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In This Article

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

Summary

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

Abstract

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within <10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Introduction

Plant histone deubiquitinases play an important role in controlling gene expression by post-translational modification of histones, specifically by erasing their monoubiquitylation marks1. So far, OTLD1 is one of the only few plant histone deubiquitinases characterized at the molecular level in Arabidopsis2,3. OTLD1 removes monoubiquitin groups from the H2B histone molecules, thereby promoting the removal or addition of euchromatic acetylation and methylation modifications of H3 histones in the target gene chromatin4,5. Moreover, OTLD1 interacts with another chromatin-modifying enzyme, the histone lysine demethylase KDM1C, to affect transcriptional suppression of the target genes6,7.

Most histone-modifying enzymes lack DNA binding capabilities, and thus cannot recognize their target genes directly. One possibility is that they cooperate with DNA-binding transcription factor proteins which bind these enzymes and direct them to their chromatin targets. Specifically, in plants, several major histone-modifying enzymes (i.e., histone methyltransferases8,9, histone acetyltransferases10, histone demethylases11, and Polycomb repressive complexes12,13,14) are known to be recruited by transcription factors. Consistent with this idea, recently, one possible mechanism for OTLD1 recruitment to the target promoters was proposed which is based on specific protein-protein interactions of OTLD1 with a transcription factor LSH1015.

LSH10 belongs to a family of the plant ALOG (Arabidopsis LSH1 and Oryza G1) proteins that function as central developmental regulators16,17,18,19,20,21,22. The fact that the members of the ALOG protein family contain DNA binding motifs23 and exhibit the capacities for transcriptional regulation22, nuclear localization19, and homodimerization24 lends further support to the notion that these proteins, including LSH10, may act as specific transcription factors during epigenetic regulation of transcription. One of the main experimental techniques used to characterize the LSH10-OTLD1 interaction in vivo is fluorescence resonance energy transfer (FRET)15.

FRET is an imaging technique for directly detecting close-range interactions between proteins within <10 nm from each other25 inside living cells. There are two main variations of the FRET approach26: sensitized emission (SE-FRET) (Figure 1A) and acceptor bleaching (AB-FRET) (Figure 1B). In SE-FRET, the interacting proteins-one of which is tagged with a donor fluorochrome (e.g., green fluorescent protein, GFP) and the other with an acceptor fluorochrome (e.g., monomeric red fluorescent protein, mRFP27,28)-non-radiatively transfer the excited state energy from the donor to the acceptor. Because no photons are emitted during this transfer, a fluorescent signal is produced that has a radiative emission spectrum similar to that of the acceptor. In AB-FRET, protein interactions are detected and quantified based on elevated radiative emission of the donor when the acceptor is permanently inactivated by photobleaching, and thus is unable to receive the non-radiative energy transferred from the donor (Figure 1). Importantly, the subcellular location of the FRET fluorescence is indicative of the localization of the interacting proteins in the cell.

The ability to deploy FRET in living tissues and determine the subcellular localization of the interacting proteins simultaneously with detecting this interaction per se, makes FRET the technique of choice for studies and initial characterization of protein-protein interactions in vivo. A comparable in vivo fluorescence imaging methodology, bimolecular fluorescence complementation (BiFC)29,30,31,32, is a good alternative approach, although, unlike FRET, BiFC may produce false positives due to spontaneous assembly of the autofluorescent BiFC reporters33, and quantification of its data is less precise.

This article shares the successful experience in implementing both SE-FRET and AB-FRET techniques and presents a protocol for their deployment to investigate the interactions between OTLD1 and LSH10 in plant cells.

Protocol

Nicotiana benthamiana, Agrobacterium tumefaciens strain EHA105, or GV3101 were used for the present study.

