For the lack of space, we cited mostly review articles, and we apologize to our colleagues whose original work was not cited. The work in the V.C. laboratory is supported by grants from NIH, NSF, USDA/NIFA, BARD, and BSF to V.C., and the S.G.L. laboratory is supported by NIH and funds from the Departments of Plant Pathology and Plant-Microbe Biology to S.G.L.

1. Plant Material
<ol>
	<li>Choice of plant species</li>
	<li>Use the plant species native to the protein of interest, i.e., one which encodes this protein for endogenous proteins or which represents the natural host for the pathogen for viral proteins. In addition, the selected plant species must be amenable to the chosen method of transient genetic transformation. 
	NOTE: The studies routinely employ Nicotiana benthamiana, which represents a good host for TMV and is transformed efficiently by the Agrobacterium-mediated genetic transformation technique, i.e., agroinfiltration (see Step 3). N. benthamiana plants also are easily grown and have large leaves, which are easily inoculated by Agrobacterium and easily analyzed by confocal microscopy.</li>
	<li>Plant growth
	<ol>
		<li>Plant N. benthamiana seeds in wet soil at high density. Keep them in a controlled environment chamber at 20-25 &#730;C under 16 h of light at ~75 &#181;mol photons m-2 s-1 and 8 h of darkness. After the diameter of euphyll reaches 0.5 cm, carefully transfer the seedlings to larger pots and continue growth in the same chamber under the same conditions.</li>
		<li>Maintain the plants until they grow to 4-8-leaf stage within 4 weeks when the biggest leaves are 4-6 cm in diameter; at this time, they can be used for agroinfiltration (see Step 3). 
		NOTE: Importantly, never use the plants if they began to flower because, at this developmental stage, the leaf architecture often varies24,25, sometimes leading to inconsistent results.</li>
	</ol>
	</li>
</ol>
2. Expression Vector Construction
<ol>
	<li>Choice of expression system
	<ol>
		<li>Choose the expression system, i.e., vectors and vector delivery method, best suited for expression of the protein of interest in the plant species studied. 
		NOTE: Occasionally, such specific combinations of tested protein/plant species may require specific vectors and vector delivery method for optimal expression and function of the protein of interest. For most dicotyledonous plants, however, select binary plasmids as expression vectors and agroinfiltration as delivery system.</li>
	</ol>
	</li>
	<li>Protein fluorescent tagging
	<ol>
		<li>Fluorescently tag each expressed protein for analysis of its subcellular distribution22, including Pd localization. 
		NOTE: Employ autofluorescent proteins, such as CFP and DsRed2, as tags. Tag the tested protein with CFP and coexpress it with a free DsRed2. CFP functions both as tag and as a virus-unrelated cargo molecule. DsRed2 functions to calibrate overall efficiency of transient expression and, because it is cell-autonomous, to identify the initially transformed cell; this latter function is important when assessing cell-to-cell movement of potentially non-cell-autonomous tested proteins. For coexpression with the Pd marker PDCB1, which also is cell-autonomous, tag the tested protein with CFP and PDCB1 with DsRed2.</li>
		<li>As a rule of thumb, fuse the autofluorescent tag to the carboxyl-terminus of the expressed protein. However, if the tagged full-length tested protein exhibits compromised Pd targeting, transfer the tag to the amino-terminus of the protein.</li>
		<li>Clone the coding sequences of the proteins to be tested into a suitable plant expression vector. 
		NOTE: There are many different plant expression vectors that allow fluorescent tagging. We use a series of pSAT plasmids26 or some of their derivatives27,28, which are suitable for cloning of the sequence of interest directly in frame with the suggested autofluorescent tags (see Step 2.2.1) in both amino- and carboxyl-terminal orientations.</li>
		<li>Transfer each expression cassette into an Agrobacterium binary vector. 
