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 membrane^3,4. Comparative proteomic approaches identified numerous PD functional proteins, including β-1,3-glucanases (BGs), callose synthases (CALSs), plasmodesmata-located proteins (PDLPs), callose-binding proteins (PDCBs), multiple C2 domains transmembrane region proteins (MCTPs)³, and leucine-rich repeat receptor-like kinases (RLK)⁵. Recently, Kirk et al. developed a tool termed plasmodesmata in silico proteome 1 (PIP1), which made it possible to predict new PD proteins in 22 plant species⁶. PD varies in permeability and structure during plant development and response to various stresses. Callose (β-1,3-glucan) deposition and hydrolysis at the neck region surrounding the PD is one of the broadly known mechanisms of PD regulation⁷.

Many pathogenic microbes, including fungi, bacteria, and viruses, can manipulate the PD dilation or structure during their infection^2,8,9. Magnaporthe oryzae, the causative agent of rice blast, deploys intracellular invasive hyphae to move from cell to cell through PD⁸. A bacterial pathogen Pseudomonas syringae pv. tomato requires an effector protein HopO1-1 for intercellular movement and spread in the host plant through interacting with and destabilizing PDLP7, thus increasing the molecular flux in neighboring cells in Arabidopsis⁹. However, plant viruses are more versatile in regulating PD during their intercellular transmission, with the viral movement protein (MP) promoting cell-to-cell movement². Owing to their important function in regulating plant development and growth, as well as their interaction with plant pathogenic microbes, PD has gained increasing attention in recent years. In Arabidopsis thaliana, there are two major types of PD functional proteins, PDLPs (1-8) and PDCBs (1-5), and many of them, e.g., PDLP5^1,10,11, PDLP1¹², PDLP6¹³, PDLP7¹⁴, and PDCB1¹⁵, were found to play a role in manipulating the PD permeability through regulation of callose deposition. However, some PDLPs were found to have a functional redundancy, e.g., knockout mutants of pdlp1 and pdlp1,2 did not affect the molecular trafficking, although double knockout mutants of pdlp1,3 and pdlp2,3 showed increased plasmodesmal permeability¹⁶. Interestingly, downregulation/knockout or over-expression of PDLP5 alone results in an increase or decrease in plasmodesmal permeability, respectively^1,17. Recently, Li et al. have found that PDLP5 and PDLP6 function at different cell interfaces¹³. These results indicate that PDLP5 might have non-redundant functions with other PDLPs.

Due to the critical function of PD in intercellular communication, we developed a protocol for deploying the plant PD proteins PDLP5 and PDCB1 and the viral cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV) as simple, convenient, and reliable PD markers for cell biology experimentation. For further verification, visualization of PD using aniline blue staining of the sampled tissues proceeded in parallel. The protocols described for PD localization of PDLP5, PDCB1, and TVCV MP can be easily adapted to analyze potential PD localization of any cellular or pathogen-derived proteins in living plants.

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 agroinfiltration experiments.
   NOTE: Do not use the plants when they begin to flower, as GFP accumulation will diminish at the inflorescence stage¹⁸.

2. Vector construction

1. Use PCR to amplify the coding sequences of interest with Q5 High-Fidelity DNA Polymerase and clone them into entry vector pDONR207 by the BP reaction¹⁹ using a commercially available BP Clonase kit.
2. Transfer the target genes in the resulting entry plasmids into the destination vector pPZP-RCS2A-nptII-DEST-EGFP-N1 by the LR reaction¹⁹ using a commercially available LR Clonase kit to produce plasmids with the EGFP tag fused to the C-terminus of the target proteins.
3. Verify all constructs by PCR and sequencing¹⁹.

3. Agroinfiltration

1. Streak Agrobacterium tumefaciens EHA105 cells containing different vectors on LB agar plates and incubated for 2 days under 28 ˚C.
2. Transfer a single colony into 2 mL of LB broth and incubate overnight at 28 ˚C with agitation (250 rpm).
3. Remove 1 mL from the overnight culture, followed by the addition of 4 mL of fresh LB broth, and reculture for 1 h under the same conditions.
4. Adjust the bacterial suspension to OD₆₀₀ = 0.1 (OD₆₀₀ = 0.2 for co-expression of two proteins) with infiltration buffer (MgCl₂, 10 mM; MES, 10 mM, pH 5.6).
5. Mix different bacterial suspensions at a ratio of 1:1 (v/v) to make a final concentration of OD₆₀₀ = 0.1. (optional).
6. Incubate the bacterial suspension at room temperature for 3 h with soft agitation.
7. Infiltrate the abaxial surface of the fully expanded leaves from different plants with a 1 mL needleless disposable syringe, and mark the infiltrated area (a darker color than the surrounding, non-infiltrated tissue) with a waterproof pen.
8. Proceed to the Confocal microscopy section of this protocol (step 5).
   NOTE: Other strains of Agrobacterium tumefaciens suitable for the transformation of plant species of interest can also be used.

