The work in the VC laboratory was supported by grants from NIH (R35GM144059), NSF (MCB 1913165 and IOS 1758046), and BARD (IS-5276-20) to VC. The funders had no role in study design, data collection, and interpretation, or the decision to publish.

The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Plant growth
<ol>
	<li>Grow Nicotiana benthamiana seeds in wet soil in a controlled environment chamber at 23 &#730;C under 16 h of light and 8 h of darkness.</li>
	<li>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.</li>
</ol>
2. Vector construction
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Verify all constructs by PCR and sequencing19.</li>
</ol>
3. Agroinfiltration
<ol>
	<li>Streak Agrobacterium tumefaciens EHA105 cells containing different vectors on LB agar plates and incubated for 2 days under 28 &#730;C.</li>
	<li>Transfer a single colony into 2 mL of LB broth and incubate overnight at 28 &#730;C with agitation (250 rpm).</li>
	<li>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.</li>
	<li>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).</li>
	<li>Mix different bacterial suspensions at a ratio of 1:1 (v/v) to make a final concentration of OD600 = 0.1. (optional).</li>
	<li>Incubate the bacterial suspension at room temperature for 3 h with soft agitation.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Aniline blue stain
<ol>
	<li>Add an aliquot of 200 &#181;L of 1% aniline blue (in 50 mM potassium phosphate buffer, pH 8.0) to a microscope slide20.</li>
	<li>Excise the infiltrated area of approximately 0.5 cm &#215; 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).</li>
	<li>Place the microscope slides with the samples in a desiccator attached to a vacuum pump and evacuate for 2 min (&#60;0.8 Pa), followed by a slow release of the pressure and incubation in the dark for 30 min at room temperature.</li>
	<li>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.</li>
</ol>
5. Confocal microscopy
<ol>
	<li>Harvest two leaves from two plants at different time points after the infiltration. Cut the infiltration zone into approximately 0.5 cm &#215; 0.5 cm slices away from the vein with a blade.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Collect 20 images for each condition, using at least two independent biological replicates.</li>
	<li>Score PD localization of the tested protein based on the diagnostic punctate appearance of the signal at the periphery of the cell21,22. 
	NOTE: When detecting the subcellular localization of an unknown protein, using at least three plants is recommended.</li>
</ol>
6. Data analysis
<ol>
	<li>Use Fiji software to split channels and add the scale bar to the images for visualization.</li>
	<li>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.</li>
	<li>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 &#181;m2 was calculated.</li>
	<li>Use two-way ANOVA with Tukey&#39;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.</li>
</ol>