		NOTE: Although pSAT-based constructs are suitable for direct biolistic delivery into plant tissues, agroinfiltration, which is the transformation method recommended here (see Step 3), requires that the expression cassette to be located between the T-DNA borders of a binary vector29. 
		NOTE: All pSAT vectors are designed to allow such transfer to the pPZP-RCS2 multigene expression binary vector26,30 by a single-step cloning using rare cutting nucleases AscI, I-PpoI, I-SceI, I-CeuI, PI-PspI and PI-TliI26. Researchers who prefer to utilize recombinatorial cloning, e.g., using heterologous lox sites31, can employ pSAT vector variants26,27.</li>
		<li>For coexpression, transfer both expression cassettes, e.g., tested protein-CFP and DsRed2 or tested protein-CFP and PDCB1-DsRed2 (see Step 2.1), to the same pPZP-RCS2 binary vector. 
		NOTE: Alternatively, Agrobacterium cultures harboring individual binary constructs can be mixed together for infiltration into N. benthamiana (see Step 3.1).</li>
	</ol>
	</li>
</ol>
3. Agroinfiltration
<ol>
	<li>Add 1 &#181;g of a pPZP-RCS2-based plasmid from Step 2.2.5 to 100 &#181;L culture of competent cells of Agrobacterium tumefaciens strain EHA105 or GV3101 prepared as described32, incubate on ice for 30 min, flash-freeze in liquid nitrogen for 1 min, and incubate at 28 &#176;C for 15 min.
	<ol>
		<li>Then, add 1 mL of LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) to the competent cell mixture and incubate at 28 &#176;C for 2 h. Spin down the cells at 3,000 &#215; g for 1 min, re-suspend them in 0.2 mL of LB, and plate them on LB agar supplemented with appropriate antibiotics (e.g., 100 mg/L spectinomycin and 50 mg/L rifampicin for pPZP-RSC2-based vectors26,30). Grow at 28 &#176;C for 48 h until individual colonies are visible.</li>
	</ol>
	</li>
	<li>Pick and plate several individual colonies onto a fresh LB agar plate (see Step 3.1), grow at 28 &#176;C for 48 h, and take a small amount of bacteria for analysis by colony PCR33 to confirm the presence of the specific binary constructs.</li>
	<li>Grow each Agrobacterium colony with the construct of interest overnight at 28 &#176;C in 5 mL of LB medium supplemented with appropriate antibiotics (see Step 3.1.1). Centrifuge the cells at 3,000 &#215; g, and re-suspend them to OD600=0.5 in agroinfiltration buffer (10 mM MgCl2, 10 mM MES (pH 5.6), 150 &#181;M acetosyringone). Incubate for 2 h at room temperature.
	<ol>
		<li>If using a mixture of binary plasmids rather than a single multigene expression plasmid for protein coexpression, mix the corresponding cell cultures at 1:1 v/v ratio before infiltration. Then incubate the cells at 28 &#176;C for 2 h.</li>
	</ol>
	</li>
	<li>Load the inoculum into a 1 mL plastic syringe and press the nozzle of the syringe (no needle) against the lower (abaxial) epidermis of the fully mature N. benthamiana leaves. Hold the leaf with a gloved finger on the adaxial face34,35.
	<ol>
		<li>Introduce the Agrobacterium in infiltration medium by slow injection. For statistical significance, infiltrate several leaves on each plant. 
		NOTE: Note that the oldest leaves are not used as they do not yield consistent expression levels.</li>
		<li>Inoculate all leaves in situ. Maintain the infiltrated plant until observation as described in Step 1.2 .</li>
	</ol>
	</li>
</ol>
4. Confocal Microscopy
<ol>
	<li>Use a blade to cut each leaf into fragments between the veins 24-48 h after the infiltration, (depending on the efficiency of transient expression of the specific tested protein); the size of each tissue slice should be such that the slice is completely covered by the cover glass (22 mm x 40 mm) used for microscopy.</li>
	<li>Place the leaf slices on the object slide with the abaxial leaf surface facing up, place a drop of water on the leaf slice and cover it with the cover glass. Immobilize the cover glass with small pieces of tape on each side.</li>
	<li>Observe the agroinfiltrated tissues under a laser scanning confocal microscope and record the resulting images.
	<ol>
		<li>First, use a 10X objective lens to identify cells with the signal, and then use 63x objective lens for more detailed observations.</li>
