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Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae
利用染色质免疫共沉淀(ChIP)技术检测稻瘟病菌中蛋白质-DNA的直接与间接相互作用   

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Abstract

Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae.

Materials and Reagents

  1. Oak Ridge Centrifuge Tubes (Thermo Fisher Scientific, NalgenetmTM, catalog number: 3118-0050 )
  2. Miracloth (EMD Millipore Corporation, catalog number: 475855-1R )
  3. Saccharomyces cerevisiae strain XK1-25: MATa trp1 (kindly provided by Dr. Jin-Rong Xu, Dept. of Botany and Plant Pathology, Purdue University)
  4. pHZ126 yeast shuttle vector (kindly provided by Dr. Jin-Rong Xu, Dept. of Botany and Plant Pathology, Purdue University, USA)
  5. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
  6. Alkali-cation yeast transformation kit (MP Biomedicals, catalog number: 2200-200 )
  7. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
  8. UltraClean Midi Prep Kit (MO BIO Laboratories, catalog number: 12700-20 )
  9. Zymoprep Yeast Plasmid Mini Prep II Kit (Epigenetics, catalog number: D2004 )
  10. Formaldehyde (Thermo Fisher Scientific, PierceTM, catalog number: 28906 )
  11. DSG (Thermo Fisher Scientific, catalog number: 20593 )
  12. CelLyticTM PN Plant Nuclear Isolation Kit (Sigma-Aldrich, catalog number: CELLYTPN1 )
  13. Nuclei isolation buffer (NIB) (included in the CelLyticTM PN Plant Nuclear Isolation Kit)
  14. Triton X-100 Solution (10% in H2O) (MBL International, catalog number: JM-2104-100 )
  15. Proteinase inhibitor cocktail (Sigma-Aldrich, catalog number: P8215 )
  16. Deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  17. Sodium butyrate (Sigma-Aldrich, catalog number: B5887 )
  18. Sepharose 4B beads (Sigma-Aldrich, catalog number: CL4B200 )
  19. ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, catalog number: A2220 )
  20. Anti-IgG agarose (Sigma-Aldrich, catalog number: A0919 )
  21. 3x FLAG® Peptide (Sigma-Aldrich, catalog number: F4799 )
  22. Glycine (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15527-013 )
  23. Proteinase K (Thermo Fisher Scientific, catalog number: EO0491 )
  24. RNase A (Thermo Fisher Scientific, catalog number: EN0531 )
  25. Wizard PCR Clean-up kit (Promega Corporation, catalog number: A9281 )
  26. Yeast nitrogen base without amino acids (Difco, catalog number: 0919-15 )
  27. Trp DO supplement (Clontech, catalog number: 630413 )
  28. Disuccinimidyl glutarate (DSG) (Thermo Fisher Scientific, catalog number: 20593)
  29. Lysing Enzymes from Trichoderma harzianum (Sigma-Aldrich, catalog number: L1412 )
  30. Hygromycin B (Thermo Fisher Scientific, GibcoTM, catalog number: 10-687-010 )
  31. SD-Trp solid medium (see Recipes)
  32. SD-Trp liquid solution (see Recipes)
  33. LB agar plates supplemented with 100 μg/ml ampicillin (see Recipes)
  34. Liquid complete medium (see Recipes)
  35. DSG cross-linking buffer (see Recipes)
  36. 1x NIB (see Recipes)
  37. NIBA (see Recipes)
  38. Nuclear lysis buffer (see Recipes)
  39. HEPES-buffered saline (HBS) (see Recipes)
  40. 0.1 M glycine HCl (pH 3.5) (see Recipes)
  41. Elution buffer (see Recipes)
  42. OM buffer (see Recipes)
  43. ST buffer (see Recipes)
  44. STC buffer (see Recipes)
  45. PTC buffer (see Recipes)
  46. Bottom medium (see Recipes)
  47. Top medium (see Recipes)

Equipment

  1. Benchtop shakers (Eppendorf, catalog number: M1354-0000 )
  2. VirTis VirSonic 50 Ultrasonic Cell Disrupter Disruptor (VirTis company)
  3. Centrifuge (Eppendorf, model: 5415D )
  4. Mini-Tube Rotators (Thermo Fisher Scientific, Fisher Scientific, catalog number: 05-450-127 )

Procedure

  1. The first step towards ChIP analysis in M. oryzae is to construct a plasmid expressing the target gene fused to 3x FLAG from its native promoter. We routinely use the yeast shuttle vector and methodology described by Zhou et al. (2011), summarized below:
    1. Vector preparation
      A yeast shuttle vector, pHZ126 (Zhou et al., 2011), is available for generating C-terminal 3x FLAG fusion constructs. In addition to 3x FLAG encoding sequences, pHZ126 carries TRP1 for selection in yeast Trp- strains, AmpR for selection in Escherichia coli (E. coli) strains, and HygR for selection based on hygromycin resistance in M. oryzae. In preparation for subcloning the target gene, 100 μg of pHZ126 vector DNA is linearized by digestion with XhoI and, after size confirmation on a 0.8% agarose gel, purified with the QIAquick gel extraction kit, following the manufacturer’s protocol.
    2. Target gene amplification.
      1. The following primer pairs are used to generate the target gene PCR product with overlapping sequences for fusing in-frame with the 3xFLAG coding sequence carried on the pHZ126 shuttle vector (Zhou et al., 2011):
        Gene-fw: 5’-TATAGGGCGAATTGGGTACTCAAATTGGTTXXXXXXXXXXX-3’
        Gene-rv: 5’-TTTATAATCACCGTCATGGTCTTTGTAGTCYYYYYYYYYYY-3’
        Note: The underlined nucleotide sequences are specific for the target gene. The non-underlined sequences are used for homologous recombination onto the yeast shuttle vector pHZ126. In order to generate an in-frame gene fusion between target gene and the 3xFLAG tag sequence on pHZ126, the nucleotide sequences in italics in the primer Gene-rv encode the last codon before the target gene stop codon. For best results, the gene specific sequences are designed with a melting temperature of 65 °C, an ideal GC content of 40-60%, and 35-40 cycles of the PCR reaction should be performed.
      2. The target gene amplicon is gel purified using the QIAquick gel extraction kit, following the manufacturer’s protocol.
    3. The target gene PCR product from step A2 is subcloned into the XhoI digested yeast shuttle vector pHZ126 by co-transformation into the yeast strain XK1-25, following the protocol of the Alkali-Cation Yeast Transformation Kit.
      Note: The non-underlined nucleotide sequences added to the Gene-fw and Gene-rv primers above facilitate recombination onto pHZ126 by aligning with vector sequences on either side of the XhoI restriction site. Thus, the PCR product does not need to be digested with XhoI.
      Screening of yeast transformants carrying the target gene-3x FLAG fusion in pHZ126 is performed on plates containing SD-Trp solid medium followed by colony PCR with gene-specific primer pairs for verification.
    4. Positive yeast colonies are propagated on a fresh SD-Trp plate, and yeast plasmid DNA is extracted using the Zymoprep Yeast Plasmid Mini Prep II kit.
    5. The yeast plasmid DNA is then transformed into competent cells of E. coli strain DH5a, using standard procedures. E. coli cells carrying the yeast plasmid DNA are selected on ampicillin-containing LB agar plates, followed by verification using colony PCR.
    6. The plasmid DNA is extracted from E. coli using the Mo Bio UltraClean Midi Plasmid Prep kit, and an aliquot is sequenced to ensure the fidelity of the target gene sequence and 3x FLAG fusion.