4. Aniline blue stain

1. Add an aliquot of 200 µL of 1% aniline blue (in 50 mM potassium phosphate buffer, pH 8.0) to a microscope slide²⁰.
2. Excise the infiltrated area of approximately 0.5 cm × 0.5 cm, away from the vein, with a blade, and transfer the leaf tissue samples to the aniline blue solution (abaxial side-up) on the microscope slide, and make sure the leaf tissue samples are submerged in the aniline blue solution, and then cover it with a cover glass (22 mm x 50 mm).
3. Place the microscope slides with the samples in a desiccator attached to a vacuum pump and evacuate for 2 min (<0.8 Pa), followed by a slow release of the pressure and incubation in the dark for 30 min at room temperature.
4. Visualize the fluorescent signal of aniline blue under a laser scanning confocal microscope with compatible software.
   NOTE: Here, the aniline blue staining method by Huang et al.²⁰ was used. In a modification of this technique, the same experiment was performed without the 2 min vacuum step, producing similar results and suggesting that vacuum drying is not essential for aniline staining. Also, the concentration of the aniline blue stain and the staining time may vary according to the source of the leaf tissue, etc.

5. Confocal microscopy

1. Harvest two leaves from two plants at different time points after the infiltration. Cut the infiltration zone into approximately 0.5 cm × 0.5 cm slices away from the vein with a blade.
   1. Place the tissue samples into a drop of sterile water (abaxial side-up) in the central part of a microscope slide, and cover them with a cover glass (22 mm x 50 mm), taking care to avoid bubbles.
2. Ensure that the excitation wavelengths for the detection of CFP, GFP, and RFP signals are 405 nm, 488 nm, and 561 nm, respectively, and the emission filters for detection were 410-602 nm for CFP, and 400-602 nm for EGFP and RFP, with the pinhole 1 AU and the Master Gain set as 769 V.
3. Visualize the fluorescent signal of autofluorescent tags in the infiltrated area using a laser scanning confocal microscope with compatible software at 1 day, 2 days, 3 days, 5 days, 7 days, and 10 days after the infiltration.
   1. Use a 10x objective lens and GFP filters to locate cells with the fluorescent signal and then switch to a 40x objective lens for visualization of subcellular localization and image recording.
4. Collect 20 images for each condition, using at least two independent biological replicates.
5. Score PD localization of the tested protein based on the diagnostic punctate appearance of the signal at the periphery of the cell^21,22.
   NOTE: When detecting the subcellular localization of an unknown protein, using at least three plants is recommended.

6. Data analysis

1. Use Fiji software to split channels and add the scale bar to the images for visualization.
2. Use Fiji software to split channels before measuring the mean grayscale value for each image to assess the GFP (for MP, PDCB1, and PDLP5) or CFP (for aniline blue staining) fluorescence intensity at different time points.
3. Use Fiji software to split channels before normalization of the images of GFP (for MP, PDCB1, and PDLP5) or CFP (for aniline blue staining). The area of the image was measured, and the PD puncta were counted manually. The number of PD puncta per 100 µm² was calculated.
4. Use two-way ANOVA with Tukey's multiple comparisons test²³ to determine the P-values between the different samples and different time points with statistical and graphing software.
   NOTE: For a single-channel fluorescence image, the grayscale value of each pixel represents the fluorescence intensity of that point²⁴. Here, the mean grayscale value was used to assess the fluorescence intensity of each sample at different time points.

Results

To facilitate studies of PD function in plant physiology and interactions with pathogens, three simple and reliable reference proteins were developed for PD localization. Two cellular PD proteins and a pathogen-derived MP protein encoded by the plant tobamovirus TVCV were selected. The subcellular localization of these proteins was visualized using an autofluorescent reporter EGFP fused to the C-terminus of each protein. In an alternative approach, PD were visualized using aniline blue staining of the PD-associated callo...

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