		<li>Use the 458 nm wavelength from an argon ion laser to excite CFP, and 543 nm to excite DsRed2. Retain all settings for image acquisition, i.e., laser intensity and photomultiplier tube (PMT) settings, between experiments. On average, examine 100-120 cells for each experimental condition.</li>
	</ol>
	</li>
</ol>
5. Identification of PLS
<ol>
	<li>Diagnose the localization of the tested protein using confocal microscope (section 4). Check if the tested protein has the characteristic peripheral punctate pattern25,36,37,38,39,40,41 and colocalizes with the Pd marker PDCB123. This indicates that the tested protein most likely contains PLS sequences.</li>
	<li>After confirmation of the PLS activity of tested protein, progressively subdivide its open reading frame (ORF) (genbank accession number BAF93925.1) into smaller fragments, autofluorescently, (e.g., CFP,) tag each of them, and analyze their subcellular localization as described in Step 4. Initially, the size of 100 amino acid residues for each subdivided fragment is recommended.</li>
	<li>Further subdivide the truncated fragments which have the PLS activity and check their subcellular localization as described in Step 4 until the smallest fragment that still localizes to Pd, i.e. is sufficient to transport the autofluorescent tag cargo to Pd, is identified. This fragment is presumed to represent the PLS sequence.</li>
	<li>Delete the putative PLS from the full-length tested protein, i.e. fuse the remaining part of the test protein without putative PLS to autofluorescent tag, and determine the subcellular localization of the resulting mutant protein as described in Step 4. If this mutant protein that lacks the putative PLS fails to localize to Pd, this will indicate that the identified PLS is not only sufficient (see Step 5.3), but also necessary for the Pd transport of the tested protein.</li>
	<li>Agroinfiltrate the leaves with a mixture of Agrobacterium cultures harboring the expression construct for the CFP-tagged PLS and free DsRed2 (see Step 3.3). Observe infiltrated tissue using confocal microscope with objective lens of 10x using the same settings as in Step 4.3.</li>
	<li>Score cells that contain both CFP and DsRed2 signals as initially infiltrated cells, and score the adjacent cells that contained only the CFP signal as those that exhibit movement. 
	NOTE: This step examines the identified PLS for its potential cell-to-cell movement ability; this is because many proteins that target Pd (including most plant viral MPs) also move through Pd to the neighboring cells. The concentration of Agrobacterium used for infiltration depends on the expression efficiency of different structures respectively. For agroinfiltration, make sure to use the cell culture dilution that ensures (i) transformation of single cells, leaving the adjacent cells untransformed, (ii) achieves similar efficiency of expression. For example, our experiments utilize OD600 of 0.01 and 0.005 for expression of CFP-tagged PLS and free DsRed2, respectively.</li>
</ol>
6. Identification of Key PLS Residues using Alanine Scanning42
<ol>
	<li>Use standard PCR protocols to amplify the PLS coding sequence employing a pair of primers designed to contain the desired mutation, i.e., substitution of the target amino acid residue with alanine, around the central region of each primer as described21. Carry out primer design and use the site-directed mutagenesis kit for site-directed mutagenesis according to the manufacturer&#39;s instructions.</li>
	<li>Purify the resulting PCR products using any standard DNA purification kit, and digest them at 37 &#176;C with DpnI to eliminate the parental (i.e., non-mutated) methylated and hemimethylated DNA. Cool this down for 5 min at room temperature (not on ice). Then transform the mixture directly into E. coli competent cells43, and identify colonies with the desired mutant constructs as described in Step 3.2.</li>
	<li>Introduce the mutant constructs into the binary vector as described in Step 2.2.4, perform agroinfiltration as described in Step 3, and assess Pd targeting as described in Steps 4 and 5.1.</li>
</ol>