  2. The next step towards ChIP is to integrate the target gene fused to 3x FLAG into the M. oryzae genome. This is achieved by transforming the plasmid DNA isolated in step A6 into M. oryzae protoplasts and selecting for hygromycin resistance. Protoplast generation and transformation, as well as screening for fungal transformants, are performed as described previously (Fernandez et al., 2012; Wilson et al., 2010) and summarized as follows:
    1. Mycelia are grown in 350 ml of liquid complete medium with continuous shaking at 150 rpm for 48 h at 24 °C, harvested with sterile Miracloth and washed with sterile distilled water.
    2. The mycelia are divided into two parts, and each is incubated in 40 ml of OM buffer at 29 °C with shaking at 75 rpm.
    3. The mycelia/OM solution is aliquoted into two Oak Ridge centrifuge tubes, and overlaid with the same volume of cold ST buffer on ice. After centrifugation at 5,000 rpm for 15 min at 4 °C in a swinging bucket rotor, protoplasts are recovered from the OM/ST interface using a 1 ml sterile pipette and pooled.
    4. After gently mixing with the same volume of cold STC buffer, the protoplasts are pelleted by centrifugation at 3,000 rpm for 10 min at 4 °C, followed by three washes in 20 ml of cold STC buffer.
    5. The protoplast pellet is resuspended in cold STC buffer to give a final concentration of 2-5 x 108 protoplasts/ml.
    6. 100 ul of protoplasts (~1 x 107) are mixed with 5 μg of plasmid DNA, and incubated at room temperature for 20 min. Protoplasts and DNA are then gently mixed with 1 ml of PTC buffer by gently inverting the tube, and the solution is incubated at room temperature for a further 20 min.
    7. To select for transformants, the protoplast/PTC solution is next mixed with 100 ml of cool but molten (45 °C) bottom medium and poured into four plates (~25 ml/plate). The plates are allowed to set, then incubated in the dark for at least 24 h at 24 °C. The bottom medium is then overlaid with the same volume of top medium containing 200 μg/ml Hygromycin B, and the plates are incubated under dark conditions until colonies appear (approx. 2 -3 weeks).
    8. Hygromycin resistant colonies are screened for target gene-3x FLAG genomic integration by PCR using gene-specific primer pairs. Expression of the target gene can be further verified by quantitative real time PCR (qRT-PCR) and immunoblot analysis using the ANTI-FLAG antibody.

  3. Preparation of fungal mycelia for ChIP
    1. Once M. oryzae strains that express the target gene fused to 3x FLAG have been verified, these can then be grown up for ChIP analysis. Fungal strains carrying the target gene-3x FLAG fusion are grown on agar plates for 5-10 days before the leading edge of the plate colony is excised and blended with 350 ml liquid complete medium (CM). The mycelia and CM mix is decanted into a suitable flask and the fungus is allowed to grow at 24 °C for 48 h with continuous shaking at 150 rpm (Figure 1A). For some studies, mycelia might be transferred to a second, defined, minimal medium for 16 h of additional growth. A wild type strain or a strain carrying the empty pHZ126 vector should be used as the experimental control.
    2. For each strain, fungal mycelia are collected using two layers of Miracloth and the harvested mycelia are washed extensively with sterile water (Figure 1B-C).


      Figure 1. Collection of fungal mycelia. A. Fungal strains carrying the target gene-3x FLAG fusion are grown in liquid complete medium at 24 °C. B. After 48 h, the mycelia are harvested by filtering the culture through two pieces of Miracloth placed in a funnel. The mycelium is washed with sterile water. C. The mycelial mat is gently patted with paper towels to remove excess water.

  4. Two-step cross-linking [modified from Nowak et al. (2005)] of fungal mycelia for ChIP
    1. First step cross-linking: 1 g of washed mycelia is suspended in 100 ml of DSG cross-linking buffer and incubated for 45 min at room temperature with continuous shaking at 100 rpm.
    2. Second step cross-linking: formaldehyde is added to a final concentration of 1% for the last 20 min of cross-linking with DSG.
    3. Terminate cross-linking: glycine is added to a final concentration of 0.125 M, and incubated at room temperature for 10 min.
    4. The mycelia are harvested with Miracloth, washed excessively with sterile distilled water, flash frozen in liquid nitrogen and stored at -80 °C until use.

  5. Isolation of crosslinked chromatin with CelLyticTM PN Plant Nuclear Isolation Kit, following the manufacturer’s protocol
    Note: From procedure E to F, perform all steps at 4 °C. All buffers and equipment used should be pre-cooled to 4 °C.
    1. Crosslinked mycelia are ground to a fine powder in liquid nitrogen using a pre-chilled mortar and pestle.
    2. 100 mg of mycelial powder is suspended in 500 μl of 1x NIB in a 1.5 ml Eppendorf tube by vigorous vortexing.
    3. The suspension is filtered through 2 layers of sterile Miracloth into a fresh 1.5 ml Eppendorf tube.
    4. The suspension is centrifuged at 1,260 x g for 10 min at 4 °C.
    5. The supernatant is discarded and the pellet is re-suspended in 500 μl of NIBA.
    6. For the lysis of cell membranes, 15 μl of 10% Triton X-100 is added to a final concentration of 0.3% and the solution is gently inverted to mix.
    7. The solution is centrifuged at 12,000 x g for 5 min at 4 °C.
    8. The supernatant is removed and the pellet resuspended in 500 μl of NIBA.
    9. The solution is centrifuged at 12,000 x g for 5 min at 4 °C, and the supernatant discarded.
    10. The resulting pellet is the crude nuclear fraction.