1. FRET vector construction

  1. Select fluorescent tags for the donor/acceptor FRET pair.
    1. Use EGFP from pPZP-RCS2A-DEST-EGFP-N115,28 (see Table of Materials) to generate the donor vector.
    2. Use mRFP from pPZP-RCS2A-DEST-mRFP-N1 (see Table of Materials) to generate the acceptor vector.
  2. Generate the donor/acceptor FRET constructs using a site-specific recombination cloning technique34, such as the Gateway recombination cloning system35.
    1. Amplify the coding sequences of the proteins of interest36 (i.e., the Arabidopsis OTLD1 and LSH10)15.
      NOTE: It is also a good idea to utilize a negative control that represents a homolog of one of the interacting proteins but is not expected to exhibit interaction; the OTLD1-LSH10 interaction study employs a homolog of LSH10, LSH4, that does not recognize OTLD1. OTLD1, LSH10, and LSH4 cDNAs are amplified by PCR using primers listed in Table 1.
    2. Clone OTLD1, LSH10, and LSH4 into the entry vector pDONR207 by the site-specific recombination cloning technique34.
    3. Use the Gateway LR Clonase II (see Table of Materials) to transfer LSH10 and LSH4 from pDONR207 into the binary destination vector pPZP-RCS2A-DEST-EGFP-N1 to generate the binary donor constructs p35S::LSH10-GFP (tested construct) and p35S::LSH4-GFP (negative control).
    4. Use the same commercially available enzyme (step 1.2.3) to transfer OTLD1 from pDONR207 into the binary destination vector pPZP-RCS2A-DEST-mRFP-N1 to generate the binary acceptor construct p35S::OTLD1-mRFP (tested construct).
    5. PCR-amplify36 mRFP from pPZP-RCS2A-DEST-mRFP-N1 using primers listed in Table 1, clone it by the recombination cloning technique into pDONR207, and then use LR Clonase II to transfer mRFP into pPZP RCS2A-DEST-EGFP-N1 to generate the binary fusion construct p35S::mRFP-GFP (positive control).
  3. Perform transformation of the donor and acceptor constructs into Agrobacterium.
    1. Add 1 µg of each plasmid from steps 1.2.3-1.2.5 to 100 µL of the culture of competent cells of Agrobacterium tumefaciens strain EHA105 or GV3101, prepared using standard protocols37 or obtained commercially, and incubate at 37 °C for 5 min.
    2. Add 1 mL of LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl; see Table of Materials) to the competent cell mixture and agitate at 200 rpm and 28° C for 1.5 h. Collect the cells by centrifugation at 3,000 × g for 1 min at room temperature.
    3. Resuspend the cells in 0.1 mL of LB medium by pipetting and spread them on LB agar supplemented with the appropriate antibiotics (e.g., 0.01% spectinomycin and 0.005% rifampicin; see Table of Materials). Grow at 28 °C for 2 days.
    4. Pick individual colonies and inoculate each of them separately into 1 mL of LB broth supplemented with the appropriate antibiotics.
    5. Grow the cells at 28 °C for 24 h and transfer 0.2 mL of the culture into a new tube. Collect the cells by centrifugation at 10,000 × g for 30 s at room temperature.
    6. Extract plasmid DNA by a standard protocol for isolating plasmids from Agrobacterium cells38 and resuspend the extracted DNA in 30 µL of water. To identify the colonies harboring the desired plasmids, use 2 µL of the DNA preparation as a template for PCR with gene-specific primers listed in Table 1. Mix 0.7 mL of the identified culture with 0.3 mL of glycerol and store at -80 °C.

2. Agroinfiltration

  1. Grow Nicotiana benthamiana plants.
    NOTE: Throughout the entire experiment, all plants must be healthy.
    1. Sow and grow N. benthamiana seeds in a pot containing wet soil at a high density.
    2. Keep the planted seeds in a growth chamber set at 23 °C with 16 h of light and 8 h of dark cycle with 150-170 µmol/m2s light intensity until the diameter of the euphyll reaches 0.5 cm.
    3. Transfer the seedlings to larger pots and allow them to grow in the same chamber with the same parameters.
      NOTE: Plants are ready for agroinfiltration when their largest leaves are 5-7 cm in diameter, usually within 4-5 weeks. In smaller plants that are too young, the effects of agroinfiltration will be too severe for the FRET analysis.
  2. Prepare bacterial cells for agroinfiltration.
    1. Grow each Agrobacterium colony containing the FRET constructs overnight in 5 mL of LB medium supplemented with the appropriate antibiotics (step 1.3.3) and 150 µM acetosyringone at 28 °C (see Table of Materials).
    2. Centrifuge the cells at 3,000 × g for 5 min at room temperature.
    3. Resuspend the cells in agroinfiltration buffer (10 mM MgCl2, 10 mM MES pH 5.6, 150 µM acetosyringone) to OD600 = 0.5.
    4. Combine the resuspended cells at a 1:1 v/v ratio between cells harboring the appropriate constructs (step 2.2.5).
    5. For the double-construct agroinfiltrations, mix the aliquots of two cultures and, for single-construct agroinfiltrations, mix the aliquots of the same culture:
      1. Tested proteins: OTLD1-mRFP + LSH10-GFP (bacteria harboring the p35S::OTLD1-mRFP and p35S::LSH10-GFP constructs).
      2. Negative control: OTLD1-mRFP + LSH4-GFP (bacteria harboring the p35S::OTLD1-mRFP and p35S::LSH4-GFP constructs).
      3. Negative control: LSH10-GFP + free mRFP (bacteria harboring the p35S::LSH10-GFP and pPZP-RCS2A-DEST-mRFP-C1 constructs).
      4. Positive control: mRFP-GFP (bacteria harboring the p35S::mRFP-GFP construct).
    6. Incubate the cells at 28 °C for 0.5-1 h.
  3. Perform agroinfiltration.
    1. Load the bacterial culture into a 1 mL needleless syringe.
    2. Gently but firmly press the nozzle of the syringe against the abaxial side of the fully expanded N. benthamiana leaves while holding the leaf with a gloved finger on the adaxial side.
    3. Infiltrate up to four spots on a leaf, three leaves per plant, two or three plants for each bacterial culture. Change gloves between samples to prevent cross-contamination.
    4. Maintain the agroinfiltrated plants in the same growth chamber under the same conditions, as described in step 2.1.2, for 24 h to 36 h. Do not keep the agroinfiltrated plants for longer than 36 h, as this will reduce the fluorescence signal.

