VN is supported by the Royal Society. JPR is an Australian Research Council Future Fellow (FT0992129). We thank Dr. Miriam Gifford for critically reading the manuscript.

Note: all steps are done at room temperature, unless stated otherwise.
1.&#160;Preparation of Plant Materials and Buffers
<ol>
	<li>For N. benthamiana
	<ol>
		<li>Grow the Agrobacterium tumefaciens strains of interest for transient expression (in this example Prf-FLAG, Prf-3xHA, Pto-FLAG and empty vector as a control) by shaking (200 rpm) in liquid culture (L medium with appropriate antibiotics) at 28 &#176;C until stationary phase.</li>
		<li>Collect and pellet agrobacteria by centrifugation (3,000 x g for 5 min). Discard supernatant and resuspend pelleted agrobacteria in infiltration buffer.</li>
		<li>Measure the amount of agrobacteria by obtaining the optical density (OD) value at an absorbance of 600 nm (Abs 600 nm). Adjust the OD of bacteria to 0.1-0.8.</li>
		<li>Infiltrate 4&#160;week&#160;old N. benthamiana plants (22 &#176;C, 16 hr light) with agrobacteria by hand (with a 1-ml needleless syringe) or by vacuum (add 0.02% v/v surfactant). 
		Note: Use the first nearly-fully expanded leaf, and the two immediately older leaves for most effective protein expression. For detection of ubiquitinated or acetylated proteins infiltrate leaves with 100 nM MG-132 or 100 ng/ml Trichostatin A, respectively, at 1 and 2 days post-infiltration.</li>
		<li>Harvest the infiltrated leaves 2-5 days post-infiltration and freeze in liquid nitrogen. Store the samples in a -80 &#176;C freezer. 
		Note: Determine the best level of expression of the protein of interest beforehand by taking samples over a time course of 5 days and detect protein levels by immunoblotting.</li>
	</ol>
	</li>
	<li>For A. thaliana stable transgenic lines
	<ol>
		<li>Grow A. thaliana seeds in 6-well plates supplemented with 5 ml of liquid MS medium (1% w/v sucrose) per well. Put three to five seeds (sterilized and stratified) of the transgenic line of interest or the untransformed control line per well. Shake at 200 rpm for two days before transferring the plate to a growth room and leave for 2 weeks (22 &#176;C, 10 hr light).</li>
		<li>Harvest samples and freeze in liquid nitrogen. 
		Note: You can use as a control a transgenic line expressing an unrelated protein with the same tag as the protein of interest.</li>
	</ol>
	</li>
	<li>Buffers
	<ol>
		<li>Prepare Buffer A. De-gas the buffer for 1-2 hr (for RLKs, other membrane bound proteins and nuclear proteins, add 1% v/v IGEPAL CA-63053). 
		Note: You can keep Buffer A at 4 &#176;C indefinitely. You can use 1% v/v Triton instead of IGEPAL CA-360.</li>
		<li>Prepare 80 ml of cold Buffer B at least 1-2 hr before extraction. 
		Note: The ideal ratio of tissue vs. extraction buffer is 1:4 (w/v).</li>
	</ol>
	</li>
</ol>
2.&#160;Protein Extraction
<ol>
	<li>Just prior to extraction prepare 80 ml of cold Buffer C. 
	Note: Add 10 &#956;M MG-132 for the detection of ubiquitinated proteins.</li>
	<li>Grind 20 g of N. benthamiana tissue (approximately 15 leaves) or A. thaliana seedlings (2-4 g per 6-well plate) in liquid nitrogen using a mortar and pestle.</li>
	<li>Add 80 ml of cold Buffer C to the 20 g of ground tissue, mix well, and thaw on ice. 
	Note: Grind all samples at the same time (protein of interest and control).</li>
	<li>Homogenize with 3 bursts of 10 sec each at full speed with a tissue homogenizer (prechilled at 4 &#176;C). Filter through a 22-25 &#956;m tissue and centrifuge at 30,000 x g for 30 min at 4 &#176;C. 
