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>

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...

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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

Universidad de Cartagena UDEC

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

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Biology

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 ...

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 ...

An Integrated Approach for Microprotein Identification and Sequence Analysis

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model organisms. However, despite this wealth of sequence information, comprehensive gene annotation efforts have proven challenging, especially for small proteins. Notably, conventional protein annotation methods were designed to intentionally exclude putative proteins encoded by short open reading frames (sORFs) less than 300 nucleotides in length to filter out the exponentially higher number of spurious noncoding sORFs throughout the genome. As a result, hundreds of functional small proteins called microproteins (&#60;100 amino acids in length) have been incorrectly classified as noncoding RNAs or overlooked entirely.
Here we provide a detailed protocol to leverage free, publicly available bioinformatic tools to query genomic regions for microprotein-coding potential based on evolutionary conservation. Specifically, we provide step-by-step instructions on how to examine sequence conservation and coding potential using Phylogenetic Codon Substitution Frequencies (PhyloCSF) on the user-friendly University of California Santa Cruz (UCSC) Genome Browser. Additionally, we detail steps to efficiently generate multiple species alignments of identified microprotein sequences to visualize amino acid sequence conservation and recommend resources to analyze microprotein characteristics, including predicted domain structures. These powerful tools can be used to help identify putative microprotein-coding sequences in noncanonical genomic regions or to rule out the presence of a conserved coding sequence with translational potential in a noncoding transcript of interest.

The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

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mRNA Interactome Capture from Plant Protoplasts

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Identification of Post-translational Modifications of Plant Protein Complexes

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

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

An Integrated Approach for Microprotein Identification and Sequence Analysis

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays