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Alberto Carbonell (ed.), Plant Argonaute Proteins: Methods and Protocols, Methods in Molecular Biology,vol. 1640, DOI 10.1007/978-1-4939-7165-7_8, © Springer Science+Business Media LLC 2017

Chapter 8

Dissecting the Subnuclear Localization Patterns of Argonaute Proteins and Other Components of the RNA- Directed DNA Methylation Pathway in Plants

Cheng-Guo Duan and Jian-Kang Zhu

Abstract

RNA-directed DNA methylation (RdDM) is a nuclear pathway which is comprised of multiple main and accessory protein components, including two plant-specific DNA-dependent RNA polymerases, Pol IV and Pol V, and argonaute (AGO) proteins. Regulation in the RdDM pathway can be achieved via multiple mechanisms, including the spatial distribution of different RdDM components. Here we describe a proto-col for dissecting the subnuclear localization of AGO proteins and other RdDM components, including nuclei extraction from seedlings, slide preparation, and subsequent immunostaining.

Key words RdDM, Argonaute, Immunostaining, Subnuclear localization, Microscopy, DNA methylation

1 Introduction

RNA-directed DNA methylation (RdDM) is a conserved epigene-tic silencing pathway in plants [1]. RdDM plays significant roles in diverse processes including transposon silencing, pathogen defense, stress response, and genome stability [2–4]. In the RdDM pathway, two plant-specific RNA polymerases, Pol IV and Pol V, and many other proteins are required [2–6]. Among these other proteins, Argonaute (AGO) proteins directly bind small RNAs to target genomic regions with high sequence complementarity to the guide small RNA [7, 8]. In plants, the AGO family contains multiple members. For example, Arabidopsis thaliana encodes ten AGOs which are classified into different clades according to their phyloge-netic origin. AGO4, AGO6, and AGO9 are thought to be involved in the RdDM pathway [9–11]. RdDM takes place in the nucleus and is regulated through the spatial segregation of different com-ponents. For example, AGO4 and AGO6 are co-localized with

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different RNA polymerases in different nuclear sub- compartments [12, 13]. Determining the subnuclear co- localization patterns of the different components is critical for understanding the complex regulation of the RdDM pathway in plants.

Here we describe an immunostaining protocol for dissecting the co-localization relationship of RdDM components in the nucleus. We use AGO (AGO4 and AGO6) proteins and the largest subunits of RNA polymerase (NRPD1 and NRPE1) proteins to test their co-localization patterns.

2 Materials

The plants used in this study include A. thaliana Col-0, Flag- AGO4, Flag-AGO6, NRPD1-Flag, and NRPE1-Flag [8, 13], which are plants expressing the tagged proteins under their respective native promoters in their respective mutant backgrounds. All plants were grown on 1% sucrose containing 1/2 Murashige and Skoog (MS) medium under a long-day photoperiod (16 h light/8 h dark). Two-week-old seedlings were collected for nuclei extraction.

1. Nuclear extraction buffer 1 (NEB1): 10 mM Tris–HCl (pH 9.5), 10 mM potassium chloride (KCl), 500 mM sucrose, 4 mM spermidine, 10 mM spermine, 0.1% Triton-X 100, 8% formaldehyde, and 0.1% 2-mercaptoethanol. Make aliquots and store at 4 °C (see Note 1).

2. NEB2: 10 mM Tris–HCl (pH 9.5), 10 mM KCl, 1.7 M sucrose, 4 mM spermine, 10 mM spermine, 0.1% Triton-X 100, 8% formaldehyde, and 0.1% 2-mercaptoethanol. Make aliquots and store at 4 °C (see Note 1).

3. 10× PBST stock solution: 1.28 M sodium chloride (NaCl), 20 mM KCl, 80 mM disodium phosphate (Na2HPO4), 20 mM monopotassium phosphate (KH2PO4), and 0.1% Triton-X 100. Prepare 10× stock in water and autoclave.

4. Blocking solution: prepare 1% bovine serum albumin (BSA) in 1× PBST. Prepare it fresh and store at 4 °C.

5. DAPI: ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific).

6. Nylon mesh: 20, 53, and 100 mm (Spectra/Mesh). 7. Razor blades. 8. Petri dishes. 9. Nail polish. 10. Glass slides (VWR Superfrost Plus) and cover glass. 11. Primary antibody: anti-Flag monoclonal antibody generated in

mouse (Sigma–Aldrich), anti-AGO4 polyclonal antibody gen-

2.1 Plant Materials and Growth Conditions

2.2 Solutions, Antibodies, and Other Materials

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erated in rabbit (Agrisera), and anti-AGO6 polyclonal anti-body generated in rabbit (Agrisera) (see Note 2).