	Note: Alternatively, homogenize with a fresh prechilled mortar and pestle.</li>
	<li>For the blocking and washing steps of the affinity matrix, prepare 20 ml of Buffer D.</li>
	<li>Block 100 ml of suitable affinity matrix using 500 ml of cold Buffer D containing 1 % BSA for 5 min at 4 &#176;C. 
	Note: Preferred affinity matrices include anti-FLAG M2 agarose, Streptavidin agarose, GFP-Trap_A and anti-HA agarose.</li>
	<li>Wash 3x with 1 ml cold Buffer D.</li>
	<li>Remove supernatant of leaf extract and filter through a 0.45 &#956;m syringe filter into a new tube on ice. 
	Note: The extract color should be green or yellow; if the sample has turned brown, discard and start over.</li>
	<li>Take an aliquot of the crude extract for immunoblot, add 5x SDS-PAGE loading buffer, vortex and denature by boiling samples for 10 min at 80-100 &#176;C in a heat block.</li>
</ol>
3.&#160;Protein Immunoprecipitation
<ol>
	<li>Split the protein extract into two 50&#160;ml tubes and add 50 &#956;l of the affinity matrix suspended in Buffer D to each tube. Incubate with gentle rotation for 2 hr at 4 &#176;C. 
	Note: Longer incubations have not been found to increase yield.</li>
	<li>Prepare 600 &#956;l (6x bead volume of affinity matrix) of elution buffer by adding to Buffer D (final concentrations): 0.5% BSA, 0.25 mg/ml FLAG peptide (for anti-FLAG M2 agarose) or 10 mM D-biotin (for Streptavidin agarose). 
	Note: Elution is not recommended in the case of GFP-Trap_A and anti-HA matrices, so proceed directly to boiling in 1x SDS-PAGE loading buffer, step 3.11.</li>
	<li>Precipitate the affinity matrix by centrifugation at 4 &#176;C (5 min at 1,000 x g).</li>
	<li>Take an aliquot of the unbound extract for immunoblot, discard the rest of the supernatant, leaving approximately 500 &#956;l in the bottom of the tube.</li>
	<li>Resuspend the slurry solution with a wide-bore tip.</li>
	<li>Transfer the mixed slurry solution from both tubes to a single 1.5&#160;ml tube. 
	Note: From now on, use 1.5 ml low-protein binding microfuge tubes.</li>
	<li>Pulse 3x for 5 sec using a benchtop centrifuge to precipitate the affinity matrix and remove the supernatant.</li>
	<li>Wash 3-5x with 1 ml cold buffer D. Between the washes precipitate the affinity matrix as previously described and discard the supernatant. In the last wash remove excess Buffer D with the needle of a syringe. 
	Note: A final wash using Buffer D with 0.1% IGEPAL can be used to further reduce nonspecific binding.</li>
	<li>Elute with 200 &#956;l of the appropriate elution buffer (for anti-FLAG M2 or Streptavidin agarose see step 3.2) 3x, 5 min each time with constant shaking.</li>
	<li>Pool the 3 eluates in a single tube. Concentrate the proteins by using 30 &#956;l of absorption resin. Vortex, let stand for 5 min and precipitate the resin (2 min at 10,000 x g). Discard the supernatant. 
	Note: For GFP-Trap_A and anti-HA agarose, skip the steps 3.9&#160;and 3.10.</li>
	<li>Add 50 &#956;l of 1x SDS-PAGE loading buffer to the precipitated affinity matrix or absorption resin. Vortex and boil for 10 min at 80-100 &#176;C in a heat block.</li>
	<li>Centrifuge the boiled affinity matrix or resin for 2 min at 10,000 x g. Aliquot 5 &#956;l of the supernatant for immunoblot, and run with other fractions onto a ~10 cm long SDS-PAGE gel (Figure 2).</li>
	<li>For separation and identification of phosphorylated proteins by mass spectrometry, load the remaining 45 &#181;l onto a ~19-cm long SDS-PAGE gel if extra separation is required (Figure 2). 
