin	Sigma-Aldrich	S4014
Rifampicin	Sigma-Aldrich	R3501
Ampicillin	Sigma-Aldrich	A0166