Exploring Plasmodesmata Function: Confocal Microscopy of Targeted Proteins

<ol>
	<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasmodesmata-localized+protein+mediates+crosstalk+between+cell-to-cell+communication+and+innate+immunity+in+Arabidopsis.">A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis.</a> The Plant Cell. 23 (9), 3353-3373 (2011).</li><li>Benitez-Alfonso, Y., Faulkner, C., Ritzenthaler, C., Maule, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata+gateways+to+local+and+systemic+virus+infection.">Plasmodesmata gateways to local and systemic virus infection.</a> Mol Plant-Microbe Interact. 23 (11), 1403-1412 (2010).</li><li>Bayer, E. M., Benitez-Alfonso, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+Channels+under+pressure.">Plasmodesmata: Channels under pressure.</a> Annu Rev Plant Biol. 75, 21 (2024).</li><li>Maule, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+Structure,+function+and+biogenesis.">Plasmodesmata: Structure, function and biogenesis.</a> Curr Opin Plant Biol. 11, 680-686 (2008).</li><li>Fernandez-Calvino, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+plasmodesmal+proteome.">Arabidopsis plasmodesmal proteome.</a> PLoS One. 6 (4), e18880 (2011).</li><li>Kirk, P., Amsbury, S., German, L., Gaudioso-Pedraza, R., Benitez-Alfonso, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+meta-proteomic+pipeline+for+the+identification+of+plasmodesmata+proteins+and+regulatory+conditions+in+diverse+plant+species.">A comparative meta-proteomic pipeline for the identification of plasmodesmata proteins and regulatory conditions in diverse plant species.</a> BMC Biol. 20, 128 (2022).</li><li>Levy, A., Erlanger, M., Rosenthal, M., Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasmodesmata-associated+&#946;-1,3-glucanase+in+Arabidopsis.">A plasmodesmata-associated &#946;-1,3-glucanase in Arabidopsis.</a> The Plant J. 49 (4), 669-682 (2007).</li><li>Kankanala, P., Czymmek, K., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+for+rice+membrane+dynamics+and+plasmodesmata+during+biotrophic+invasion+by+the+blast+fungus.">Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus.</a> The Plant Cell. 19 (2), 706-724 (2007).</li><li>Aung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+bacteria+target+plant+plasmodesmata+to+colonize+and+invade+surrounding+tissues.">Pathogenic bacteria target plant plasmodesmata to colonize and invade surrounding tissues.</a> The Plant Cell. 32 (3), 595-611 (2020).</li><li>Cui, W., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+callose+synthases+CalS1/8+regulate+plasmodesmal+permeability+during+stress.">Arabidopsis callose synthases CalS1/8 regulate plasmodesmal permeability during stress.</a> Nat Plants. 2 (5), 160 (2016).</li><li>Liu, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytosphinganine+affects+plasmodesmata+permeability+via+facilitating+PDLP5-stimulated+callose+accumulation+in+Arabidopsis.">Phytosphinganine affects plasmodesmata permeability via facilitating PDLP5-stimulated callose accumulation in Arabidopsis.</a> Mol Plant. 13 (1), 128-143 (2020).</li><li>Caillaud, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasmodesmal+protein+PDLP1+localizes+to+haustoria-associated+membranes+during+downy+mildew+infection+and+regulates+callose+deposition.">The plasmodesmal protein PDLP1 localizes to haustoria-associated membranes during downy mildew infection and regulates callose deposition.</a> PLoS Pathog. 10 (11), e1004496 (2014).</li><li>Li, Z., Su-Ling, L., Christian, M., Walley, J. W., Aung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata-located+protein+6+regulates+plasmodesmal+function+in+Arabidopsis+vasculature.">Plasmodesmata-located protein 6 regulates plasmodesmal function in Arabidopsis vasculature.</a> The Plant Cell. , (2024).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+PDLP7+modulated+plasmodesmata+function+is+related+to+BG10-dependent+glucosidase+activity+required+for+callose+degradation.">Arabidopsis PDLP7 modulated plasmodesmata function is related to BG10-dependent glucosidase activity required for callose degradation.</a> Sci Bull. , (2024).</li><li>Simpson, C., Thomas, C., Findlay, K., Bayer, E., Maule, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+GPI-anchor+plasmodesmal+neck+protein+with+callose+binding+activity+and+potential+to+regulate+cell-to-cell+trafficking.">An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking.</a> The Plant Cell. 21 (2), 581-594 (2009).</li><li>Thomas, C. L., Bayer, E. M., Ritzenthaler, C., Fernandez-Calvino, L., Maule, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+targeting+of+a+plasmodesmal+protein+affecting+cell-to-cell+communication.">Specific targeting of a plasmodesmal protein affecting cell-to-cell communication.</a> PLoS Biol. 6 (1), e7 (2008).</li><li>Lim, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata+localizing+proteins+regulate+transport+and+signaling+during+systemic+acquired+immunity+in+plants.">Plasmodesmata localizing proteins regulate transport and signaling during systemic acquired immunity in plants.</a> Cell Host Microbe. 19 (4), 541-549 (2016).