	Note: A blank lane can be included between all samples to reduce contamination of samples.</li>
</ol>
4.&#160;Protein Digestion
<ol>
	<li>Stain the SDS-PAGE gel with colloidal Coomassie Brilliant Blue (cCBB) stain. 
	Note: Minimize handling of the gel to reduce contamination with keratins. Make sure that none of the equipment or tools have been used for immunoblotting or other activities that might have left residual protein contamination.</li>
	<li>Destain the gel with copious washing in water, preferably O/N. Cut the band of interest from the gel with a clean razor blade. 
	Note: Align the gel with the immunoblot if necessary to locate the correct area. As phosphorylation can delay migration of proteins on SDS-PAGE cut the area immediately above and below the band of interest.</li>
	<li>Dice the gel slice into cubes of 2-4 mm (this ensures that the gel will be covered by solutions in the tube without blocking pipette tips). Place in a tube.</li>
	<li>Estimate the volume required to keep the gel well submerged. Use this amount for derivatization, incubation with protease and peptide extraction, and then use double to triple volumes for washing. 
	Note: In a typical digest with approximately 100 &#956;l of the cut gel pieces, use 500 &#956;l solution for washing, 2 x 180 &#956;l for dehydration, 150 &#956;l for reduction, 120 &#956;l for alkylation, and 120 &#956;l for digestion.</li>
	<li>Wash the gel pieces 2 x 20 min (or until destained) by adding 50% acetonitrile (in 50 mM ammonium bicarbonate, ABC). Pipette off the solution taking care not to remove gel pieces.</li>
	<li>Dehydrate with 100% acetonitrile for 5 min and remove free liquid. 
	Note: The gel should appear shrunken and white. If the blue color persists, incubate longer in acetonitrile/ABC and/or at elevated temperature (45-55 &#176;C).</li>
	<li>Reduce protein disulfide bonds by adding 10 mM DTT in water and incubate for 30-45 min at 56 &#186;C with shaking. Remove free liquid.</li>
	<li>Alkylate cysteine residues by addition of 55 mM chloroacetamide (in 50 mM ABC) for 20-30 min (room temperature, dark). Remove free liquid.</li>
	<li>Wash gel pieces 2 x 10 min with 50% acetonitrile (in 50 mM ABC). Remove free liquid.</li>
	<li>Dehydrate gel pieces with 100% acetonitrile for 5 min and remove free liquid.</li>
	<li>For the tryptic digest, add 40 &#956;l of trypsin working solution (100 ng of trypsin in digestion buffer: 50 mM ABC, 5% acetonitrile for an average Coomassie-stained band). Allow gel pieces to rehydrate for 10 min. Add enough of digestion buffer to cover gel pieces if necessary. Incubate at 37 &#186;C O/N.</li>
	<li>To stop the digestion and extract the peptides from the gel pieces, add 5% formic acid (in 50% acetonitrile) (add the same volume as the digestion volume). Sonicate for 5-10 min. Transfer the supernatant to new tube. Repeat the extraction 3x.</li>
	<li>Dry the peptides by lyophilization (preferred) or a vacuum concentrator (such as Speed-Vac or Savant). Store at -20 &#186;C.</li>
	<li>Submit your sample for mass spectrometry. 
	Note: We submitted our sample to the mass spectrometry facility at The Sainsbury Laboratory (Norwich, UK). Protocols at mass spectrometry facilities vary considerably, but typically peptides will be dissolved in 0.2% trifluoroacetic acid with 2% acetonitrile before loading onto a liquid chromatography system coupled to electro-spray tandem mass spectrometry.</li>
</ol>

