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

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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. 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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, decorating and visualizing these structures. This need is addressed here by selecting three known PD-localizing proteins: Arabidopsis PDLP5 and PDCB1, and MP from the tobamoviral pathogen TVCV. These proteins were tagged at their C-termini with the autofluorescent reporter EGFP, and the ability of the resulting fusion proteins to sort to PD was determined following their transient expression in leaves of Nicotiana benthamiana and confocal microscopy analyses of the expressing tissues. For general visualization of PD in these tissues, the standard aniline blue staining method was utilized.
Plant viral MPs are thought to fall into two major groups in regard to their effects on PD structure and integrity: MPs that increase PD permeability reversibly and without any structural alterations in PD and MPs that gate PD by modifying their structure. MPs of tobamoviruses, such as TVCV, belong to the first group MP group, positioning them as attractive markers of PD for subcellular localization studies27,28.
These experiments positioned PDLP5-EGFP and MP-EGFP as reliable and reproducible biological markers of PD, with the efficiency of PD detection comparable to that of the aniline blue dye. The PD localization of PDLP5-EGFP and MP-EGFP was detected throughout the entire observation period of 10 dpi, with the optimal time for observing the characteristic PD localization pattern between 2 dpi and 3 dpi. Although expression from the 35S promoter may result in post-transcriptional silencing of the expressed gene, this often occurs after prolonged expression in stably transformed transgenic plants. On the other hand, the use of this strong constitutive promoter is important for the easy detection of the marker protein. If the reference protein expression declines after 7-10 dpi, shorter periods, i.e., 2-3 dpi, should be used. Significant differences in the overall subcellular localization pattern between PDLP5 and MP were not detected here, except that MP accumulated in the cell nucleus in addition to PD. Conversely, PDCB1 is not recommended for use as a PD marker per se due to the complexity of its subcellular localization pattern, which includes substantial localization to the ER.
The ease of detection of the EGFP fluorescence signal also depended on the growth stage of the plant; specifically, in N. benthamiana, the lowest EGFP signal accumulation was noted at the inflorescence stage18.
This study aimed to develop the PD localization markers maximally exclusive to other subcellular compartments. Thus, their use as references for the localization of proteins of interest with additional subcellular localization capabilities should be combined with the expression of other subcellular markers, such as those specific to the ER, mitochondria, plasma membrane, etc. In this case, the inclusion of additional Agrobacterium cells for the expression of these markers should be taken into account when calculating the final concentration of agroinfiltration cultures, i.e., OD600 of the liquid cultures. Obviously, the cell concentration and plant growth times should be adapted for maximal expression and PD localization pattern detection in other plant species.

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.