12. Secondary antibody: Alexa Fluor® 488-conjugated goat anti- rabbit IgG (H + L) secondary antibody (Thermo Fisher Scientific) and Alexa Fluor® 568-conjugated donkey anti-mouse IgG (H + L) secondary antibody (Thermo Fisher Scientific) (see Note 2).

3 Methods

Figure 1 shows a diagram for the main experimental steps regard-ing nuclei extraction and slide preparation (see Note 3). Centrifugations are done in a microcentrifuge.

1. Plant growth: seeds are sterilized and sown on 1/2 MS medium. After 2-day vernalization at 4 °C, seeds are grown on a chamber with photoperiod of 16 h light/8 h dark at 23 °C for 2 weeks.

2. Homogenization: collect about 2 g of seedlings from 1/2 MS medium and transfer seedlings to petri dish on ice (see Note 4). Add 2 mL of NEB1 pre-chilled at 4 °C into the samples and chop seedlings with a razor blade to get a fine homogenate.

3. Fixing: add equal volume of 8% formaldehyde (prepared in NEB1 buffer) to the homogenate. Use wide-bore pipette tips to mix the homogenate gently. Cover the petri dish and leave it at 4 °C for 30–60 min to fix the homogenate.

4. Filtration: use wide-bore pipette tips to transfer the fixed homogenate onto one sheet of 100 μm nylon filter. Sequentially filter the homogenate through the 53 μm and 20 μm filters. Split the homogenate into 1.5 mL tubes.

5. Centrifuge the homogenate for 3 min at 2500 (600 × g) at 4 °C. Discard the supernatant and gently resuspend the pellet in 300 μL of NEB1. Layer 300 μL of resuspended pellet on the top of the 300 μL NEB2 in a 1.5-mL tube (see Note 5).

6. Centrifuge the assembly at top speed (10,000–13,000 rpm, 10,000–16,000 × g) at 4 °C for 1 h to pellet the nuclei.

7. Carefully remove the two layers of supernatant and gently resuspend the nuclei pellet in 40 μL of NEB1 to obtain the nuclei preparation.

8. Pipette 3–5 μL of nuclei onto a slide and mix with 3 μL of DAPI. Cover with a coverslip to check the quality of nuclei under a microscope.

9. Pipette 3 μL of nuclei onto the slide and spread into a thin film of 20 × 20 mm square by a piece of coverslip (see Note 6).

3.1 Nuclei Extraction and Slide Preparation

Immunostaining for Protein Sub–Nuclear Localization

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10. Air-dry the slide at room temperature and store the dried slide on the cardboard slide storage book at 4 °C.

1. Use PAGO4::Flag-AGO4 and PAGO6::Flag-AGO6 seedlings [8] to prepare the slides.

2. Put air-dried slides into a square plate. Cover the area of nuclei with 200 μL of 4% formaldehyde (prepared in PBST) to refix the nuclei for 30 min on the bench. Wash the slide with 1× PBST for 3–5 min.

3.2 Subnuclear Localization of AGO4 and AGO6

Fig. 1. A workflow for nuclei extraction and slide preparation. Diagram showing experimental steps regarding nuclei extraction and slide preparation to analyze the subnuclear localization of AGOs and other components of the A. thaliana RdDM pathway

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3. Create a humid chamber by placing several layers of wet paper towel on the bottom of a square plate. Place the slide on the top of the wet towels and drop 200 μL of blocking solution to the nuclei area. Place a piece of plastic sheet on top of the blocking solution. Incubate the covered plate at 37 °C for 30 min.

4. Wash slides with 1× PBST for 3–5 min in a new square plate. 5. Transfer the slides to a new humid chamber. Drop 100 μL of

primary antibody in blocking solution (1:200 for anti-Flag) to the nuclei area and place a piece of plastic sheet on top of the solution. Keep the covered plate on the bench in the dark overnight.

6. Wash slides with 1× PBST for 3–5 min in a new square plate. 7. Transfer the slides to a new humid chamber. Drop 200 μL of

blocking solution and place a piece of plastic sheet on top of the solution. Incubate the covered plate at 37 °C for 30 min.

8. Rinse the slides with 1× PBST briefly. Drop 100 μL of Alexa Fluor® 568-conjugated donkey anti-mouse secondary anti-body (1:400) in 1× PBST to nuclei area and place a piece of plastic sheet on top of the PBST solution. Put the covered plate inside a 37 °C incubator for 2 h.

9. Wash the slides with 1× PBST for 3–5 min in an empty square plate.

10. Drop 5 μL of ProLong® Gold Antifade Mountant with DAPI in the center of the nuclei area and apply a coverslip. Seal the slide with clear nail polish for microscope observation.