  6. Chromatin shearing by sonication
    1. The crude nuclear fraction from step E10 is resuspended in 300 μl of pre-cooled nuclear lysis buffer.
    2. Chromatin is extracted from nuclei and sheared using the VirTis VirSonic 50 Ultrasonic Cell Disrupter Disruptor. Samples are kept on ice throughout the procedure. To generate chromatin fragments in the optimal size range of 200 bp-1,000 bp (with an average fragment size of 500 bp), a Microtip power setting of 4 in cycles of 20 sec “on” and 20 sec “off” during a total treatment time of 6 min can be used for M. oryzae nuclei.
    3. Samples are centrifuged at 12,000 x g for 5 min at 4 °C, and the supernatant containing the nuclear lysate and sheared chromatin is transferred to a fresh tube.
    Note: In a preliminary series of experiments, the optimal sonication parameters for shearing chromatin can be empirically determined for the VirTis VirSonic 50 Ultrasonic Cell Disrupter Disruptor by checking the size of chromatin DNA fragments, following sonication, on a 1.5% agarose gel. The power level setting or the total treatment time of sonication can be subsequently adjusted until chromatin DNA is fragmented to the appropriate size.

  7. Chromatin immunoprecipitation
    1. The nuclear lysate containing sheared chromatin is precleared by incubating with 30 μl Sepharose 4B beads with gentle rotation for 4 h at 4 °C.
    2. The beads are centrifuged at 5,000 x g for 30 sec at 4 °C.
    3. The supernatant is collected into 3 x 100 μl aliquots in fresh Eppendorf tubes. One sample is saved until step H1 to serve as the input chromatin control. All samples are placed on ice until use.
    4. The affinity resin is prepared according to the manufacturer's instructions.
      1. Briefly, 20 µl ANTI-FLAG M2 Affinity Gel is removed to a sterile 1.5 ml tube. The gel is washed with 0.5 ml HBS twice followed by 0.5 ml 0.1 M glycine HCl (pH 3.5) once. This is followed by three washes with 0.5 ml HBS. Between washing steps the resin is pelleted by spinning at 5,000 x g for 30 sec at 4 °C.
      2. 20 µl Anti-IgG Agarose resin is prepared in the same way as above and is used to determine the background of non-specific chromatin binding to the gel.
    5. 100 µl of cleared nuclear lysate from Step G3 is placed in separate tubes containing the ANTI-FLAG or Anti-IgG agarose resin and incubated overnight at 4 °C with gentle agitation.
    6. The resin is washed four times in 0.5 ml HBS buffer containing 0.1% TritonX-100. Following these wash steps the beads are pelleted by spinning at 5,000 x g for 30 sec at 4 °C.
    7. To elute the protein-DNA complex, 100 μl Elution buffer is added to the resin pellet and incubated for 30 min at 4 °C with gentle agitation.
    8. The sample is centrifuged at 5,000 x g for 30 sec at 4 °C and the supernatant transferred to a fresh tube.

  8. Reverse cross-linking
    Note: In addition to the eluate from the ANTI-FLAG and Anti-IgG agarose resin, the input chromatin saved at step G3 is also included in the following steps.
    1. To digest proteins, Proteinase K is added to each sample at a final concentration of 0.2 mg/ml and the solution is incubated at 45 °C for 2 h.
    2. To reverse the covalent association of the epitope tagged protein with its direct or indirect DNA target, NaCl is added to a final concentration of 0.2 M and the solution is incubated at 65 °C overnight.

  9. Analysis of ChIPed DNA
    1. Contaminating RNA is removed from the ChIPed sample by incubating with RNase A (final concentration is 0.1 mg/ml) at room temperature for 30 min.
    2. DNA is purified using the Wizard PCR Clean-up kit and eluted into 30 μl ddH2O.
    3. Quantitative real-time PCR (qPCR) is used to determine if known or suspected DNA target sequences are enriched in samples following ChIP. Using specific primers for the gene of interest (i.e., MoSOD1 in Figure 2), DNA enrichment values following ChIP are determined by subtracting the background signal of the Anti-IgG agarose treated sample from the ChIP ANTI-FLAG signal, and normalizing against the input control. For example, Fernandez and associates (2014) demonstrated that MoJmjC physically associates with the MoSOD1 promoter in a MoSIR2 dependent manner (Figure 2) such that MoSOD1 DNA is more highly enriched following ChIP by JmjCFLAG in a ∆sir2 mutant background than following ChIP by JmjCFLAG in a wild type (WT) background.


      Figure 2. Quantification of ChIPed DNA. Three biological replications were performed per strain. Indirect binding of MoJmjC to MoSod1 DNA was demonstrated by ChIP following two-step cross-linking using Guy11 wild type (WT) and ∆sir2 deletion strains carrying the JMJCFLAG allele. The ANTI-FLAG M2 Affinity Gel was used in parallel with the Mouse Anti-IgG agarose. The quantification of eluted MoSod1 DNA was performed at least in triplicate by qPCR using specific primers designed in the MoSod1 promoter region (Fernandez et al., 2014). qPCR values obtained from ANTI-FLAG M2 Affinity Gel immunoprecipitation were adjusted for non-specific chromatin binding and precipitation using Anti-IgG agarose values and normalized against the input controls. The fold enrichment of MoSod1 DNA in WT JMJCFLAG ChIP samples relative to ∆sir2 JMJCFLAG ChIP samples is shown. Error bars are the standard deviation. Bars with different letters are significantly different (Student’s t-test P ≤ 0.05).