</li><li>Sheludko, Y. V., Sindarovska, Y. R., Gerasymenko, I. M., Bannikova, M. A., Kuchuk, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+several+Nicotiana+species+as+hosts+for+high-scale+Agrobacterium-mediated+transient+expression.">Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression.</a> Biotechnol Bioeng. 96 (3), 608-614 (2007).</li><li>Walhout, A. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY+recombinational+cloning:+Application+to+the+cloning+of+large+numbers+of+open+reading+frames+or+ORFeomes.">GATEWAY recombinational cloning: Application to the cloning of large numbers of open reading frames or ORFeomes.</a> Methods Enzymol. 328, 575-592 (2000).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dsRNA-induced+immunity+targets+plasmodesmata+and+is+suppressed+by+viral+movement+proteins.">dsRNA-induced immunity targets plasmodesmata and is suppressed by viral movement proteins.</a> The Plant Cell. 35 (10), 3845-3869 (2023).</li><li>Roberts, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+the+frequency+and+architecture+of+plasmodesmata+during+the+sink-source+transition+in+tobacco+leaves.">Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves.</a> Protoplasma. 218, 31-44 (2001).</li><li>Yuan, C., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasmodesmal+localization+signal+of+TMV+MP+is+recognized+by+plant+synaptotagmin+SYTA.">The plasmodesmal localization signal of TMV MP is recognized by plant synaptotagmin SYTA.</a> mBio. 9 (4), e01314-e01318 (2018).</li><li>Ward, S. J., Ramirez, M. D., Neelakantan, H., Walker, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+prevents+the+development+of+cold+and+mechanical+allodynia+in+paclitaxel-treated+female+C57Bl6+mice.">Cannabidiol prevents the development of cold and mechanical allodynia in paclitaxel-treated female C57Bl6 mice.</a> Anesth Analg. 113 (4), 947-950 (2011).</li><li>Erkkil&#228;, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widefield+fluorescence+lifetime+imaging+of+protoporphyrin+IX+for+fluorescence-guided+neurosurgery:+An+ex+vivo+feasibility+study.">Widefield fluorescence lifetime imaging of protoporphyrin IX for fluorescence-guided neurosurgery: An ex vivo feasibility study.</a> J Biophotonics. 12 (6), e201800378 (2019).</li><li>Levy, A., Zheng, J. Y., Lazarowitz, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tobamovirus+Turnip+vein+clearing+virus+30-kilodalton+movement+protein+localizes+to+novel+nuclear+filaments+to+enhance+virus+infection.">The tobamovirus Turnip vein clearing virus 30-kilodalton movement protein localizes to novel nuclear filaments to enhance virus infection.</a> J Virol. 87 (11), 6428-6440 (2013).</li><li>Yuan, C., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+functional+plasmodesmal+localization+signal+in+a+plant+viral+cell-to-cell-movement+protein.">Identification of a functional plasmodesmal localization signal in a plant viral cell-to-cell-movement protein.</a> mBio. 7 (1), e2052-e2015 (2016).</li><li>Kumar, G., Dasgupta, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability,+functions+and+interactions+of+plant+virus+movement+proteins:+what+do+we+know+so+far.">Variability, functions and interactions of plant virus movement proteins: what do we know so far.</a> Microorganisms. 9 (4), 695 (2021).</li><li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+nondestructive+cell-to-cell+and+systemic+movement+of+plant+viruses.">The ins and outs of nondestructive cell-to-cell and systemic movement of plant viruses.</a> Crit Rev Plant Sci. 23 (3), 195-250 (2004).</li></ol>

The authors declare no competing interests.

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, ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABT AC 1 phase motor</td>
			<td>BRANDTECH</td>
			<td>&#160;ABF63/4C-7RQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens EHA105</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Contamination control&#160;</td>
			<td>CCI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II Enzyme mix</td>
			<td>Invitrogen</td>
			<td>#11789020</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Enzyme mix</td>
			<td>Invitrogen</td>
			<td>#11791020</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0.1.</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotiana benthamiana</td>
			<td>Plant species</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pDONR207</td>
			<td>Invitrogen</td>
			<td>#12213013</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>#M0491S</td>
			<td></td>
		</tr>
	</tbody>
</table>

confocal microscopy analysis of protein sorting to plasmodesmata in nicotiana benthamiana

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.

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.

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...

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, ...

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Research

JoVE Journal

Biology

1.1K Views.  State University of New York. 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. T...

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

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.

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.

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.