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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 – a transcription factor that moves from inner cell layers to epidermis of the plant leaf13 – 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.

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Protocol

1. Plant Material

  1. Choice of plant species
  2. 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.
  3. Plant growth
    1. Plant N. benthamiana seeds in wet soil at high density. Keep them in a controlled environment chamber at 20-25 ˚C under 16 h of light at ~75 µmol 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.
    2. 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.

2. Expression Vector Construction

  1. Choice of expression system
    1. 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.
  2. Protein fluorescent tagging
    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. 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).

3. Agroinfiltration

  1. Add 1 µg of a pPZP-RCS2-based plasmid from Step 2.2.5 to 100 µL 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 °C for 15 min.
    1. 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 °C for 2 h. Spin down the cells at 3,000 × 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 °C for 48 h until individual colonies are visible.
  2. Pick and plate several individual colonies onto a fresh LB agar plate (see Step 3.1), grow at 28 °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.
  3. Grow each Agrobacterium colony with the construct of interest overnight at 28 °C in 5 mL of LB medium supplemented with appropriate antibiotics (see Step 3.1.1). Centrifuge the cells at 3,000 × g, and re-suspend them to OD600=0.5 in agroinfiltration buffer (10 mM MgCl2, 10 mM MES (pH 5.6), 150 µM acetosyringone). Incubate for 2 h at room temperature.
    1. 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 °C for 2 h.
  4. 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.
    1. 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.
    2. Inoculate all leaves in situ. Maintain the infiltrated plant until observation as described in Step 1.2 .

4. Confocal Microscopy

  1. 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.
  2. 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.
  3. Observe the agroinfiltrated tissues under a laser scanning confocal microscope and record the resulting images.
    1. First, use a 10X objective lens to identify cells with the signal, and then use 63x objective lens for more detailed observations.
    2. 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.

5. Identification of PLS

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.

6. Identification of Key PLS Residues using Alanine Scanning42

  1. 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's instructions.
  2. Purify the resulting PCR products using any standard DNA purification kit, and digest them at 37 °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.
  3. 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.

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Results

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

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Discussion

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...

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Disclosures

No conflicts of interest declared.

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
Confocal laser scanning microscope (CLSM)ZeissLSM5Any CLSM with similar capabilities is appropriate
Zen software for confocal microscope imagingZeiss2009 versionThe software should be compatible with the CLSM used
Quickchange II site-directed mutagenesis kit Agilent200523
AcetosyringoneSigma-AldrichD134406
MESSigma-Aldrich69892
Syringes without needlesBD309659
MgCl2FisherScientificM33-500
Spectinomycin Sigma-AldrichS4014
RifampicinSigma-AldrichR3501
Ampicillin Sigma-AldrichA0166

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