Recipes

  1. SD-Trp solid medium
    1 M sorbitol
    0.67% (w/v) yeast nitrogen base without amino acids
    0.074% (w/v) Trp DO supplement
    1.5% agar
    2% (w/v) glucose
    Sterilized by autoclaving at 121 °C for 20 min
    Note: Filter sterilized glucose should be added after autoclaving at a temperature below 55 °C.
  2. SD-Trp liquid solution
    1 M sorbitol
    0.67% (w/v) yeast nitrogen base without amino acids
    0.074% (w/v) Trp DO supplement
    2% (w/v) glucose
    Sterilize by autoclaving at 121 °C for 20 min
    Note: Filter sterilized glucose should be added after autoclaving at a temperature below 55 °C.
  3. LB agar plates supplemented with 100 μg/ml ampicillin
    1% bacto tryptone
    0.5% yeast extract 1% NaCl
    1.5% bacto agar
    Sterilized by autoclaving at 121 °C for 20 min, and ampicillin is added when the liquid has cooled below 55 °C
  4. Liquid complete medium
    10 g/L glucose
    2 g/L peptone
    1 g/L yeast extract
    1 g/L casamino acids
    0.1% (v/v) trace elements
    0.1% (v/v) vitamin supplement
    6 g/L NaNO3
    0.5 g/L KCl
    0.5 g/L MgSO4
    1.5 g/L KH2PO4
    Adjust PH 6.5
    Sterilized by autoclaving at 121 °C for 30 min
  5. DSG cross-linking buffer
    20 mM HEPES (pH 7.4)
    1 mM EDTA
    1 mM phenylmethylsulfonyl fluoride (PMSF)
    2 mM disuccinimidyl glutarate
    DSG cross-linking buffer should be freshly prepared. HEPES and EDTA are sterilized by autoclaving at 121 °C for 20 min. PMSF and DSG should be added just before use. PMSF is dissolved in isopropanol or ethanol. DSG is dissolved in DMSO to make a stock solution of 0.5 M for immediate use
  6. 1x NIB
    1x NIB is diluted from 4x NIB with deionized H2O. Then DTT is added to a final concentration of 1 mM. 4x NIB is provided with the CelLyticTM PN Plant Nuclear Isolation Kit
    Note: The DTT should be added just before use.
  7. NIBA
    Add proteinase inhibitor cocktail with a ratio of 1:100 to 1x NIB
    Note: The proteinase inhibitor cocktail should be added just before use.
  8. Nuclear lysis buffer
    50 mM HEPES (pH 7.5)
    150 mM NaCl
    1 mM EDTA
    1% Triton X-100
    0.1% deoxycholate
    0.1% SDS
    10 mM sodium butyrate
    1 mM PMSF
    1% (v/v) proteinase inhibitor cocktail
    Sterilized by autoclaving at 121 °C for 30 min
    PMSF and the proteinase inhibitor cocktail should be added just before use
  9. HEPES-buffered saline (HBS)
    10 mM HEPES (pH 7.5)
    150 mM NaCl
    Sterilized by autoclaving at 121 °C for 30 min
  10. 0.1 M glycine HCl (pH 3.5)
    0.1 M glycine dissolved in ddH2O
    Adjust pH with HCl to 3.5
  11. Elution buffer
    50 mM Tris-HCl (pH 7.5)
    200 μg/ml 3x FLAG peptide
    150 mM NaCl
    Dissolve 3x FLAG peptide in autoclave sterilized 1x TBS (PH 7.5) solution, and store at -20 °C
  12. OM buffer
    1.2 M MgSO4
    10 mM NaPO4 (pH 5.8)
    0.75% (w/v) glucanex
    Adjust pH 5.5 with 1 M Na2HPO4, and filter sterilize 
    OM buffer should be prepared fresh
  13. ST buffer
    0.6 M sorbitol
    0.1 M Tris-HCl (pH 7.0)
    Sterilized by autoclaving at 121 °C for 30 min
  14. STC buffer
    1.2 M sorbitol
    0.1 M Tris-HCl (pH 7.5)
    10 mM CaCl2
    Sterilized by autoclaving at 121 °C for 30 min
  15. PTC buffer
    60% PEG 4000
    10 mM Tris-HCl (pH 7.5)
    10 mM CaCl2
    Filter sterilize
    PTC buffer should be prepared freshly.
  16. Bottom medium
    10 g/L glucose
    273 g/L sucrose
    2 g/L peptone
    1 g/L yeast extract
    1 g/L casamino acids
    0.1% (v/v) trace elements
    0.1% (v/v) vitamin supplement
    6 g/L NaNO3
    0.5 g/L KCl
    0.5 g/L MgSO4
    1.5 g/L KH2PO4
    1.5% agar
    Adjust PH 6.5 and sterilize by autoclaving at 121 °C for 20 min
  17. Top medium
    10 g/L glucose
    2 g/L peptone
    1 g/L yeast extract
    1 g/L casamino acids
    0.1% (v/v) trace elements
    0.1% (v/v) vitamin supplement
    6 g/L NaNO3
    0.5 g/L KCl
    0.5 g/L MgSO4
    1.5 g/L KH2PO4
    1% agar
    Adjust PH 6.5 and sterilize by autoclaving at 121 °C for 20 min

Acknowledgments

This work was supported by the National Science Foundation (IOS-1145347) and USDA-NIFA (2014-67013-21559). This protocol was adapted and modified from Fernandez et al. (2014).

References

  1. Fernandez, J., Marroquin-Guzman, M., Nandakumar, R., Shijo, S., Cornwell, K. M., Li, G. and Wilson, R. A. (2014). Plant defence suppression is mediated by a fungal sirtuin during rice infection by Magnaporthe oryzae. Mol Microbiol 94(1): 70-88.
  2. Fernandez, J., Wright, J. D., Hartline, D., Quispe, C. F., Madayiputhiya, N. and Wilson, R. A. (2012). Principles of carbon catabolite repression in the rice blast fungus: Tps1, Nmr1-3, and a MATE-family pump regulate glucose metabolism during infection. PLoS Genet 8(5): e1002673.
  3. Marroquin-Guzman, M. and Wilson, R. A. (2015). GATA-dependent glutaminolysis drives appressorium formation in Magnaporthe oryzae by suppressing TOR inhibition of cAMP/PKA signaling. PLoS Pathog 11(4): e1004851.
  4. Nowak, D. E., Tian, B. and Brasier, A. R. (2005). Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation. Biotechniques 39(5): 715-725.
  5. Wilson, R. A., Gibson, R. P., Quispe, C. F., Littlechild, J. A. and Talbot, N. J. (2010). An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus. Proc Natl Acad Sci U S A 107(50): 21902-21907.
  6. Zhou, X., Li, G. and Xu, J. R. (2011). Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi. Methods Mol Biol 722: 199-212.