3. Confocal microscopy

  1. Prepare microscope slides for fluorescence visualization.
    1. After 24-36 h of the infiltration, use a razor blade to cut each agroinfiltrated leaf into small pieces (2 mm x 4 mm) between the veins.
    2. Place the leaf pieces on a glass slide with the abaxial leaf surface facing up. Place a drop of water on the leaf pieces and cover them with the cover glass. Slightly tap the cover glass to remove air bubbles.
    3. Turn on the microscope and laser (see Table of Materials). Place the slide into the microscope stage holder for imaging under the specific FRET parameters (steps 3.2 and 3.3).
    4. Begin the observations using a 10x objective lens to identify cells that exhibit both the GFP and mRFP signals, and then use a 40x objective lens for subsequent detailed observations.
      NOTE: Importantly, SE-FRET and AB-FRET usually are performed on the same tissue sample, allowing the use of the same channel settings (step 3.2) except for the FRET channel, which is toggled on/off for the SE-FRET and AB-FRET observations, respectively (steps 3.2.2.3 and 3.3.1).
  2. Set up the parameters for SE-FRET (Figure 1A).
    1. Open the Multi-Dimensional Acquisition (MDA) tool.
    2. Establish a set of three confocal channels in the same field of view (Supplementary Figure 1).
      1. Set the donor channel (the GFP channel) for excitation and emission of the donor fluorochrome with the 405 nm excitation laser and 400-597 nm emission filter.
      2. Set the acceptor channel (the mRFP channel) for excitation and emission of the acceptor fluorochrome with the 561 nm excitation laser and 400-597 nm emission filter.
        NOTE: The emission filter for mRFP was set at 400-597 nm to separate the mRFP signal from the FRET signal at 597-617 nm (step 3.2.2.3) and, therefore, reduce the FRET-independent mRFP emission.
      3. Set the FRET channel for excitation of the donor and emission of the acceptor fluorochromes with the 405 nm excitation laser and 597-617 nm emission filter.
    3. Set the donor excitation intensity at a minimum level to observe FRET while avoiding photobleaching, reducing the SE-FRET efficiency.
      NOTE: This excitation intensity is experimentally selected before conducting the FRET procedure to avoid photobleaching. It varies depending on leaf thickness, age, and time after overexpression.
    4. Excite the donor and scan for cells containing the acceptor's expected fluorescence signal.
    5. Select the region that contains the fluorescence signal of interest.
    6. Acquire a SE-FRET image sequence by pressing the Snap button.
      1. Image 10-15 cells expressing the mRFP-GFP construct (positive control) first; adjust the focus, zoom, and smart gain parameters to focus on the area of interest to be captured (Supplementary Figure 2).
      2. Using the same settings, image 10-15 cells, each expressing OTLD1-mRFP, free mRFP, LSH10-GFP, or LSH4-GFP separately.
        NOTE: These images are acquired by the "PixFRET" plug-in of ImageJ (see Table of Materials), which was used for the FRET data analyses (step 3.4.1) to determine the spectral bleed-through (SBT) values for the acceptors and the donors; these images are used by the software to generate the SE-FRET images for the OTLD1-mRFP + LSH10-GFP, OTLD1-mRFP + LSH4-GFP, and LSH10-GFP + free mRFP protein pairs (step 3.2.6.3).
      3. Also, using the same settings, image 10-15 cells co-expressing OTLD1-mRFP + LSH10-GFP, OTLD1-mRFP + LSH4-GFP, and LSH10-GFP + free mRFP protein pairs.
  3. Set up parameters for AB-FRET (Figure 1B).
    1. Utilize the donor and acceptor channel parameters set for SE-FRET (step 3.2.2) but turn off the FRET channel.
    2. Set the parameters for photobleaching of the acceptor (mRFP) (Supplementary Figure 3).
      1. Ensure that bleaching starts after five images. Allow 200 iterations for each area bleach. Keep 100% laser intensity at 561 nm.
      2. Maintain a bleaching duration of 45 s. Ensure a scan speed of 512 x 512 pixels at 400 Hz.
    3. Draw the region of the cell to be bleached; for example, for nuclear interactions, regions of interest are drawn around the entire area of the cell nucleus39.
    4. Activate bleaching by pressing the Start experiment button; activating this function will perform the photobleaching and acquire the AB-FRET image sequence.
  4. Analyze the FRET data.
    1. For analyzing SE-FRET data, use the "PixFRET" plug-in for the ImageJ software to generate corrected images of the SE-FRET efficiency after subtracting SBT40 (step 3.2.6.2).
    2. For analyzing the AB-FRET data, calculate %AB-FRET as the percent increase in GFP emission after mRFP photobleaching using the following formula41: %AB-FRET = [(GFPpost - GFPpre) / GFPpre] x 100, where GFPpost is GFP emission after mRFP photobleaching, and GFPpre is GFP emission before mRFP photobleaching.
    3. When reviewing the FRET images, pay attention to the subcellular localization of the FRET signal.
      NOTE: In many cases, these cellular compartments (e.g., nucleus, chloroplasts, ER, etc.) can be easily identified and, as an additional benefit of the FRET technique, provide important clues to the biological function of the interacting proteins.

