Confocal Microscopy Analysis of Protein Sorting to Plasmodesmata in Nicotiana benthamiana

Summary

This protocol describes the selection of optimal plasmodesmal markers for confocal microscopy-based analyses of protein targeting to plasmodesmata during virus-plasmodesmata interactions or plasmodesmal transport.

Abstract

Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, macromolecules, as well as invading viruses. Plasmodesmata play fundamental roles in the regulation of intercellular communication, contributing to plant development, environmental responses, and interactions with viral pathogens. Discovering plasmodesmal localization of plant or viral proteins could provide useful functional information about the protein and is important for understanding the mechanisms of plant-virus interactions. To facilitate these studies, we describe a protocol for confocal microscopy-based analysis of different plasmodesmal targeting proteins to select the best plasmodesmal marker for studying the virus-plasmodesmata interactions or plasmodesmal transport. Specifically, the analyses of these events are illustrated using the cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV), the Arabidopsis Plasmodesmata-Localized Protein 5 (PDLP5) and Plasmodesmata Callose-Binding Protein 1 (PDCB1). The protein plasmodesmal localization data are analyzed in parallel with the global visualization of plasmodesmata using aniline blue staining of the sampled tissues. These approaches can be easily adapted to analyze the plasmodesmal localization of any cellular or pathogen proteins in planta.

Introduction

Plasmodesmata (PD) play a fundamental role in controlling plant development, environmental responses, and interactions with viral pathogens through the regulation of intercellular communication^1,2. PD initially forms during cytokinesis, with hundreds of PD inserted into the new cell between the two daughter cells, thus supplying the channels for cell-to-cell communication^3,4. PD is a membrane-rich structure, containing the endoplasmic reticulum (ER)-derived membrane, a trans-PD desmotubule, in the central part of the pores that are lined by the plasma ....

Protocol

The details of the reagents and the equipment used in this study are listed in the Table of Materials.

1. Plant growth

1. Grow Nicotiana benthamiana seeds in wet soil in a controlled environment chamber at 23 ˚C under 16 h of light and 8 h of darkness.
2. After about 2 weeks, carefully transfer the seedlings with the peat pellets around their roots to larger pots and continue growth under the same conditions for 4-5 weeks for the .......

Discussion

Any cell biological studies of plant intercellular communication and cell-to-cell transport during normal plant development and morphogenesis, as well as during plant-pathogen interactions, necessitate the detection and monitoring of the sorting of proteins-both endogenous and pathogen-encoded-to plant intercellular connections, the plasmodesmata (PD). These experiments would be substantially facilitated by using reference proteins, whether endogenous or pathogen-derived, that faithfully and consistently localize to PD, .......

Materials

Name	Company	Catalog Number	Comments
ABT AC 1 phase motor	BRANDTECH	ABF63/4C-7RQ
Agrobacterium tumefaciens EHA105
Contamination control	CCI
Gateway BP Clonase II Enzyme mix	Invitrogen	#11789020
Gateway LR Clonase II Enzyme mix	Invitrogen	#11791020
GraphPad Prism 8.0.1.	GraphPad Software Inc.
Image J	National Institutes of Health and the Laboratory for Optical and Computational Instrumentation
Laser scanning confocal microscope	Zeiss	LSM 900
Nicotiana benthamiana	Plant species
pDONR207	Invitrogen	#12213013
Q5 High-Fidelity DNA Polymerase	NEB	#M0491S