1. Use PNRPD1::NRPD1-Flag and PNRPE1::NRPE1-Flag seedlings [13] to prepare the slides. Immunostain the slides first with anti-Flag primary antibody (1:200 dilution) and then label them with Alexa Fluor® 568-conjugated donkey anti-mouse secondary antibody (1:200 dilution) according to the procedure described above. Perform the second immunolabel-ing as follows.

2. Apply the blocking solution to the slides (see step 3 in Subheading 3.2).

3. Wash the slides (see step 4 in Subheading 3.2). 4. Incubate the slides with second primary antibody (see step 5 in

Subheading 3.2). Use anti-AGO4/anti-AGO6 as second pri-mary antibodies (1:200 dilution for anti-AGO4 and 1:100 dilution for anti-AGO6).

5. Wash the slides (see step 6 in Subheading 3.2). 6. Apply the blocking solution to the slides (see step 7 in

Subheading 3.2).

3.3 Subnuclear Colocalization of AGO4 and AGO6 Together with NRPD1 and NRPE1

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7. Incubate the slides with secondary antibody (see step 8 in Subheading 3.2). Use Alexa Fluor® 488-conjugated goat anti- rabbit secondary antibody.

8. Wash the slides (see step 9 in Subheading 3.2). 9. Drop 6 μL of ProLong® Gold Antifade Mountant with DAPI

in the center of nuclei area, and cover with a coverslip. Seal the slide with clear nail polish for observation under a microscope.

4 Notes

1. Prepare NEB1 and NEB2 fresh every time needed. Ideally, all reagents and solutions should be freshly prepared before the experiment.

2. The dilution ratio for each primary and secondary antibody has to be optimized empirically.

3. All the steps during nuclei extraction are performed on ice. 4. Avoid bringing agar with roots when transferring seedlings

from MS medium to petri dishes. The presence of excessive agar in seedlings will result in bad quality nuclei.

5. For nuclei extraction, if the nuclei pellet looks still green after density gradient extraction with NEB2, additional washes with NEB1 are recommended.

6. To maintain the film thin while spreading the nuclei on the slide via coverslip, you only need to do it once toward one orientation for each slide (Fig. 1).

Acknowledgments

This work was supported by the Chinese Academy of Sciences and by National Institutes of Health Grant R01GM070795 (to J.–K. Z).

References

1. Ghildiyal M, Zamore PD (2009) Small silenc-ing RNAs: an expanding universe. Nat Rev Genet 10(2):94–108. doi:10.1038/nrg2504

2. Matzke MA, Mosher RA (2014) RNA- directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet 15(6):394–408. doi:10.1038/nrg3683

3. Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation

patterns in plants and animals. Nat Rev Genet 11(3):204–220. doi:10.1038/nrg2719

4. Zhang H, Zhu JK (2011) RNA-directed DNA methylation. Curr Opin Plant Biol 14(2):142–147. doi:10.1016/j.pbi.2011.02.003

5. Pikaard CS, Haag JR, Ream T, Wierzbicki AT (2008) Roles of RNA polymerase IV in gene silencing. Trends Plant Sci 13(7):390–397. doi:10.1016/j.tplants.2008.04.008

Cheng-Guo Duan and Jian-Kang Zhu

135

6. Wierzbicki AT, Haag JR, Pikaard CS (2008) Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135(4):635–648. doi:10.1016/j.cell.2008.09.035

7. Mallory A, Vaucheret H (2010) Form, func-tion, and regulation of ARGONAUTE pro-teins. Plant Cell 22(12):3879–3889. doi:10.1105/tpc.110.080671

8. Havecker ER, Wallbridge LM, Hardcastle TJ, Bush MS, Kelly KA, Dunn RM, Schwach F, Doonan JH, Baulcombe DC (2010) The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22(2):321–334. doi:10.1105/tpc.109.072199

9. Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299(5607):716–719. doi:10.1126/science.1079695

10. Zheng X, Zhu J, Kapoor A, Zhu JK (2007) Role of Arabidopsis AGO6 in siRNA accumu-

lation, DNA methylation and transcriptional gene silencing. EMBO J 26(6):1691–1701. doi:10.1038/sj.emboj.7601603

11. Olmedo-Monfil V, Duran-Figueroa N, Arteaga- Vazquez M, Demesa-Arevalo E, Autran D, Grimanelli D, Slotkin RK, Martienssen RA, Vielle-Calzada JP (2010) Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464(7288):628–632. doi:10.1038/nature08828

12. Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear process-ing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126(1):93–106. doi:10.1016/j.cell.2006.05.032

13. Pontes O, Li CF, Costa Nunes P, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA pro-cessing center. Cell 126(1):79–92. doi:10.1016/j.cell.2006.05.031

Immunostaining for Protein Sub–Nuclear Localization