简介

染色质免疫沉淀(ChIP)是一种强大的技术,用于分析细胞中的蛋白质-DNA相互作用。已建立了稳健的ChIP程序用于研究蛋白质和DNA之间的直接相互作用。然而,在体内检测间接蛋白-DNA相互作用是具有挑战性的。最近,我们使用ChIP来分析推定的组蛋白去甲基化酶MoJmjC和在稻瘟病真菌Magnaporthe oryzae中超氧化物歧化酶1编码基因MoSOD1的启动子之间的间接蛋白质-DNA相互作用( oryzae )(Fernandez ,,2014)。我们用3x FLAG表位标记MoJmjC(Fernandez等人,2014),而不是更大和更常用的GFP表位,以减轻空间位阻。我们还采用了使用DSG和甲醛的两步交联策略,而不是更常用于分析直接蛋白质-DNA相互作用的一步甲醛交联方法,以便更好地捕获间接的MoJm - MoSOD1 DNA相互作用。此外,我们已经表明两步交联适用于GATA转录因子,Asd4及其同源结合位点之间的直接蛋白-DNA相互作用的ChIP分析(Marroquin-Guzman和Wilson,2015)。在这里,我们提供详细的协议的染色质免疫沉淀,与通用的两步交联,在M。 oryzae 。

材料和试剂

  1. Oak Ridge离心管(Thermo Fisher Scientific,Nalgenetm TM,目录号:3118-0050)
  2. Miracloth(EMD Millipore Corporation,目录号:475855-1R)
  3. 酿酒酵母菌株XK1-25:MATa trp1(由Dr. Jin-Rong Xu博士和普渡大学植物病理学部提供)
  4. pHZ126酵母穿梭载体(由Dr. Jin-Rong Xu博士和美国普渡大学植物病理学部提供)
  5. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  6. 碱性阳离子酵母转化试剂盒(MP Biomedicals,目录号:2200-200)
  7. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
  8. UltraClean Midi Prep试剂盒(MO BIO Laboratories,目录号:12700-20)
  9. Zymoprep Yeast Plasmid Mini Prep II Kit(Epigenetics,目录号:D2004)
  10. 甲醛(Thermo Fisher Scientific,Pierce TM,目录号:28906)
  11. DSG(Thermo Fisher Scientific,目录号:20593)
  12. CelLytic PN植物核分离试剂盒(Sigma-Aldrich,目录号:CELLYTPN1)
  13. 核分离缓冲液(NIB)(包含在CelLytic TM supper PN植物核分离试剂盒中)
  14. Triton X-100溶液(10%,在H 2 O中)(MBL International,目录号:JM-2104-100)
  15. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8215)
  16. 脱氧胆酸盐(Sigma-Aldrich,目录号:D6750)
  17. 丁酸钠(Sigma-Aldrich,目录号:B5887)
  18. Sepharose 4B珠(Sigma-Aldrich,目录号:CL4B200)
  19. ANTI-FLAG M2 Affinity Gel(Sigma-Aldrich,目录号:A2220)
  20. 抗IgG琼脂糖(Sigma-Aldrich,目录号:A0919)
  21. 3x FLAG 肽(Sigma-Aldrich,目录号:F4799)
  22. 甘氨酸(Thermo Fisher Scientific,Invitrogen TM,目录号:15527-013)
  23. 蛋白酶K(Thermo Fisher Scientific,目录号:EO0491)
  24. RNase A(Thermo Fisher Scientific,目录号:EN0531)
  25. Wizard PCR Clean-up试剂盒(Promega Corporation,目录号:A9281)
  26. 无氨基酸的酵母氮源(Difco,目录号:0919-15)
  27. Trp DO supplement(Clontech,目录号:630413)
  28. 戊二酸二琥珀酰亚胺酯(DSG)(Thermo Fisher Scientific,目录号:20593)
  29. 来自哈茨木霉的裂解酶(Sigma-Aldrich,目录号:L1412)
  30. 潮霉素B(Thermo Fisher Scientific,Gibco TM ,目录号:10-687-010)
  31. SD-Trp固体培养基(参见配方)
  32. SD-Trp液体溶液(参见配方)
  33. 补充有100μg/ml氨苄青霉素(参见Recipes)的LB琼脂平板
  34. 液体完全培养基(见配方)
  35. DSG交联缓冲液(参见配方)
  36. 1x NIB(请参阅配方)
  37. NIBA(请参阅食谱)
  38. 核裂解缓冲液(见配方)
  39. HEPES缓冲盐水(HBS)(参见配方)
  40. 0.1 M甘氨酸HCl(pH 3.5)(参见配方)
  41. 洗脱缓冲液(参见配方)
  42. OM缓冲区(参见配方)
  43. ST缓冲区(参见配方)
  44. STC缓冲区(请参阅配方)
  45. PTC缓冲区(参见配方)
  46. 底部介质(参见配方)
  47. 顶级媒介(见配方)

设备

  1. 台式振荡器(Eppendorf,目录号:M1354-0000)
  2. VirTis VirSonic 50超声波细胞破碎仪(VirTis公司)
  3. 离心机(Eppendorf,型号:5415D)
  4. 微型管旋转器(Thermo Fisher Scientific,Fisher Scientific,目录号:05-450-127)

程序

  1. 在 M中进行ChIP分析的第一步。 oryzae 是构建表达与来自其天然启动子的3x FLAG融合的靶基因的质粒。我们常规使用Zhou等人(2011)描述的酵母穿梭载体和方法,概述如下:
    1. 矢量准备。
      酵母穿梭载体,pHZ126(Zhou等人, 2011),可用于产生C末端3x FLAG融合构建体。 ?除了3x FLAG编码序列之外,pHZ126携带TRP1 在用于在大肠杆菌(大肠杆菌)菌株中选择的酵母Trp - 菌株中选择Amp 基于潮霉素抗性的HygR选择 ?in M。 oryzae 。在准备亚克隆靶基因时, pHZ126载体DNA通过用XhoI消化和在大小后线性化 在0.8%琼脂糖凝胶上确认,用QIAquick凝胶纯化 提取试剂盒,按照制造商的协议。
    2. 靶基因扩增
      1. 以下引物对用于产生靶基因PCR 产物具有重叠序列用于与3xFLAG框内融合 编码序列在pHZ126穿梭载体上进行(Zhou等人, 2011):
        Gene-fw:5'-TATAGGGCGAATTGGGTACTCAAATTGGTT XXXXXXXXXXX -3'
        Gene-rv:5'-TTTATAATCACCGTCATGGTCTTTGTAGTC YYY YYYYYYYY -3'
        注意:带下划线的核苷酸序列是靶标特异性的 基因。非下划线序列用于同源重组 ?到酵母穿梭载体pHZ126上。为了生成帧内 在pHZ126上靶基因和3xFLAG标签序列之间的基因融合, 引物Gene-rv中的斜体字的核苷酸序列编码 目的基因终止密码子前的最后一个密码子。为了获得最佳结果,该基因 ?特异性序列设计为具有65℃的熔解温度, ?理想的GC含量为40-60%,和35-40个循环的PCR反应应该 ?
      2. 使用QIAquick凝胶提取试剂盒,按照制造商的方案凝胶纯化靶基因扩增子。
    3. 将来自步骤A2的靶基因PCR产物通过共转化到酵母中亚克隆到XhoI消化的酵母穿梭载体pHZ126上 ?菌株XK1-25,按照碱性阳离子酵母的方案 转化试剂盒。
      注意:未加下划线的核苷酸序列 添加到上述Gene-fw和Gene-rv引物上方便于重组 通过与XhoI的任一侧上的载体序列比对而在pHZ126上 ?限制性位点。因此,PCR产物不需要消化 与XhoI。
      筛选携带靶的酵母转化体 在含有SD-Trp的平板上进行pHZ126中的基因-3x FLAG融合 固体培养基,随后用基因特异性引物对进行菌落PCR 验证。
    4. 将阳性酵母菌落在新鲜培养基上繁殖 SD-Trp板,使用Zymoprep提取酵母质粒DNA 酵母质粒Mini Prep II试剂盒
    5. 然后酵母质粒DNA 转化到E的感受态细胞中。大肠杆菌菌株DH5α 程序。 E。选择携带酵母质粒DNA的大肠杆菌细胞 ?含氨苄青霉素的LB琼脂平板上,随后使用进行验证 菌落PCR
    6. 从E中提取质粒DNA。大肠杆菌 Mo Bio UltraClean Midi Plasmid Prep试剂盒,并将等分试样进行测序 确保靶基因序列和3x FLAG融合的保真度。

