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. Author manuscript; available in PMC: 2021 Nov 30.
Published in final edited form as: Methods Mol Biol. 2019;2026:21–28. doi: 10.1007/978-1-4939-9612-4_2

Rapid Examination of Phytochrome–Phytochrome Interacting Factor (PIF) Interaction by In Vitro Coimmunoprecipitation Assay

Inyup Paik 1, Enamul Huq 1,2
PMCID: PMC8630752  NIHMSID: NIHMS1757734  PMID: 31317400

Abstract

Phytochromes are red/far-red light receptors that perceive and transduce light signals to regulate various physiological responses. A large number of phytochrome interacting proteins have been identified using genetic and biochemical approaches. A direct interaction between phytochrome and its interacting proteins, therefore, defines one of the critical steps to initiate light signaling cascades in plants. Thus it is important to thoroughly examine the light-dependent interaction between phytochromes and putative phytochrome-interacting proteins. In this chapter, a protocol for rapid and simple light dependent in vitro coimmunoprecipitation between phytochromes and phytochrome interacting factors is described. In principle, this protocol can be adapted for other putative phytochrome interacting proteins.

Keywords: Protein–protein interaction, Phytochrome interacting factors, In vitro coimmunoprecipitation assay, TnT system

1. Introduction

Phytochromes (phys) are red and far-red light receptors with pivotal roles in plant growth and development. They utilize a chromophore to perceive light and switch between two interconvertible conformers (Pr and Pfr) in response to far-red and red light, respectively. Phytochromes are synthesized in the cytosol and red light perception by phytochromes leads to a translocation of phytochromes into the nucleus [1, 2] and triggers light signaling cascade primarily by direct interaction with multiple phytochrome interacting proteins [3]. A variety of phytochrome interacting proteins were identified including multiple transcription factors, translational regulators, protein kinases, phosphatases, E3 ligases, other photoreceptors, and hormone signaling regulators [4–14].

Among them, the basic helix–loop–helix transcription factors called phytochrome interacting factors (PIFs) are well conserved in plants and have been shown to function as master regulators of transcription in response to light and environmental cues [15].

Myriad of evidence have indicated that physical interaction between phytochromes and their binding proteins triggers multiple biochemical responses including but not limited to conformational changes, sequestration, phosphorylation, and degradation of the phytochrome interacting proteins [8, 16–19]. Many of these phytochrome interacting proteins exert conformer specific bindings indicating the complex light regulation of the binding between phytochrome and its interacting proteins.

The biological significance of physical interaction between phytochrome and its binding proteins is critical; therefore, it is essential to examine direct phytochrome-protein interaction in vitro. However, the high molecular weight of the phytochromes (~125 kDa) and the requirement of the chromophore make it laborious to purify functional phytochromes for simple in vitro interaction assay. In this chapter, we provide a detailed protocol for simple and rapid light dependent co-immunoprecipitation assay between phytochrome and PIFs, a bHLH class of transcription factors from Arabidopsis, by utilizing a T&T quick-coupled transcription/translation system (Promega, Madison, WI).

The T&T quick-coupled transcription/translation system provides a rapid method for synthesis of phytochrome and its binding proteins simply by mixing and incubating template DNA in the T&T reaction. This system also allows assembly of holoproteins by expressing the apoprotein in the TnT reaction and incubation with a chromophore in case of phytochromes. Eukaryotic nature of the T&T mixture enables putative post-translational modifications of the protein of interest and helps proper folding of the proteins. On the other hand, the system only allows for relatively small amount of translation product. Therefore, it may not be adequate for large-scale biochemical analysis.

2. Materials

2.1. Expression of Protein Using the TnT System and 35S-Methionine Labeling

  1. TnT® Quick Coupled Transcription/Translation Systems (Promega, Madison, WI; Catalog #L1170).

  2. 35S-Methionine (PerkinElmer; Catalog #NEG709A).

  3. Plasmid DNA (e.g., pET17b) or PCR-generated DNA template that contains coding sequence for the proteins of interest cloned under the control of the T7 or SP6 RNA polymerase promoter. See the TnT® manual for details.

  4. 6× SDS gel loading buffer: 300 mM Tris–HCl, pH 6.8, 12% SDS, 0.6% Bromophenol blue, 60% Glycerol, 600 mM DTT.

  5. 2× SDS gel loading buffer: Add 1 mL 6× SDS gel loading buffer and 2 mL H2O.

  6. 4× lower gel buffer: 1.5 M Tris–HCl, pH 8.8, 0.4% SDS.

  7. 4× upper gel buffer: 0.5 M Tris–HCl, pH 6.8, 0.4% SDS.