The authors declare that they have no competing interests (financial or other).

Elucidating the mechanism of receptor activation by PAMPs and effectors can contribute greatly to our understanding of plant immunity. In the last 20 years, genetic and yeast two-hybrid screens have been instrumental for the discovery of PRRs and NB-LRR proteins. More recently, mass spectrometry-based protocols have been established for the identification of proteins regulated differentially during immune signaling55-58, their PTMs11,33,59,60, the composition of immune complexes61 and effector targets62. Here we describe a straightforward protocol for the identification of PTMs regulating the activation of immune complexes.
In comparison with previously described protocols, this protocol allows the detailed identification of dynamic changes of PTMs. Protocols of large-scale proteomics approaches can identify PTMs of proteins but are not able to reveal the site plasticity of PTMs due to limited amounts of protein. In the case of protein phosphorylation, large-scale proteomics approaches typically only identify the predominant phosphorylation sites11,33,59. The detailed characterization of a protein phosphorylation status requires a substantial amount of protein that can be obtained only through partial purification of the target protein47. The protocol described here couples a protein purification approach that yields a substantial amount of the target protein, with mass spectrometry analysis of the purified protein. Following this protocol, the single phosphorylation event of Pto was attributed predominantly to Ser-198 as previously described52 but some mass spectrometry spectra also supported a single phosphorylation event on Thr-195 or Thr-199. When double phosphorylation events of Pto were observed, the predominant combination of phosphorylated amino acids was Ser-198 and Thr-199 although combinations on other sites were also observed (Table 1). These results clearly demonstrate the phosphorylation site plasticity of protein kinases and the ability of the described protocol to characterize in details all possible phosphorylation sites.
The most critical steps of this protocol are: 1) sufficient extraction of proteins; 2) protection of PTMs; and 3) sufficient amount of target protein. First, for sufficient extraction of proteins, it is important to grind the tissue in liquid nitrogen and subsequently to use a tissue homogenizer as described in the protocol. If a tissue homogenizer is not available, a mortar and pestle can be used. It is also important to use a ratio of one to three (or four) of grams of tissue to volume of extraction buffer. This tissue to buffer ratio and the high strength buffer that we suggest will ensure that the pH will remain neutral during the extraction process. We found that this is of particular importance for A. thaliana and tomato extractions52. Protection of PTMs can be achieved by including in all the buffers the appropriate inhibitors of enzymes that can remove PTMs. It is also critical to perform all steps at 4 &#176;C and to prechill buffers and instruments. Higher yields of target protein can be achieved by using plants expressing the protein in sufficient amounts and by using high amounts of tissue (approximately 20 g). The most crucial step for obtaining sufficient amounts of target protein is concentrating the target protein by direct immunoprecipitation. The significance of this step is highlighted by our failure to identify PTMs of Pto after a Prf immunoprecipitation due to the limited amount of coimmunoprecipitated Pto (Figure 2B). In contrast, direct immunoprecipitation of Pto yielded substantially more measurable peptides (Figure 2C) leading to approximately 80% coverage of the protein sequence and identification of PTMs (Table 1). We also strongly recommend the use of epitope-tags for protein immunoprecipitation. In comparison to antibodies raised against native proteins epitope-tags affinity matrix can yield higher amounts of partially purified protein. By using an antibody raised against native Pto protein51 (Figure 2A) for immunoprecipitation, we were able to identify only single phosphorylation of the two predominant phosphorylation sites of Pto.
This protocol has primarily been developed for partial purification of N. benthamiana cytoplasmic protein and subsequent identification of their phosphorylation sites. However, using the modifications described herein, this protocol can be easily adapted for A. thaliana proteins and membrane bound proteins. The main objective of this protocol is to yield sufficient amounts of the target protein for the identification of PTMs and not to achieve the highest purity of the complex. If higher purity of the complex is required a second step of purification can be added to this protocol52. In that case, a first step will allow elution of the target protein from the affinity matrix without the use of high salts or acid and the subsequent step may entail a matrix, which requires harsh elution conditions. It is important to highlight that we have only tested this protocol for the identification of phosphorylation sites and that we are currently testing its ability for detailed identification of additional PTMs.
Our representative results clearly demonstrate the phosphorylation site plasticity of protein kinases and the ability of the described protocol to characterize phosphorylation sites. Most importantly, they highlight that identification of phosphorylation sites relying in low amount of proteins by mass spectrometry and by single point mutations of auto-phosphorylation sites can give confusing results. We show here and previously47 that double phosphorylation of Pto on Ser-198 and Thr-199 is associated with the activation of the Prf/Pto complex. In contrast, previously published results47,63 have shown that single point mutations of Ser-198 and Thr-199 to alanine, which prevent phosphorylation on these sites, are capable of signaling suggesting that phosphorylation of these sites is not a prerequisite for complex activation. These results can now be explained by phosphorylation of the secondary site Thr-195. Similar insight into the phosphorylation site plasticity of other protein kinase can be obtained following this protocol. Furthermore, a combined approach using phosphorylation sites point mutations, proteins that can inhibit specific kinases (effectors) coupled with protein immunoprecipitation and mass spectrometry analysis will lead to a better understanding of the evolutionary significance of the phosphorylation site plasticity of protein kinases.