  2. 朝向ChIP的下一步是将融合到3x FLAG的靶基因整合到M中。 oryzae 基因组。这通过将步骤A6中分离的质粒DNA转化为M来实现。 oryzae 原生质体并选择潮霉素抗性。如先前所述(Fernandez等人,2012; Wilson等人,2010)进行原生质体产生和转化以及筛选真菌转化体,并总结为如下:
    1. 菌丝体在350ml具有连续的液体完全培养基中生长 在24℃下以150rpm振荡48小时,用无菌Miracloth收获 并用无菌蒸馏水洗涤
    2. 将菌丝体分成两部分,并且每种在29℃下在75ml的OM缓冲液中以75rpm振摇孵育。
    3. 将菌丝体/OM溶液等分到两个Oak Ridge离心机中 管,并在冰上用相同体积的冷ST缓冲液覆盖。后 ?在4℃下在摇摆桶中以5,000rpm离心15分钟 转子,使用1ml从OM/ST接口回收原生质体 无菌移液管并合并。
    4. 与其轻轻混合后 体积的冷STC缓冲液,原生质体沉淀 在4℃下以3,000rpm离心10分钟,然后洗涤三次 ?在20ml冷STC缓冲液中。
    5. 将原生质体沉淀重悬浮于冷STC缓冲液中,得到2-5×10 8原生质体/ml的终浓度。
    6. 将100μl原生质体(?1×10 7个)与5μg质粒DNA混合, ?并在室温下温育20分钟。原生质体和DNA是 然后通过轻轻倒转管子与1ml PTC缓冲液轻轻混合, 并将溶液在室温下再温育20分钟
    7. 为了选择转化体,接下来是原生质体/PTC溶液 与100ml冷却但熔融(45℃)的底部介质混合并倾倒 (?25ml /板)。然后使板固化 在黑暗中在24℃下孵育至少24小时。底部培养基是 然后用相同体积的含有200μg/ml的顶部培养基覆盖 潮霉素B,并将平板在黑暗条件下温育直至 菌落出现(约2-3周)。
    8. 潮霉素抗性 通过PCR筛选菌落用于靶基因-3x FLAG基因组整合 ?使用基因特异性引物对。靶基因的表达可以是 通过定量实时PCR(qRT-PCR)和免疫印迹进一步验证 使用ANTI-FLAG抗体进行分析。

  3. 制备用于ChIP的真菌菌丝体
    1. 一旦M。表达与3x FLAG融合的靶基因的oryzae 菌株 已经验证,然后可以长大以用于ChIP分析。真菌 ?携带靶基因-3x FLAG融合体的菌株在琼脂上生长 板前5-10天平板菌落的前缘 切除并与350ml液体完全培养基(CM)混合。菌丝体 ?并将CM混合物倒入合适的烧瓶中,并允许真菌 在24℃下以150rpm连续振荡生长48小时 1A)。对于一些研究,菌丝体可能被转移到第二, 用于额外生长16小时。野生型 菌株或携带空pHZ126载体的菌株应用作 实验控制
    2. 对于每个菌株,真菌菌丝体 收集使用两层Miracloth和收获的菌丝体 用无菌水充分洗涤(图1B-C)

      图1。 真菌菌丝体的收集 A.携带靶标的真菌菌株 基因-3x FLAG融合体在24℃下在液体完全培养基中生长。乙。 48小时后,通过过滤培养物收获菌丝体 两件Miracloth放置在漏斗。菌丝体用洗涤 无菌水。 C.用纸巾轻轻地拍打菌丝垫 ?去除多余的水。

  4. 用于ChIP的真菌菌丝体的两步交联[由Nowak等人(2005)修改]。
    1. 第一步交联:将1g洗涤过的菌丝体悬浮于100ml 的DSG交联缓冲液并在室温下孵育45分钟 ?以100rpm连续振荡
    2. 第二步交联: 在最后20分钟内加入甲醛至最终浓度为1% ?与DSG交联。
    3. 终止交联:加入甘氨酸至终浓度为0.125M,并在室温下温育10分钟。
    4. 用Miracloth收获菌丝体,用过量洗涤 无菌蒸馏水中,在液氮中快速冷冻并储存 -80°C,直到使用。

  5. 按照制造商的方案,用CelLytic TM TM PN植物核分离试剂盒分离交联的染色质。
    注意:从步骤E到F,在4°C执行所有步骤。所有缓冲液和设备应预先冷却至4°C。
    1. 使用预冷的研钵和杵将交联的菌丝体在液氮中研磨成细粉。
    2. 通过剧烈涡旋将100mg菌丝体粉末悬浮在1.5ml Eppendorf管中的500μl1x NIB中。
    3. 将悬浮液通过2层无菌Miracloth过滤到新鲜的1.5ml Eppendorf管中
    4. 将悬浮液在4℃下以1,260×g离心10分钟
    5. 弃去上清液,将沉淀物重新悬浮于500μlNIBA中
    6. 对于细胞膜的裂解,加入15μl10%Triton X-100 至终浓度为0.3%,并将溶液温和地倒转 混合
    7. 将溶液在4℃下以12,000×g离心5分钟
    8. 除去上清液,将沉淀重悬于500μlNIBA中
    9. 将溶液在4℃下以12,000xg离心5分钟,弃去上清液。
    10. 所得沉淀是粗核级分。