  8. 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel:
    1. Lower gel (total 10 mL): Add 3.4 mL 30% acrylamide–bis solution, 2.5 mL 4× lower gel buffer, 5 μL tetramethylethylenediamine (TEMED), 50 μL 10% ammonium persulfate (APS), 4.05 mL H2O.
    2. Upper gel (total 5 mL): Add 0.83 mL 30% acrylamide–bis solution, 1.25 mL 4× lower gel buffer, 5 μL TEMED, 50 μL 10% APS, 2.87 mL H2O.

  9. Fixing solution (total 100 mL): Add 10 mL of glacial acetic acid to a mixture of 50 mL of methanol and 40 mL H2O.

  10. 10% glycerol.

  11. Phycocyanobilin (PCB): Dissolve PCB (Frontier Scientific, Catalog# P14137) in DMSO to 600 μM final concentration. Protect from light and store at −20 °C.

  12. Whatman 3 MM filter paper.

  13. Standard equipment to run and dry SDS-PAGE gels.

  14. Typhoon imaging system (GE Healthcare Life Sciences).

2.2. Coimmunoprecipitation

  1. Antibody for the immunoprecipitation of the bait protein; we used GAL4-TA antibody (C-10; Santa Cruz; Catalog #sc-1663).

  2. Dynabeads™ Protein A for Immunoprecipitation (Thermo Fisher Scientific; Catalog #10002D).

  3. Magnetic rack.

  4. 10× PBS buffer: To make 1 L add 25.6 g Na2HPO4 · 7 H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.

  5. 50 mg/mL BSA (bovine serum albumin).

  6. 10% NP-40 (Tergitol, Sigma; Catalog #127087-87-0).

  7. Water.

  8. 2× PBS binding buffer (2× PBS, 1 μg/mL BSA, 0.1% NP-40): To make 50 mL, add 10 mL 10× PBS, 1 mL 50 mg/mL BSA, 05 mL 10% NP-40, 38.5 mL water.

  9. 1× PBS binding buffer (PBS, 1 μg/mL BSA, 0.1% NP-40): To make 50 mL, add 5 mL 10× PBS, 1 mL 50 mg/mL BSA, 0.5 mL 10% NP-40, 43.5 mL water; in the following, 1× PBS binding buffer always refers to PBS binding buffer containing BSA.

  10. 1× PBS binding buffer without BSA (PBS, 0.1% NP-40): To make 50 mL, add 5 mL 10× PBS, 0.5 mL 10% NP-40, 44.5 mL water; in the following, it is explicitly mentioned if 1× PBS binding buffer without BSA is to be used.

3. Methods

3.1. Expression of Proteins with T&T Reaction and 35S-Methionine Labeling

3.1.1. T&T Reaction

  1. Quickly thaw TnT® Quick Coupled Transcription/Translation mix on tap water and place it on ice. Store rest of mix in aliquots at −80 °C. Avoid freeze–thaw cycle more than once.

  2. Prepare DNA that contains T7/SP6 promoter (see Note 1).

  3. Prepare T&T reactions for prey and bait proteins as following in 1.5 mL test tubes: 40 μL T&T quick master mix, 2 μL 35S-methionine, 1 μg template plasmid DNA, nuclease-free water to 50 μL total volume.

  4. Incubate tubes in 30 °C for 60–90 min.

3.1.2. Preparing Prey (Phytochrome) Protein

  1. Add 49 μL 2× PBS buffer to T&T prey mix, resulting in 98 μL T&T prey mix.

  2. Add 2 μL of the 600 μM phycocyanobilin (PCB) to the 98 μL T&T prey mix to a final concentration of 12 μM PCB (in the green safe light).

  3. Incubate for 1 h on ice in the dark.

  4. Save 10 μL of the translation product for input control.

3.1.3. Preparing Bait (GAD-PIFs) Protein

  1. Dilute the bait T&T mix with 50 μL 2× PBS buffer, resulting in 100 μL T&T bait mix.

  2. Attach the GAD antibody to the Dynabeads: add 5 μL (1 μg) antibody to 20 μL beads; incubate for 30 min at 4 °C.

  3. Collect Dynabeads on magnetic rack. Wash twice with 500 μL 1× PBS binding buffer and resuspend into 40 μL 1× PBS binding buffer.

  4. Add 40 μL beads to the bait T&T mix, incubate for 2 h at 4 °C, wash twice with 500 μL 1× PBS binding buffer and resuspend the pellet into 40 μL 1× PBS binding buffer.

  5. Save 10 μL of the translation product for input control.

3.2. In Vitro Coimmunoprecipitation

3.2.1. Light Treatment and Immunoprecipitation

  1. Combine 12.5 μL of prepared T&T bait and 12.5 μL of original T&T prey in a total volume of 50–75 μL (make up rest of the volume with 1× PBS buffer) (see Note 2).