Immune pathways rely on various post-translational modifications (PTMs) of proteins to rapidly transduce signals and activate immune responses1. PTMs are rapid, reversible, and highly specific chemical alterations of protein structure that can affect protein conformation, activity, stability, localization, and protein-protein interactions2-4. In plants, more than 300 types of PTMs have been identified including ubiquitination, sumoylation, sulfation, glycosylation, and phosphorylation2,5. A growing body of evidence highlights the importance of PTMs in different aspects of plant immunity1,5,6. Protein phosphorylation, the reversible attachment of a phosphate group to a serine, threonine&#160;or tyrosine residue is a regulator of many cellular functions and not surprisingly the most highly studied PTM in plant defense signaling cascades5-11. Phosphorylation-dependent signaling is an integral part of plant defense activation initiated after extracellular perception of microbes by transmembrane receptors, or intracellular recognition by multidomain resistance proteins5,8.
Upon invading plants, microbes are detected by plasma membrane receptors called pattern recognition receptors (PRRs)12. Known PRRs are either receptor-like proteins (RLPs) or receptor-like kinases (RLKs), both carrying a ligand-binding ectodomain and a single-pass transmembrane domain. In contrast to RLPs, RLKs have an intracellular kinase domain13-15. The ectodomain of PRRs binds to conserved microbe elicitor molecules called pathogen-associated molecular patterns (PAMPs) leading, in the case of RLKs, to autophosphorylation and transphosphorylation within the intracellular domain6,16. Downstream of perception-induced phosphorylation of PRRs, subsequent phosphorylation of cytoplasmic proteins including kinases17,18, E3 ligases19, mitogen-activated protein kinases (MAPKs)20,21, calcium-dependent protein kinases (CDPK)22,23, and transcription factors24,25 leads to PAMP-triggered immunity (PTI).
In addition to the extracellular perception by PRRs, intracellular recognition of pathogens is achieved through cytoplasmic receptors. These multidomain resistance (R) proteins contain a variable N-terminus domain, a central nucleotide-binding (NB) motif, and C-terminal leucine-rich repeats (LRR) and therefore called NB-LRR proteins26,27. R proteins directly or indirectly recognize pathogen-derived virulence molecules called effectors, also known to target PRRs and other nodes of the immune system. Recognition induces strong defenses leading to effector-triggered immunity (ETI)28-31. NB-LRR proteins have a requisite ATP-binding motif within the NB domain30,32, but they lack a kinase domain. Phosphorylation of conserved domains of NB-LRRs has been reported in a large-scale proteomic survey33, but its relevance for ETI is unclear. Similarly to PTI, activation of NB-LRR proteins leads to phosphorylation of cytoplasmic proteins including MAPKs34-37 and CDPKs38-40. Most importantly, effector recognition can lead to phosphorylation of accessory proteins that interact directly with the NB-LRR proteins41. Some examples include RIN4 (Arabidopsis thaliana) and Pto (tomato, Solanum lycopersicum) proteins that interact with NB-LRR proteins, RPM142,43 and Prf44, respectively. During infection by Pseudomonas syringae bacteria, the effector protein AvrB induces RIN4 phosphorylation, most likely by the receptor-like kinase RIPK45,46. Similarly, in tomato the P. syringae effectors AvrPto and AvrPtoB induce phosphorylation of Pto47. In contrast to RIN4 which lacks a kinase domain and must be trans-phosphorylated, Pto is an active kinase capable of auto-phosphorylation48 and trans-phosphorylation of substrates49,50. While Pto requires its kinase activity for effector-dependent initiation of signaling, kinase-dead Pto mutants are still able to signal in an effector-independent way51. We recently explained these observations by demonstrating that the Prf/Pto complex is oligomeric, containing multiple Prf and Pto molecules52 and that Pto molecules within the same complex can trans-phosphorylate each other47. We proposed a model in which one molecule of Pto (sensor) interacts with the effector protein, causing a conformation change to the NB-LRR protein (Prf) that in turn activates a second Pto molecule (helper) within the complex. Subsequently, the helper Pto molecule trans-phosphorylates the sensor Pto leading to full activation of the resistance complex47.
These examples demonstrate that the identification of immune complex components and their potential PTMs upon effector recognition can lead to a better understanding of how signals are transduced from effector perception to downstream targets. Here we describe a protein purification method for NB-LRR-interacting proteins and the subsequent identification of their PTMs. We use Nicotiana benthamiana and the tomato Prf/Pto complex as a model, but the same protocol can easily be applied to RLKs from N. benthamiana and A. thaliana53 with small modifications as we describe within the protocol.