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 callose deposits. The experiments showed that both PDLP5 and MP exhibited punctate PD localization, with MP also localized in the cell nucleus (Figure 1A), as previously noted25. In contrast, PDCB1 appeared to partition between PD and ER (Figure 1A). For researchers less experienced in interpreting the subcellular localization of GFP-tagged proteins, Figure 1A also illustrates the characteristic nucleocytoplasmic pattern of EGFP expressed in plant cells. To confirm the ER signal of PDCB1, we co-expressed PDCB1 with an ER marker and found that the PDCB1 signal overlapped the signal of the ER marker (Figure 1B). To assess the efficiency of the PD localization of the tested proteins, the percentage of cells expressing the fluorescent signal that exhibited PD localization of this signal was calculated (Table 1).
For the identification of subcellular localization by employing reference proteins, it is useful to understand the time scale of the reference protein expression and potential changes of its localization. Figure 2 shows such kinetics for MP, PDLP5, and PDCB1 when PD themselves were visualized by aniline blue staining. Both MP and PDLP5 began to accumulate at PD already at one-day post infiltration (dpi), and this accumulation reached an apparent maximum at 2 dpi, remaining stable for 5 more days, after which the signal intensity decreased, most likely due to protein degradation. The time course of the PDCB1 subcellular sorting was more complex: for the first 3 dpi, PDCB1 exhibited a comparably strong ER signal, which almost completely obscured PD-specific signal localization, whereas at 5 dpi, this latter signal became more pronounced (Figure 2), albeit in fewer cells. Interestingly, the emergence of the clear PD localization pattern of PDCB1 after 5 dpi coincided with substantially fewer cells showing the EGFP signal (Supplementary Figure 1). Thus, PDLP5 and MP can function as PD reference proteins with little background in most other cellular compartments, with an optimal visualization time window of 2-5 dpi. PDCB1, on the other hand, is not as suitable as a PD marker.
It was then verified that the PD markers retain their subcellular localization specificity in the presence of other PD proteins. To this end, MP-EGFP or PDLP5-EGFP was co-expressed with a PD-localizing26&#160;MP of another plant virus, Tobacco mosaic virus (TMV) tagged with CFP. Figure 3 shows that, in both cases, the co-expressed proteins exhibited a clear PD localization pattern. Similarly, PD-specific histological aniline blue staining of the MP-EGFP- or PDLP5-EGFP-expressing tissues detected no interference with the PD localization of the EGFP and aniline blue signals (Figure 3).
PD localization studies often benefit from a more quantitative estimation of the reference protein&#39;s overall expression and PD signal. This quantification approach is illustrated in Figure 4 and Figure 5. For the quantification of general expression, the signal intensity of PDLP5 and MP was measured. Both proteins showed strong signals at all the visualization time points during the 10 dpi period, with maximal signal intensity at 3 dpi for PDLP5 and at 2 dpi for MP (Figure 4). There were no statistically significant differences in signal intensity between PDLP5 and MP at any of the visualization time points. The PDCB1 signal intensity exhibited greater fluctuation between time points, with the strongest signal observed at 7 dpi (Figure 4), although it was difficult to collect statistically significant data at this time point due to the scarcity of expressing cells. Thus, the first 3 dpi represent the optimal timing for detecting PDCB1 expression. As expected, aniline blue staining samples exhibited a more stable signal than the transiently expressed proteins (Figure 4).
The PD puncta formed by PDLP5 and MP were then quantified. PDLP5 showed a stable number of PD puncta throughout the observation period, with no statistically significant differences between any two of the tested time points (Figure 5). The highest number of PD puncta formed by MP was detected at 2 dpi, and they remained relatively stable until the end of the observation period. Comparing the numbers of PD puncta between PDLP5 and MP revealed no statistically significant differences at most sampling times (Figure 5). The number of PD puncta stained with aniline blue also remained stable, although it slightly decreased at 10 dpi. The numbers of PD puncta decorated by aniline blue, PDLP5, or MP were similar to each other with statistical significance (Figure 5), consistent with the PD specificity of these markers. The numbers of PD puncta for PDCB1 were not analyzed because, at earlier time points, the PD signal was obscured by the ER signal, and at later time points, it was observed in only a relatively few cells (see Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67378/67378fig01.jpg" /> 
Figure 1: Plasmodesmata (PD) localization of TVCV MP, PDLP5, and PDCB1 transiently expressed in Nicotiana benthamiana and aniline blue staining of PD. (A) PD localization of MP-EGFP, PDLP5-EGFP, and PDCB1-EGFP and aniline staining. EGFP signal is in green, aniline blue signal is in blue, and plastid autofluorescence is in red. (B) Co-expression of MP-EGFP, PDLP5-EGFP, and PDCB1-EGFP with the endoplasmic reticulum (ER) marker tagged with mRFP. Images were recorded at 2 days post infiltration (dpi) and are single confocal sections representative of two independent experiments (N = 20 images from 2 plants). EGFP signal is in green, mRFP signal is in purple, and plastid autofluorescence is in red. Yellow arrows indicate PDCB1 localized at the ER. Scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67378/67378fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67378/67378fig02.jpg" /> 
