Genome-Wide Analysis of Chromatin Accessibility in Arabidopsis Infected with Pseudomonas syringae

  • Yogendra Bordiya
  • Hong-Gu KangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)


Changes in chromatin accessibility are an important aspect of the molecular changes that occur in eukaryotic cells responding to stress, and they appear to play a critical role in stress-induced transcriptional activation/reprogramming and epigenetic changes. In plants, pathogen infection has been shown to induce rapid and drastic transcriptional reprogramming; growing evidence suggests that chromatin remodeling plays an essential role in this phenomenon. The recent development of genomic tools to assess chromatin accessibility presents a significant opportunity to investigate the relationship between chromatin dynamicity and gene expression. In this protocol, we have adopted a popular chromatin accessibility assay, DNase-seq, to measure chromatin accessibility in Arabidopsis infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). DNase-seq provides information on chromatin accessibility through the sequencing of DNA fragments generated by DNase I digestion of open chromatin, followed by mapping these sequences on a reference genome. Of the two popular DNase-seq approaches, we based our method on the Stamatoyannopoulos protocol, which involves two DNase cleavages rather than a single cleavage, followed by size fractionation. Please note that this two-cleavage approach is widely accepted and has been used extensively by ENCODE (Encyclopedia of DNA Elements) project, a public research consortium investigating cis- and trans-elements in the transcriptional regulation in animal cells. To enhance the quality of the chromatin accessibility assay, we modified this protocol by including two centrifugation steps for nuclear enrichment and size fractionation and an extra washing step for removal of chloroplasts and Pst. The outcomes obtained by this approach are also discussed.

Key words

Biotic stress Pseudomonas syringae Arabidopsis Chromatin accessibility DNA footprinting DNase-seq DNase I hypersensitive site (DHS) 



We thank D’Maris Dempsey and Angela H. Kang for critical comments on the manuscript. This work is supported by Texas State University-Faculty Startup Program, Multi-disciplinary Internal Research Grant and National Science Foundation Grant (IOS-1553613) to H.G.K.


  1. 1.
    Tao Y et al (2003) Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae. Plant Cell 15(2):317–330CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Rivas S (2011) Nuclear dynamics during plant innate immunity. Plant Physiol 158(1):87–94CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Gross DS, Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57:159–197CrossRefPubMedGoogle Scholar
  4. 4.
    Galas DJ (2001) The invention of footprinting. Trends Biochem Sci 26(11):690–693CrossRefPubMedGoogle Scholar
  5. 5.
    Metzker ML (2010) Sequencing technologies—the next generation. Nat Rev Genet 11(1):31–46CrossRefPubMedGoogle Scholar
  6. 6.
    Tsompana M, Buck MJ (2014) Chromatin accessibility: a window into the genome. Epigenetics Chromatin 7(1):33CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jiang J (2015) The ‘dark matter’ in the plant genomes: non-coding and unannotated DNA sequences associated with open chromatin. Curr Opin Plant Biol 24:17–23CrossRefPubMedGoogle Scholar
  8. 8.
    Encode Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489(7414):57–74Google Scholar
  9. 9.
    Hesselberth JR et al (2009) Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods 6(4):283–289CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Boyle AP et al (2008) High-resolution mapping and characterization of open chromatin across the genome. Cell 132(2):311–322CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhang W et al (2012) High-resolution mapping of open chromatin in the rice genome. Genome Res 22(1):151–162CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhang W et al (2012) Genome-wide identification of regulatory DNA elements and protein-binding footprints using signatures of open chromatin in Arabidopsis. Plant Cell 24(7):2719–2731CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sullivan AM et al (2014) Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana. Cell Rep 8(6):2015–2030CrossRefPubMedGoogle Scholar
  14. 14.
    Pajoro A et al (2014) Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development. Genome Biol 15(3):R41CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kang HG et al (2008) CRT1, an Arabidopsis ATPase that interacts with diverse resistance proteins and modulates disease resistance to Turnip Crinkle Virus. Cell Host Microbe 3(1):48–57CrossRefPubMedGoogle Scholar
  16. 16.
    Zhang W, Jiang J (2015) Genome-wide mapping of DNase I hypersensitive sites in plants. Methods Mol Biol 1284:71–89CrossRefPubMedGoogle Scholar
  17. 17.
    Langmead B et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Boyle AP et al (2008) F-Seq: a feature density estimator for high-throughput sequence tags. Bioinformatics 24(21):2537–2538CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Clarke JD (2009) Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc 2009(3):pdb.prot5177CrossRefPubMedGoogle Scholar
  21. 21.
    Hiroshi Nikaido MV (1985) Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49(1):1–32Google Scholar
  22. 22.
    Ellison RT, Giehl TJ (1991) Killing of Gram-negative bacteria by lactofernn and lysozyme. J Clin Invest 88(4):1080–1091CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of BiologyTexas State UniversitySan MarcosUSA

Personalised recommendations