<table border="0" cellpadding="0" cellspacing="0" width="1108">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:296px;">MgCl2</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:181px;">M8266</td>
			<td style="width:436px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
			<td>toxic - 1 M stock in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 1mL sterile</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe needle</td>
			<td>Terumo</td>
			<td>NN-2525R&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silwet L-77 (surfactant)</td>
			<td>Lehle Seeds</td>
			<td>VIS-01</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MG-132 (Z-Leu-Leu-Leu-al)</td>
			<td>Sigma</td>
			<td>C2211</td>
			<td>1 M stock in DMSO, store at -80 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MS salts</td>
			<td>Sigma</td>
			<td>M5524</td>
			<td>oxidizing, toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Sigma</td>
			<td>16104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>adjust pH with 1 N HCl to make 1 M Tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>5 M stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td>toxic - 0.5 M stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>National Diagnostics</td>
			<td>EC-606</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGEPAL CA-630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PVPP (polyvinylpolypyrrolidone)</td>
			<td>Sigma</td>
			<td>P5288</td>
			<td>do not confuse with PVP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DTT (DL-dithiothreitol)</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>toxic - 1 M stock, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plant protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>do not freeze/thaw too many times</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF (phenylmethylsulfonyl fluoride)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>corrosive, toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calyculin A</td>
			<td>Cell Signaling Technology</td>
			<td>9902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Fluoride (NaF)</td>
			<td>Sigma</td>
			<td>S7920</td>
			<td>toxic - 1 M stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Molybdate (Na2MoO4)</td>
			<td>Sigma</td>
			<td>S6646</td>
			<td>0.5 M stock</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;">Sodium orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>450243</td>
			<td>toxic - Prepare a 200 mM solution of sodium orthovanadate. Adjust the pH to 10.0 using either 1N NaOH or 1N HCl. The starting pH of the sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 the solution will be yellow. Boil the solution until it turns colorless (approximately 10 minutes). Cool to room temperature.Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Okadaic acid</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-3513</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kinematica Polytron tissue homogeniser</td>
			<td>Fisher Scientific</td>
			<td>08-451-320&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Miracloth</td>
			<td>Merck Millipore</td>
			<td>475855-1R&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-FLAG M2 agarose</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin-agarose</td>
			<td>Thermo-Scientific</td>
			<td>20347</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GFP-Trap_A</td>
			<td>Chromotek</td>
			<td>gta-20</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-HA-Agarose</td>
			<td>Sigma</td>
			<td>A2095</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.45 &#956;m filter</td>
			<td>VWR</td>
			<td>513-1902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FLAG peptide</td>
			<td>Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-biotin</td>
			<td>Sigma</td>
			<td>47868</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">StrataClean resin</td>
			<td>Agilent</td>
			<td>400714</td>
			<td>this absorption resin is recommended for protein precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini-PROTEAN Tetra Cell</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PROTEAN II XL Cell</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>this stain is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein low-binding tubes (LoBind)</td>
			<td>Eppendorf</td>
			<td>0030 108.116</td>
			<td>these tubes are recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium bicarbonate (ABC)</td>
			<td>Sigma</td>
			<td>9830</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>VWR</td>
			<td>83639</td>
			<td>toxic, flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-chloroacetamide</td>
			<td>Sigma</td>
			<td>22790</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Promega</td>
			<td>V5280</td>
			<td>irritant, sensitizing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma</td>
			<td>14265</td>
			<td>toxic, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water-bath sonicator</td>
			<td>Ultravawe</td>
			<td>Ultra BT Ultrasonic Bath</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LTQ-Orbitrap XL</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trichostatin A</td>
			<td>Sigma</td>
			<td>T8552</td>
			<td>toxic</td>
		</tr>
	</tbody>
</table>