  6. 通过超声处理的染色质剪切
    1. 将来自步骤E10的粗核级分重悬于300μl预冷却的核裂解缓冲液中
    2. 染色质从核中提取并使用VirTis剪切 VirSonic 50超声波细胞破碎仪。将样品保存在冰上 ?整个过程。生成染色质片段 最佳大小范围为200 bp -1,000 bp(平均片段大小为 ?500bp),20秒"开"和20秒的循环中的Microtip功率设置为4 在6分钟的总处理时间内"断开"可以用于M. oryzae 细胞核。
    3. 将样品以12,000xg离心5分钟 在4℃,和含有核裂解物的上清液剪切 染色质转移到新管中。
    注意:在初步的一系列实验中,剪切染色质的最佳超声处理参数可以通过在1.5%琼脂糖凝胶上超声处理后检查染色质DNA片段的大小,对VirTis VirSonic 50超声波细胞破碎仪破碎仪进行经验确定。随后可以调整功率水平设置或声处理的总处理时间,直到染色质DNA片段化为适当大小。

  7. 染色质免疫沉淀。
    1. 包含剪切的染色质的核裂解物通过预澄清 与30μlSepharose 4B珠孵育,在4℃轻轻旋转4小时 ?C。
    2. 将珠子在5,000xg下在4℃下离心30秒
    3. 将上清液收集成新鲜的3×100μl等分试样 Eppendorf管。一个样本被保存,直到步骤H1作为输入 ?染色质控制。所有样品置于冰上直至使用。
    4. 亲和树脂根据制造商的说明书制备。
      1. 简言之,将20μl抗-FLAG M2亲和凝胶移除至无菌1.5 ?ml管。将凝胶用0.5ml HBS洗涤两次,然后用0.5ml 0.1洗涤 ?M甘氨酸HCl(pH 3.5)洗涤一次。然后用0.5洗涤三次 ml HBS。在洗涤步骤之间,通过在4℃下以5000×g旋转30秒来使树脂沉淀。
      2. 制备20μl抗IgG琼脂糖树脂 ?以与上述相同的方式并且用于确定背景 非特异性染色质与凝胶结合。
    5. 100μl清除 将来自步骤G3的核裂解物置于含有 抗-FLAG或抗IgG琼脂糖树脂,并在4℃下与之温育过夜 ?温和搅拌。
    6. 树脂在0.5ml HBS中洗涤四次 含有0.1%TritonX-100的缓冲液。在这些洗涤步骤后,珠 ?通过在4℃下以5,000xg旋转30秒沉淀
    7. 至 洗脱蛋白质-DNA复合物,向其中加入100μl洗脱缓冲液 树脂沉淀并在4℃下温和搅拌孵育30分钟
    8. 将样品在4℃以5,000xg离心30秒,将上清液转移到新管中。

  8. 反向交联。
    注意:除了来自抗-FLAG和抗IgG琼脂糖树脂的洗脱液外,在步骤G3保存的输入染色质也包括在以下步骤中。
    1. 为了消化蛋白质,在最后向每个样品中加入蛋白酶K. 浓度为0.2mg/ml,将溶液在45℃温育2小时 h。
    2. 为了逆转被标记的表位的共价缔合 蛋白与其直接或间接DNA靶标,将NaCl加入最终 ?浓度为0.2M,溶液在65℃下保温 过夜。

  9. 分析ChIP DNA
    1. 通过与ChIPed样品温育,从ChIPed样品中除去污染的RNA RNA酶A(终浓度为0.1mg/ml)在室温下反应30分钟 min。
    2. 使用Wizard PCR Clean-up试剂盒纯化DNA,并洗脱至30μlddH 2 O中。
    3. 定量实时PCR(qPCR)用于确定是否已知或 在ChIP后,样品中富集了可疑的DNA靶序列。 使用特定的引物用于感兴趣的基因( MoSOD1 2),通过扣除来测定ChIP后的DNA富集值 抗IgG琼脂糖处理的样品的背景信号 ChIP ANTI-FLAG信号,并相对于输入控制进行归一化。对于 例如,Fernandez和同事(2014)证明了MoJmjC 在MoSIR2 依赖中与MoSOD1启动子物理缔合 方式(图2),使得MoSOD1 DNA随后更高度富集 ?ChIP by JmjC FLAG 在Δ sir2 JmjC FLAG 在野生型(WT)背景中

      图2. ?ChIPed DNA。每个菌株进行三次生物复制。 通过ChIP证明MoJmjC与MoSod1 DNA的间接结合 在使用携带JMJC 标签的Guy11野生型(WT)和Δ 缺失菌株的两步交联后 等位基因。抗-FLAG M2亲和力 ?凝胶与小鼠抗IgG琼脂糖平行使用。的 至少一式三份地进行洗脱的MoSod1 DNA的定量 ?通过qPCR使用在MoSod1启动子区中设计的特异性引物 (Fernandez等人,2014年)。从ANTI-FLAG M2获得的qPCR值 针对非特异性调整亲和凝胶免疫沉淀 染色质结合和沉淀使用抗IgG琼脂糖值和 相对于输入控件进行标准化。 MoSod1 DNA的折叠富集 ?在WT JMJCFLAG ChIP样品中相对于Δ sir2 JMJC FLAG ChIP样品 显示。误差棒是标准偏差。酒吧用不同的字母 显着不同(Student's t检验p≤0.05)。