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<li>Oparka, K. J., Prior, D. A. M., Santa-Cruz, S., Padgett, H. S., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gating+of+epidermal+plasmodesmata+is+restricted+to+the+leading+edge+of+expanding+infection+sites+of+tobacco+mosaic+virus+%28TMV%29%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV).</a> Plant J. 12, 781-789 (1997).</li>
<li>Crawford, K. M., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li>
<li>Kotlizky, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+dysfunctional+movement+protein+of+Tobacco+mosaic+virus+interferes+with+targeting+of+wild-type+movement+protein+to+microtubules%5BTitle%5D)+AND+%22Mol.+Plant-Microbe+Interact%22%5BJournal%5D)">A dysfunctional movement protein of Tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.</a> Mol. Plant-Microbe Interact. 14, 895-904 (2001).</li>
<li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+transient+genetic+transformation+of+Arabidopsis+leaves+by+biolistic+bombardment%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment.</a> Nat. Protoc. 4, 71-77 (2009).</li>
<li>Giesman-Cookmeyer, D., Lommel, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alanine+scanning+mutagenesis+of+a+plant+virus+movement+protein+identifies+three+functional+domains%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains.</a> Plant Cell. 5, 973-982 (1993).</li>
<li>Ausubel, F. M., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAusubel%2C+FM+%22Current+Protocols+in+Molecular+Biology%22">Current Protocols in Molecular Biology</a>. , Wiley-Interscience. New York. (1987).</li>
<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.tandfonline.com/doi/abs/10.1080/07352680490452807">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses.</a> Crit. Rev. Plant Sci. 23, 195-250 (2004).</li>
<li>Lee, M. W., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+expression+assay+by+agroinfiltration+of+leaves%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Transient expression assay by agroinfiltration of leaves.</a> Methods Mol. Biol. 323, 225-229 (2006).</li>
<li>Burch-Smith, T. M., Schiff, M., Liu, Y., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+virus-induced+gene+silencing+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Efficient virus-induced gene silencing in Arabidopsis.</a> Plant Physiol. 142, 21-27 (2006).</li>
<li>Tian, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+fluorescent+tagging+of+full-length+Arabidopsis+gene+products+in+planta%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta.</a> Plant Physiol. 135, 25-38 (2004).</li>
<li>Tian, G. W., Chen, M. H., Zaltsman, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pollen-specific+pectin+methylesterase+involved+in+pollen+tube+growth%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">A pollen-specific pectin methylesterase involved in pollen tube growth.</a> Dev. Biol. 294, 83-91 (2006).</li>
<li>Hunter, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+nuclear+localization+signals+mediate+nuclear+localization+of+the+GATA+transcription+factor+AreA%5BTitle%5D)+AND+%22Eukaryot.+Cell%22%5BJournal%5D)">Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA.</a> Eukaryot. Cell. 13, 527-538 (2014).</li>
</ol>