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 purification of tagged proteins from stable transgenic A. thaliana lines or after transient expression of proteins in N. benthamiana. As illustrated in Figure 1 the purification of the targeted protein is coupled with mass spectrometry to allow identification of interacting proteins and the PTMs of the targeted protein. The protocol has been designed for purification of proteins involved in plant immunity and identification of their PTMs but can be applied to purification of any tagged plant protein.
As an example we use the purification of the Prf/Pto complex from N. benthamiana. The transgenic N. benthamiana line 38-1254, expressing 35S:Pto, was transiently transformed with 35S:Prf-FLAG and the complex was immunoprecipitated with anti-FLAG M2 agarose (Figure 2A). The immunoblots (IB) in Figure 2A illustrate that most of the targeted protein (Prf) was immunoprecipitated. Pto and the interacting effector protein AvrPtoB were copurified with Prf and detected using antibodies and mass spectrometry analysis (Figures 2A and 2B). We found that the Pto that copurified with Prf migrated slower on SDS-PAGE gel when coexpressed with the effector constructs 35S:AvrPto and 35S:AvrPtoB (Figure 2A). Both effector proteins induced slower migration of the Prf-interacting Pto (Figure 2A). This effector recognition-dependent slow migration of Pto was previously attributed to phosphorylation as it could be removed by treatment with phosphatase44. To detect the phosphorylation sites contributing to the slow migration of Pto, we subjected the Prf copurifying Pto to mass spectrometry analysis. Despite the high expression of Pto and its easy detection with immunoblots we could retrieve peptides covering only 57% of Pto sequence and we failed to identify any phosphorylation sites (Figure 2B).
Subsequently, we changed strategies to target the Pto protein with anti-FLAG. N. benthamiana WT plants were transiently transformed with 35S:Prf-3HA and 35S:Pto-FLAG, with or without 35S:AvrPto and 35S:AvrPtoB. The total amount of Pto protein, comprising both the Prf-complexed and the free forms, was immunoprecipitated with anti-FLAG M2 agarose and subjected to SDS-PAGE fractionation, in-gel tryptic digestion and mass spectrometric analysis (Figure 2C). With this approach we identified Pto peptides spanning approximately 80% of its sequence and an unknown 100 kDa interacting protein. We identified a set of single and double phosphorylation sites for Pto peptides 187-202 and 188-202 (Table 1). Ser-198 and Thr-199 were the predominant phosphorylation sites but other phosphorylation sites were also detected (Table 1). Most importantly double phosphorylation on Ser-198 and Thr-199 was identified only in the presence of either effector protein (Table 1). We recently identified a similar set of Pto phosphorylation sites and illustrated the significance of the double phosphorylation event for signaling47. Following the protocol described here we were able to identify dynamic changes in the phosphorylation state of the target protein.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51095/51095fig1highres.jpg" src="/files/ftp_upload/51095/51095fig1.jpg" /> 
Figure 1. General experimental design. The target protein for immunoprecipitation (IP) is tagged and expressed in planta, transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana. Following protein extraction the target protein is immunoprecipitated with an affinity matrix. The proteins are separated on an acrylamide gel by electrophoresis. Gel bands are excised. Proteins are extracted from the gel bands and subjected to mass spectrometry. Phosphorylation sites and interacting proteins are identified. <a href="https://www.jove.com/files/ftp_upload/51095/51095fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51095/51095fig2highres.jpg" src="/files/ftp_upload/51095/51095fig2.jpg" /> 
Figure 2. Prf and Pto immunoprecipitation. A). The construct 35S:Prf-FLAG was transiently expressed in 38-12 line (35S:Pto) along with empty vector (EV), 35S:AvrPto or 35S:AvrPtoB. Three days post-infiltration Prf was immunoprecipitated using anti-FLAG affinity matrix. Immunoblots (IB) for Prf, AvrPtoB and Pto were performed. As pointed by arrows at the bottom of Pto immunoblot panel, AvrPto and AvrPtoB induce a slower migration of Pto. B). The construct 35S:Prf-FLAG was transiently expressed in 38-12 line (35S:Pto) along with empty vector (EV), 35S:AvrPto or 35S:AvrPtoB. Three days post-infiltration Prf was immunoprecipitated using anti-FLAG agarose and separated on SDS-PAGE. The gel was stained with colloidal Coomassie Brilliant Blue (cCBB) and the visible Prf, Pto and AvrPtoB proteins bands were excised from the gel and analysed by mass spectrometry. The percentage peptide coverage of Pto and Prf sequences identified by mass spectrometry is indicated. C). The constructs 35S:Prf-3xHA and 35S:Pto-FLAG were transiently expressed along with 35S:AvrPtoB, 35S:AvrPto or empty vector (EV) in N. benthamiana. Three days post-infiltration the total amount of Pto-FLAG was immunoprecipitated using anti-FLAG agarose and resolved on SDS-PAGE. The visible Pto and Pto-interacting protein bands were excised from the gel and analysed by mass spectrometry. Pto and the 100 kDa Pto-interacting protein band are clearly visible on the colloidal Coomassie Brilliant Blue (cCBB) stained-gel, whereas Prf is slightly less visible (as indicated by arrows). IP: Immunoprecipitation; IB: Immunoblot; CBB: Coomassie Brilliant Blue staining; cCBB: colloidal Coomassie Brilliant Blue staining. <a href="https://www.jove.com/files/ftp_upload/51095/51095fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Prf</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="2">EV</td>