Results

Figure 2 illustrates the typical results of a SE-FRET experiment, in which the cell nuclei were simultaneously recorded in three channels (i.e., donor GFP, acceptor mRFP, and SE-FRET). These data were used to generate images of SE-FRET efficiency coded in a pseudo-color scale. On this scale, the transition from blue to red corresponds to an increase in FRET efficiency, a measure of protein-protein proximity from 0% to 100%. In this representative experiment, the SE-FRET signal was recorded i...

Discussion

This FRET protocol is simple and easy to reproduce; it also requires minimal supply investment and utilizes standard equipment for many modern laboratories. Specifically, five main technical features distinguish the versatility of this procedure. First, the FRET constructs are generated using site-specific recombination, a cloning approach that is easy to use, produces accurate results, and saves time compared to traditional restriction enzyme-based cloning. Second, N. benthamiana plants are simple to grow, prod...

Disclosures

No conflicts of interest were declared.

Acknowledgements

The work in V.C.'s laboratory is supported by grants from NIH (R35GM144059 and R01GM50224), NSF (MCB1913165 and IOS1758046), and BARD (IS-5276-20) to V.C.

Materials

NameCompanyCatalog NumberComments
Acetosyringone (3′,5′-Dimethoxy-4′-hydroxyacetophenone)Sigma-Aldrich#D134406-1G
Bacto AgarBD Biosciences#214010
Bacto tryptonBD Biosciences#211705
Bacto yeast extractBD Biosciences#212750 
Confocal laser scanning microscope (CLSM)ZeissLSM900Any CLSM with similar capabilities is suitable
EHA105VWR104013-310We use the stock in the Citovsky bacterial lab stock collection
Gateway BP Clonase II Invitrogen#11789100
Gateway LR Clonase IIInvitrogen#11791020
GV3101VWR104013-296We use the stock in the Citovsky bacterial lab stock collection
ImageJhttps://imagej.nih.gov/ij/download.html
MESSigma-Aldrich#69889-10G
MgCl2Sigma-Aldrich#63068-250G
NaClSigma-Aldrich#S5886-500G
Nicotiana benthamiana seedsHerbalistics PtyRA4 or LABWe use the stock in the Citovsky seed lab stock collection
pDONR207Invitrogen#12213013
pPZP-RCS2A-DEST-EGFP-N1 N/ARefs. 15, 28
pPZP-RCS2A-DEST-mRFP-C1N/AGenerated based on the pPZP-RCS2A-DEST-EGFP-C1 construct (see refs. 15, 28)
pPZP-RCS2A-DEST-mRFP-N1 N/AGenerated based on the pPZP-RCS2A-DEST-EGFP-N1 construct
RifampicinSigma-Aldrich#R7382-5G
SpectinomycinSigma-Aldrich#S4014-5G
Syringes without needlesBD309659
Zen software for CLSM imagingZeissZEN 3.0 versionThe software should be compatible with the CLSM used

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