Figure 2: Time course of PD localization of TVCV MP, PDLP5, and PDCB1 transiently expressed in Nicotiana benthamiana and of aniline blue staining of PD. Images were recorded at 2 dpi and are single confocal sections representative of two independent experiments (N = 20 images from 2 plants for all systems, except 7 dpi and 10 dpi for PDCB1-EGFP, in which fewer cells exhibited the signal). The EGFP signal is in green, and the aniline blue signal is in blue. Scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67378/67378fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67378/67378fig03.jpg" /> 
Figure 3: TVCV MP and PDLP5 retain their PD localization following co-expression with a PD-localizing TMV MP and costaining with aniline blue. Images were recorded at 2 dpi and are single confocal sections representative of N &#8805; 10 images from 4-6 plants. The EGFP signal is in green, the CFP signal and aniline blue signal are in blue, and the plastid autofluorescence is in red. Scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67378/67378fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67378/67378fig04.jpg" /> 
Figure 4: Time course of fluorescence intensity of TVCV MP, PDLP5, and PDCB1 transiently expressed in Nicotiana benthamiana and aniline blue PD staining. Quantitative data were collected from the experiments described in Figure 2. Error bars represent the standard deviation of the mean from multiple experiments. The individual data points are indicated as the mean for each measurement. Differences between mean values assessed by two-way ANOVA with Tukey&#39;s multiple comparisons test are statistically significant for the P-values. *P &#60; 0.05, **P &#60; 0.01, and ***P &#60; 0.001, ****P &#60; 0.0001; ns, not statistically significant. Blue bars, aniline staining; light green bars, MP-EGFP, dark green bars, PDLP5-EGFP; yellow bars, PDCB1-EGFP. <a href="https://www.jove.com/files/ftp_upload/67378/67378fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67378/67378fig05.jpg" /> 
Figure 5: Time course of PD puncta formation by TVCV MP and PDLP5 transiently expressed in Nicotiana benthamiana and by aniline blue staining of PD. Quantitative data were collected from the experiments described in Figure 2. Error bars represent the standard deviation of the mean from multiple experiments. The individual data points are indicated as the mean for each measurement. Differences between mean values assessed by two-way ANOVA with Tukey&#39;s multiple comparisons test are statistically significant for the P-values. *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, ****P &#60; 0.0001. Blue bars, aniline staining; light green bars, MP-EGFP, dark green bars, PDLP5-EGFP. <a href="https://www.jove.com/files/ftp_upload/67378/67378fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Experiment I</td>
		</tr>
		<tr>
			<td></td>
			<td>Total cells</td>
			<td>Cells with PD localization</td>
			<td>Cells with mixed signal</td>
			<td>Cells with PD localization (%)</td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>40</td>
			<td>40</td>
			<td>4</td>
			<td>100</td>
		</tr>
		<tr>
			<td>MP-EGFP</td>
			<td>40</td>
			<td>40</td>
			<td>4</td>
			<td>100</td>
		</tr>
		<tr>
			<td>PDCB1-EGFP</td>
			<td>40</td>
			<td>3</td>
			<td>38</td>
			<td>7.5</td>
		</tr>
		<tr>
			<td>PDLP5-EGFP</td>
			<td>40</td>
			<td>40</td>
			<td>0</td>
			<td>100</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Experiment II</td>
		</tr>
		<tr>
			<td></td>
			<td>Total cells</td>
			<td>Cells with PD localization</td>
			<td>Cells with mixed signal</td>
			<td>Cells with PD localization (%)</td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>40</td>
			<td>40</td>
			<td>8</td>
			<td>100</td>
		</tr>
		<tr>
			<td>MP-EGFP</td>
			<td>40</td>
			<td>39</td>
			<td>8</td>
			<td>97.5</td>
		</tr>
		<tr>
			<td>PDCB1-EGFP</td>
			<td>40</td>
			<td>12</td>
			<td>31</td>
			<td>30</td>
		</tr>
		<tr>
			<td>PDLP5-EGFP</td>
			<td>40</td>
			<td>39</td>
			<td>2</td>
			<td>97.5</td>
		</tr>
	</tbody>
</table>
Table 1: Efficiency of PD localization.
Supplementary Figure 1: PD localization of TVCV MP, PDLP5, and PDCB1 transiently expressed in Nicotiana benthamiana. Images were recorded at 5 dpi (A), 7 dpi (B), and 10 dpi (C). The EGFP signal is green, and the plastid autofluorescence is red. Scale bars = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67378/Figure S1.zip" target="_blank">Please click here to download this File.</a>

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

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.

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, ...

confocal-microscopy-analysis-protein-sorting-to-plasmodesmata

Universidad San Sebastian

Research

JoVE Journal

Biology

1.9K Views.  State University of New York. 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.

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

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

Autofluorescence Imaging to Evaluate Red Algae Physiology

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

The present protocol describes step-by-step autofluorescence imaging and evaluation of phycobiliprotein changes in red algae based on spectral analysis. This is a label-free and non-destructive method to evaluate cellular adaptation to extreme habitats, when only scarce material is available and cells grow slowly, or not at all, under laboratory conditions.

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...