  2. Place test tubes on ice in a way that the light can reach to the solutions in the tubes (see Note 3).

  3. Treat tubes with far-red (10 μmol m−2 s−1) and red (7 μmol m−2 s−1) light for 5 min to convert phyB to the inactive Pr and the active Pfr state, respectively.

  4. Incubate for 3 h at 4 °C with gentle rotation in the dark (see Note 4).

  5. Wash twice with 1 mL 1× PBS binding buffer in green safe light using the magnetic rack, then once with 1× PBS binding buffer without BSA. Wash by inverting tubes upside down for 10–20 times and make sure the Dynabead pellets are resuspended completely.

  6. Resuspend the pellet in 25 μL 1× PBS binding buffer without BSA and 5 μL 6× SDS gel loading buffer.

3.2.2. Analysis of Coimmunoprecipitation with SDS-PAGE

  1. Heat pellets from coimmunoprecipitation at 65 °C for 10 min. Heat previously prepared input controls with 10 μL of 2× SDS gel loading buffer.

  2. Load samples onto a 10% SDS-PAGE gel. Percentage of the PAGE gel should be adjusted according to the size of the protein product used in the co-immunoprecipitation assay. Use 100 V/20 mA to run the gel until the blue dye runs off the gel. This typically takes up to 2 h.

  3. Gel fixation: shake the gel in fixing solution for 30 min followed by shaking in 10% glycerol for 10 min (see Note 5).

  4. Place the gel on two sheets of Whatman 3 MM filter paper. Cover with plastic wrap and dry on a gel dryer. Dry gel under vacuum at 80 °C for 2 h.

  5. Expose the dried gel on a PhosphorImaging screen overnight (see Note 6). Scan the image the next morning using a PhosphorImager (see Note 7). An example of the results produced is shown in Fig. 1.

  6. Use Image J (https://imagej.nih.gov/ij/) software to quantify protein bands detected from the assay.

Fig. 1.

Fig. 1

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In vitro coimmunoprecipitation assay between phytochromes and PIFs. (a) Schematic diagram showing the bait and prey constructs used for the experiment. PIFs were cloned into pET17b/GAD vector as described in the method section. Empty vector containing GAD only was used as a negative control. Phytochromes are transcribed/translated followed by incubation with phycocyanobilin to reconstitute the holoprotein. (b) PIF1, PIF3, and PIF4 show red light-dependent (Pfr conformer-specific) interactions with phytochromes. apo Apoprotein, Pr Pr conformer of phytochrome, Pfr Pfr conformer of phytochrome, AV/GE missense mutations in phyA, AV/GR missense mutations in phyB. (c) Quantification of the coimmunoprecipitation results. Coimmunoprecipitated proteins were quantified using Image J software. From Huq E, Al-Sady B, Hudson M, Kim C, Apel K, Quail PH (2004) PHYTOCHROME-INTERACTING FACTOR 1 is a critical bHLH regulator of chlorophyll biosynthesis. Science 305:1937–1941. Reprinted with permission from AAAS

Acknowledgments

We thank members of the Huq laboratory for critical reading of the manuscript. This work was supported by grants from the National Institute of Health (NIH) (GM-114297) and National Science Foundation (MCB-1543813) to E.H.

Footnotes

1.

For phytochrome cloning, we used the pET3B vector (Novagen, Catalog# 69419-3), which contains the T7 promoter. For baits cloning, PIFs in this case, we used modified pET17b/GAD vector [11]. pET17b/GAD vector harbors N-terminal GAL4 activation domain (GAD) that can be used for immunoprecipitation. Any type of tag (e.g., FLAG, MYC, HA) that is compatible with the protein of interest can be used for the experiment.

2.

The total volume of the coimmunoprecipitation sample is small such that only the bottom of the 1.5 mL tube is occupied by the solution. It is important to briefly spin down tubes and keep the solutions at the tip of the tubes during the 3 h incubation.

3.

Use small ice buckets and put tubes on the ice parallel so that the light can reach into the tubes from the clear side of the 1.5 mL tubes.

4.

Gentle, non-upside-down rotation is preferred, since total immunoprecipitation reaction is only about 50 μL.

5.

Shaking in 10% glycerol prevents gels from cracking during the procedure. Gel drying makes radioisotope signals sharper; however, you need to carefully adjust drying condition to prevent cracking and masking (or damaging) signals from co-IP results.

6.

Dried gels still could be sticky on the surface and damage the phosphorscreen. This can be prevented by wrapping the gel with a plastic wrap.

7.

We used Typhoon FLA phosphorimager for the imaging. We typically used scanning parameters as follows: Laser: 650 nm, Filter: IP, PMT: 950 V, Pixel size: 25 nm.

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