			<td rowspan="2">AvrPto</td>
			<td rowspan="2">AvrPtoB</td>
		</tr>
		<tr>
			<td>Pto peptide 188-202</td>
		</tr>
		<tr>
			<td>GTELDQ[pT195]HLSTVVK</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
		</tr>
		<tr>
			<td>GTELDQTHL[pS198]TVVK</td>
			<td>10</td>
			<td>7</td>
			<td>5</td>
		</tr>
		<tr>
			<td>GTELDQTHLS [pT199]VVK</td>
			<td>8</td>
			<td>3</td>
			<td>4</td>
		</tr>
		<tr>
			<td>G[pT190]ELDQTHLS[pT199]VVK</td>
			<td>0</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>GTELDQTHL[pS198][pT199]VVK</td>
			<td>0</td>
			<td>8</td>
			<td>4</td>
		</tr>
		<tr>
			<td>GTELDQTHLSTVVK</td>
			<td>46</td>
			<td>26</td>
			<td>48</td>
		</tr>
		<tr>
			<td>Pto peptide 187-202</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>KGTELDQ[pT195]HLS TVVK</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
		<tr>
			<td>KGTELDQTHL[pS198]TVVK</td>
			<td>17</td>
			<td>17</td>
			<td>12</td>
		</tr>
		<tr>
			<td>KGTELDQTHLS[pT199]VVK</td>
			<td>4</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>KGTELDQ[pT195]HLS [pT199]VVK</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>KGTELDQTHL[pS198][pT199]VVK</td>
			<td>0</td>
			<td>7</td>
			<td>5</td>
		</tr>
		<tr>
			<td>KGTELDQTHLSTVVK</td>
			<td>21</td>
			<td>37</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Peptides 187-202 and 188-202</td>
			<td>83</td>
			<td>88</td>
			<td>50</td>
		</tr>
		<tr>
			<td>Percentage of peptides with two phosphorylation events </td>
			<td>0%</td>
			<td>26.9%</td>
			<td>11.2%</td>
		</tr>
		<tr>
			<td>Pto Sequence Coverage</td>
			<td>78%</td>
			<td>79%</td>
			<td>82%</td>
		</tr>
	</tbody>
</table>
Table 1. Double phosphorylation of a Pto peptide upon activation of signaling. 35S:Prf-3xHA and 35S:Pto-FLAG were transiently expressed along with empty vector (EV), 35S:AvrPto or 35S:AvrPtoB in N. benthamiana. Three days post-infiltration the total amount of Pto-FLAG protein was immunoprecipitated using anti-FLAG affinity matrix and resolved on SDS-PAGE. The visible bands of Pto on the colloidal Coomassie brilliant blue stained gel were excised and analysed with mass spectrometry. The number of Pto peptides identified with 0, 1, and 2 phosphorylation events is indicated.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Table of buffers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L medium</td>
			<td>Tryptone</td>
			<td>10 g/L</td>
		</tr>
		<tr>
			<td></td>
			<td>Yeast extract</td>
			<td>5 g/L</td>
		</tr>
		<tr>
			<td></td>
			<td>NaCl</td>
			<td>5 g/L</td>
		</tr>
		<tr>
			<td></td>
			<td>D-glucose</td>
			<td>1 g/L</td>
		</tr>
		<tr>
			<td>Agro-infiltration buffer</td>
			<td>MgCl2</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>MES</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Adjust to pH 5.5</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Acetosyringone (optional) </td>
			<td>150 &#956;M</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td>Tris-HCl pH 7.5</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>NaCl</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>EDTA</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>EGTA</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Glycerol</td>
			<td>5% (v/v)</td>
		</tr>
		<tr>
			<td>Buffer B</td>
			<td>Buffer A</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>PVPP</td>
			<td>2% (w/v)</td>
		</tr>
		<tr>
			<td>Buffer C</td>
			<td>Buffer B</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Dithiothreitol (DTT)</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Plant Protease Inhibitor Cocktail</td>
			<td>1% (v/v)</td>
		</tr>
		<tr>
			<td></td>
			<td>Phenylmethylsulfonyl fluoride (PMSF) </td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Calyculin A</td>
			<td>50 nM</td>
		</tr>
		<tr>
			<td></td>
			<td>Sodium fluoride (NaF)</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Sodium molybdate (Na2MoO4)</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Sodium Orthovanadate</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Okadaic acid</td>
			<td>100 nM</td>
		</tr>
		<tr>
			<td>Buffer D</td>
			<td>Buffer C without PVPP</td>
			<td></td>
		</tr>
		<tr>
			<td>5x SDS-PAGE loading buffer</td>
			<td>Tris-HCl pH 6.8</td>
			<td>60 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>SDS</td>
			<td>2%</td>
		</tr>
		<tr>
			<td></td>
			<td>Glycerol</td>
			<td>0.15%</td>
		</tr>
		<tr>
			<td></td>
			<td>Bromophenol blue</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td></td>
			<td>DTT (add just before use)</td>
			<td>50 mM</td>
		</tr>
	</tbody>
</table>
Table 2. Buffer and media recipes.

Watch this Scientific Journal Video about Identification of Post-translational Modifications of Plant Protein Complexes at JoVE.com

Identification of Post-translational Modifications of Plant Protein Complexes

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 ...

identification-post-translational-modifications-plant-protein

Research

JoVE Journal

Biology

23.7K Views.  University of Warwick. 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.

Video: Identification of Post-translational Modifications of Plant Protein Complexes

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...