食谱

  1. SD-Trp固体培养基
    1 M山梨醇
    0.67%(w/v)无氨基酸的酵母氮源 0.074%(w/v)Trp DO补充剂
    1.5%琼脂 2%(w/v)葡萄糖 在121℃高压灭菌20分钟,灭菌处理
    注意:过滤灭菌葡萄糖应在高于55℃的高压灭菌后加入。
  2. SD-Trp液体溶液
    1 M山梨醇
    0.67%(w/v)无氨基酸的酵母氮源 0.074%(w/v)Trp DO补充剂
    2%(w/v)葡萄糖 通过在121℃下高压灭菌20分钟灭菌
    注意:过滤灭菌葡萄糖应在高于55℃的高压灭菌后加入。
  3. 补充有100μg/ml氨苄青霉素的LB琼脂平板 1%细菌胰蛋白酶
    0.5%酵母提取物1%NaCl
    1.5%细菌琼脂 通过在121℃下高压灭菌灭菌20分钟,并且当液体冷却至低于55℃时加入氨苄青霉素。
  4. 液体完全培养基
    10g/L葡萄糖 2 g/L蛋白胨
    1g/L酵母提取物
    1 g/L酪蛋白氨基酸
    0.1%(v/v)痕量元素
    0.1%(v/v)维生素添加剂
    6g/L NaNO 3 3/
    0.5g/L KCl
    0.5g/L MgSO 4 4/h 1.5g/L KH 2 PO 4 sub/
    调整PH 6.5
    在121℃高压灭菌30分钟
  5. DSG交联缓冲区
    20mM HEPES(pH7.4) 1mM EDTA
    1mM苯甲基磺酰氟(PMSF)
    2mM二琥珀酰亚胺基戊二酸酯 DSG交联缓冲液应该新鲜制备。 HEPES和EDTA通过在121℃高压灭菌20分钟灭菌。 PMSF和DSG应在使用前添加。将PMSF溶解在异丙醇或乙醇中。将DSG溶解于DMSO中以制备0.5M的储备溶液,立即使用
  6. 1x NIB
    1×NIB用去离子H 2 O从4×NIB稀释。然后加入DTT至终浓度为1mM。 4x NIB与CelLytic TM PN植物核分离试剂盒一起提供。
    注意:DTT应在使用前添加。
  7. NIBA
    加入蛋白酶抑制剂混合物,比例为1:100至1x NIB
    注意:蛋白酶抑制剂鸡尾酒应在使用前添加。
  8. 核裂解缓冲液
    50mM HEPES(pH7.5) 150mM NaCl 1mM EDTA
    1%Triton X-100 0.1%脱氧胆酸盐 0.1%SDS
    10 mM丁酸钠 1mM PMSF
    1%(v/v)蛋白酶抑制剂混合物
    在121℃高压灭菌30分钟
  9. HEPES缓冲盐水(HBS)
    10mM HEPES(pH7.5) 150mM NaCl 在121℃高压灭菌30分钟
  10. 0.1M甘氨酸HCl(pH 3.5) 溶于ddH 2 O中的0.1M甘氨酸 用HCl调节pH至3.5
  11. 洗脱缓冲液
    50mM Tris-HCl(pH7.5) 200μg/ml 3×FLAG肽
    150mM NaCl 将3×FLAG肽溶解于高压灭菌的1×TBS(PH7.5)溶液中,并在-20℃下保存。
  12. OM缓冲区
    1.2M MgSO 4 10mM NaPO 4(pH 5.8)
    0.75%(w/v)葡聚糖
    用1M Na 2 HPO 4调节pH 5.5,并过滤灭菌。
    OM缓冲液应准备新鲜。
  13. ST缓冲区
    0.6M山梨醇 0.1M Tris-HCl(pH 7.0)
    在121℃高压灭菌30分钟
  14. STC缓冲区
    1.2M山梨醇 0.1M Tris-HCl(pH7.5)
    10mM CaCl 2
    在121℃高压灭菌30分钟
  15. PTC缓冲区
    60%PEG 4000
    10mM Tris-HCl(pH7.5) 10mM CaCl 2
    过滤灭菌
    PTC缓冲液应该新鲜配制。
  16. 底部媒介
    10g/L葡萄糖 273g/L蔗糖 2 g/L蛋白胨
    1g/L酵母提取物
    1 g/L酪蛋白氨基酸
    0.1%(v/v)痕量元素
    0.1%(v/v)维生素添加剂
    6g/L NaNO 3 3/
    0.5g/L KCl
    0.5g/L MgSO 4 4/h 1.5g/L KH 2 PO 4 sub/
    1.5%琼脂 调整PH 6.5,并在121℃高压灭菌20分钟,灭菌
  17. 顶端媒体
    10g/L葡萄糖 2 g/L蛋白胨
    1g/L酵母提取物
    1 g/L酪蛋白氨基酸
    0.1%(v/v)痕量元素
    0.1%(v/v)维生素添加剂
    6g/L NaNO 3 3/
    0.5g/L KCl
    0.5g/L MgSO 4 4/h 1.5g/L KH 2 PO 4 sub/
    1%琼脂
    调整PH 6.5,并在121℃高压灭菌20分钟,灭菌

致谢

这项工作是由国家科学基金会(IOS-1145347)和美国农业部NIFA(2014-67013-21559)的支持。该协议从Fernandez等人(2014)修改和修改。

参考文献

  1. Fernandez,J.,Marroquin-Guzman,M.,Nandakumar,R.,Shijo,S.,Cornwell,K.M.,Li,G.and Wilson,R.A。(2014)。 植物防御抑制是由水稻感染过程中的真菌沉默调节蛋白介导的 Magnaporthe oryzae 。 Mol Microbiol 94(1):70-88
  2. Fernandez,J.,Wright,J. D.,Hartline,D.,Quispe,C. F.,Madayiputhiya,N.and Wilson,R.A。(2012)。 稻瘟病菌中碳分解代谢物抑制的原理:Tps1,Nmr1-3和MATE-家族泵在感染期间调节葡萄糖代谢。 PLoS Genet 8(5):e1002673。
  3. Marroquin-Guzman,M.和Wilson,R.A。(2015)。 GATA依赖性谷氨酰胺分解通过抑制TOR抑制驱动Magnaporthe oryzae中的吸附形成of cAMP/PKA signaling。 PLoS Pathog 11(4):e1004851。
  4. Nowak,D.E.,Tian,B.and Brasier,A.R。(2005)。 通过染色质免疫沉淀鉴定NF-κB基因网络的两步交联方法。 a> Biotechniques 39(5):715-725
  5. Wilson,R.A.,Gibson,R.P.,Quispe,C.F.,Littlechild,J.A。和Talbot,N.J。(2010)。 NADPH依赖性遗传开关调节稻瘟病真菌的植物感染。 Proc Natl Acad Sci USA 107(50):21902-21907。
  6. Zhou,X.,Li,G.and Xu,J.R。(2011)。 在丝状真菌中产生GFP融合和表位标记构建体的有效方法 Methods Mol Biol 722:199-212。
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引用:Li, G., Marroquin-Guzman, M. and Wilson, R. A. (2015). Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae. Bio-protocol 5(21): e1643. DOI: 10.21769/BioProtoc.1643.
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