No conflicts of interest declared.

This protocol has four core constituents: the concept of identifying a sequence that is both necessary and sufficient for targeting to Pd, systematic division of the protein of interest into fragments that are progressively reduced in length, fusing the tested fragments to an autofluorescent protein that serves both as tag and as macromolecular cargo, and functional assay for Pd targeting in living plant tissues following transient expression of the tested fusion proteins. Note that Agrobacterium-mediated transient expre...

Plasmodesmata (Pd) function as conduits for intercellular transport of key regulators of plant development and morphogenesis, ranging from transcription factors to mRNA and small RNA molecules. Furthermore, this macromolecular transport capacity of Pd is utilized by most plant viruses for their intercellular spread during infection; to move through Pd, plant viruses have evolved specialized proteins, termed movement proteins (MPs), that specifically target to Pd1,2,3,4,5,6,7. Molecular pathways of Pd transport most likely are intimately interconnected with the specific sequences that target the transported proteins into these pathways. Thus, identification of these Pd localization signals (PLSs) may be diagnostic of the corresponding Pd transport pathway. This is by analogy of Pd transport8, for example, to different nuclear import pathways, which can be specific for different nuclear localization signal (NLS) sequences9,10. Conceptually, both NLSs and PLSs represent non-cleavable subcellular targeting sequences that are necessary and sufficient for targeting. However, unlike NLSs11, the sequence information about PLSs is severely limited. Specifically, only four protein sequences involved in Pd targeting have been reported, with all of them derived from endogenous plant proteins. The first one is represented by a homeobox domain of KN112 &#8211; a transcription factor that moves from inner cell layers to epidermis of the plant leaf13 &#8211; and its KNOX homologs14. The second one also is from a transcription factor, Dof, which contains a putative PLS described as the intercellular trafficking (IT) motif15. The third sequence is from the PDLP1 plasmodesmata-resident type 1 membrane protein, and it is represented by a transmembrane domain16. Finally, the fourth Pd targeting sequence was recently reported for glycosylphosphatidylinositol (GPI)-anchored proteins and it is represented by the glycosylphosphatidylinositol (GPI) modification signal17.
Interestingly, until very recently, no PLSs have been reported for viral MPs. Previous studies indicated the presence of putative PLS sequences in plant viral MPs18,19, but no true PLS, i.e., a minimal amino acid sequence both necessary and sufficient for Pd targeting of an unrelated cargo molecule (e.g., CFP) has been identified in a viral MP. Yet one of these proteins, MP of the Tobacco mosaic virus (TMV), was the first for which Pd localization and transport have been demonstrated20.
To address this gap, we developed an experimental strategy to identify TMV MP PLS. This strategy was based on three concepts. (i) We defined PLS as a minimal amino acid sequence that is both necessary and sufficient for protein targeting to Pd21. (ii) Because TMV MP first targets Pd and then translocates through these channels22, we aimed at uncoupling these two activities and identifying the bona fide PLS, which functions only for Pd targeting, and not for the subsequent transport. (iii) We analyzed the identified PLS for amino acid residues important for its Pd targeting activity, whether structurally or functionally. Using this approach, we delineated a 50-amino acid residue sequence at the amino-terminus of TMV MP that acts as bona fide PLS. This was done by producing a series of TMV MP fragments that saturated the entire length of the protein, tagging their carboxyl-termini with CFP and transiently expressing them in plant tissues. Pd localization of each of the tested fragments was determined by coexpressing them with a Pd marker protein, PDCB1 (Pd callose binding protein 1)23. The smallest fragment that still localized to Pd, but did not traverse Pd, was considered to represent PLS. Finally, the PLS was alanine-scanned to determine the key amino acid residues required for its structure and/or function.
Whereas here we illustrate this approach by describing identification of TMV MP PLS, it may be employed to discover PLSs in any other Pd-targeted proteins, whether encoded by plant pathogens or by the plants themselves; this is because our method does not take advantage of any unique features of viral MPs with regards to their ability to target to Pd.

<table><tbody><tr><td>Confocal laser scanning microscope (CLSM)</td><td>Zeiss</td><td>LSM5</td><td>Any CLSM with similar capabilities is appropriate</td></tr><tr><td>Zen software for confocal microscope imaging</td><td>Zeiss</td><td>2009 version</td><td>The software should be compatible with the CLSM used</td></tr><tr><td>Quickchange II site-directed mutagenesis kit&amp;nbsp;</td><td>Agilent</td><td>200523</td><td></td></tr><tr><td>Acetosyringone</td><td>Sigma-Aldrich</td><td>D134406</td><td></td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>69892</td><td></td></tr><tr><td>Syringes without needles</td><td>BD</td><td>309659</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>FisherScientific</td><td>M33-500</td><td></td></tr><tr><td>Spectinomycin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S4014</td><td></td></tr><tr><td>Rifampicin</td><td>Sigma-Aldrich</td><td>R3501</td><td></td></tr><tr><td>Ampicillin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A0166</td><td></td></tr></tbody></table>

identification of plasmodesmal localization sequences in proteins in planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

The representative data, which faithfully illustrate the results expected from the described protocols and identify the TMV MP PLS, are adapted from Yuan et al.21. Figure 1A first summarizes major constructs expressing the full-length TMV MP (1-268), TMV MP PLS (comprising the first 50 amino acid residues of the protein, 1-50), and its alanine scanning V4A derivatives fused to CFP (generated as described in Steps 2.2, 5.2 and 6) whereas <s...

Watch this Scientific Journal Video about Identification of Plasmodesmal Localization Sequences in Proteins In Planta at JoVE.com

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

identification-plasmodesmal-localization-sequences-proteins

Research

JoVE Journal

Immunology and Infection

8.2K Views.  State University of New York. Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Video: Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Environment

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Biology

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Exploring Plasmodesmata Function: Confocal Microscopy of Targeted Proteins

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

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.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Summary

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Confocal Microscopy Analysis of Protein Sorting to Plasmodesmata in Nicotiana benthamiana