aniline blue staining to visualize plasmodesmata in agroinfiltrated plant leaves using confocal microscopy

Exploring Plasmodesmata Function: Confocal Microscopy of Targeted Proteins

Plasmodesmata (PD) play a fundamental role in controlling plant development, environmental responses, and interactions with viral pathogens through the regulation of intercellular communication1,2. 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 communication3,4. 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 membrane3,4. Comparative proteomic approaches identified numerous PD functional proteins, including &#946;-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 (&#946;-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 infection2,8,9. 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.&#160;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., PDLP51,10,11, 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, respectively1,17. 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.

Author Spotlight: Microscopic Analysis of Protein Localization at Plasmodesmata in Plants

Confocal Microscopy Analysis of Protein Sorting to Plasmodesmata in Nicotiana benthamiana

My research is focused on the plant various movement, from cell to cell. And I'm trying to understand the mechanisms of the various plant interactions during the local spread after infection. Recently, we found that plasmodesmata-localized protein 5 can move from cell to cell in nicotiana benthamiana.This result will help us to understand the mechanisms of plasmodesmata functions in the future. Knowing a protein's subcellular localization is quite important for learning its function. This protocol provides good plasmodesmata-localized microbes, PDLP5 and TVCV MP, that can be used during protein localization studies.

Aniline Blue Staining to Visualize Plasmodesmata in Agroinfiltrated Plant Leaves Using Confocal Microscopy

To begin, obtain Nicotiana benthamiana leaves infiltrated with the agrobacterium tumefacien cells harboring desired vectors. For visualization of plasmodesmata, add an aliquot of 200 microliters of 1%aniline blue onto a microscope slide. Then, using a blade, excise the infiltrated leaf area away from the vein and transfer it to the aniline blue solution on the slide with the abaxial side up.Ensure the leaf tissue sample is fully submerged before covering it with a cover glass. Place the slide with the sample in a desiccator attached to a vacuum pump. Evacuate for two minutes at less than 0.8 pascal.Then, slowly release the pressure and incubate the slide in the dark for 30 minutes at room temperature. Now, visualize the fluorescent signal of aniline blue under a laser scanning confocal microscope.

aniline-blue-staining-to-visualize-plasmodesmata-agroinfiltrated

Research

JoVE Journal

Biology

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.

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.

Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, ...

Agroinfiltration of Plant Leaf for Studying Plasmodesmal Protein Localization

To begin, grow Nicotiana benthamiana seeds in wet soil within a controlled environment chamber set at 23 degrees Celsius, maintaining 16 hours of light, followed by eight hours of darkness. After transferring the two-weeks-old seedlings to larger pots, grow the plants under the same conditions for four to five weeks to prepare them for agroinfiltration experiments. Streak Agrobacterium to Mephasins EHA105 cells containing desired vectors onto LB agar plates and incubate them at 28 degrees Celsius.After two days, transfer a single colony from the plate into two milliliters of LB broth and incubate overnight at 28 degrees Celsius with agitation at 250 revolutions per minute. Then, remove one milliliter from the overnight culture and add four milliliters of fresh LB broth to reculture for one hour under the same conditions. Pellet the cells by centrifugation.After that, determine the cell count of the bacterial suspension at 600 nanometers and adjust the optical density to 0.1 or 0.2 using infiltration buffer. Incubate the bacterial suspension at room temperature for three hours with gentle agitation. Next, infiltrate the abaxial surface of fully expanded leaves from different plants using a one-milliliter needleless disposable syringe.Mark the infiltrated area, which will appear darker than the surrounding non-infiltrated tissue, with a waterproof pen.

Confocal Microscopy of Targeted Proteins to Investigate Plasmodesmal Localization in Agroinfiltrated Plants

To begin, obtain Nicotiana benthamiana plant infiltrated with the Agrobacterium tumefaciens cells harboring desired vectors. For protein localization studies, harvest two leaves from two plants at different time points after the infiltration. Using a blade cut the infiltration zone into slices away from the vein.Place the tissue sample with the abaxial side up on a drop of sterile water at the center of a microscope slide. Cover the slide with a cover glass ensuring no bubbles are trapped. Visualize the fluorescent signal of autofluorescent tags in the infiltrated area with a laser scanning confocal microscope.Collect 20 images for each condition using at least two independent biological replicates. Score plasmodesmal localization of the tested protein based on the diagnostic punctate appearance of the signal at the cell periphery. TVCV MP-EGFP and PDL5-EGFP proteins demonstrated clear punctate localization to plasmodesmata.While PDCB1-EGFP showed localization to both plasmodesmata and endoplasmic reticulum on day one, post infiltration. Both MP and PDLP5 began accumulating at plasmodesmata on day one post infiltration, reaching maximum intensity on day two and remained stable for five days. PDCB1 exhibited strong endoplasmic reticulum localization until day three, after which plasmodesmal localization became visible in fewer cells.