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Measuring Callose Deposition, an Indicator of Cell Wall Reinforcement, During Bacterial Infection in Arabidopsis

  • Lin Jin
  • David M. MackeyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

Abstract

The plant cell wall responds dynamically during interaction with various pathogens. Upon recognition of “nonself” components, plant cells deploy a variety of immune responses including cell wall fortification. Callose, a β-(1, 3)-d-glucan polymer, is a component of the material deposited at the site of infection between the plasma membrane and the preexisting cell wall that is hypothesized to serve as a physical barrier and platform for directed antimicrobial compound deposition. The defense-associated function of callose deposition is supported by its induction during pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) and its inhibition by defense suppressing virulence effectors. Thus, callose deposition is a commonly monitored read-out in plant defense. This protocol describes the use of aniline blue staining and fluorescent microscopy to measure callose deposition in bacteria-infected or elicitor-challenged Arabidopsis leaf tissues.

Key words

Callose PTI Aniline blue Arabidopsis Pseudomonas syringae Flg22 

Notes

Acknowledgments

This protocol is adapted from Kim and Mackey, 2008, MiMB [22]. Funding for this work was provided by the US Department of Agriculture (National Institute of Food and Agriculture, grant #2015-11870612), the Korean Rural Development Administration Next-Generation BioGreen Program (System and Synthetic Agro-Biotech Center and grant nos. PJ009088 and PJ011091), and the Ohio Agricultural Research and Development Center of the Ohio State University.

References

  1. 1.
    Ellinger D, Voigt CA (2014) Callose biosynthesis in Arabidopsis with a focus on pathogen response: what we have learned within the last decade. Ann Bot 114(6):1349–1358CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Underwood W (2012) The plant cell wall: a dynamic barrier against pathogen invasion. Front Plant Sci 3:85CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Eggert D, Naumann M, Reimer R, Voigt CA (2014) Nanoscale glucan polymer network causes pathogen resistance. Sci Rep 4:4159CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ellinger D, Naumann M, Falter C, Zwikowics C, Jamrow T, Manisseri C, Somerville SC, Voigt CA (2013) Elevated early callose deposition results in complete penetration resistance to powdery mildew in Arabidopsis. Plant Physiol 161(3):1433–1444CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Jacobs AK, Lipka V, Burton RA, Panstruga R, Strizhov N, Schulze-Lefert P, Fincher GB (2003) An arabidopsis callose synthase, GSL5, Is required for wound and papillary callose formation. Plant Cell 15(11):2503–2513CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC (2003) Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science 301(5635):969–972CrossRefPubMedGoogle Scholar
  7. 7.
    Luna E, Pastor V, Robert J, Flors V, Mauch-Mani B, Ton J (2011) Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact 24(2):183–193CrossRefPubMedGoogle Scholar
  8. 8.
    Geng X, Cheng J, Gangadharan A, Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressor of Arabidopsis defense. Plant Cell 24(11):4763–4774CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    DebRoy S, Thilmony R, Kwack YB, Nomura K, He SY (2004) A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants. Proc Natl Acad Sci USA 101(26):9927–9932CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ham JH, Majerczak D, Ewert S, Sreerekha MV, Mackey D, Coplin D (2008) WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants. Mol Plant Pathol 9(5):633–643CrossRefPubMedGoogle Scholar
  11. 11.
    Fabro G, Steinbrenner J, Coates M, Ishaque N, Baxter L, Studholme DJ, Korner E, Allen RL, Piquerez SJ, Rougon-Cardoso A, Greenshields D, Lei R, Badel JL, Caillaud MC, Sohn KH, Van den Ackerveken G, Parker JE, Beynon J, Jones JD (2011) Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity. PLoS Pathog 7(11):e1002348CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Guo M, Tian F, Wamboldt Y, Alfano JR (2009) The majority of the type III effector inventory of Pseudomonas syrinage pv. tomato DC3000 can suppress plant immunity. Mol Plant Microbe Interact 22(9):1069–1080CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Currier HB (1957) Callose substance in plant cells. Am J Bot 44(6):478–488CrossRefGoogle Scholar
  14. 14.
    Smith MM, McCully ME (1978) A critical evaluation of the specificity of aniline blue induced fluorescence. Protoplasma 95(3):229–254CrossRefGoogle Scholar
  15. 15.
    Wood PJ, Fulcher RG (1983) Dye interactions. A basis for specific detection and histochemistry of polysaccharides. J Histochem Cytochem 31(6):823–826CrossRefPubMedGoogle Scholar
  16. 16.
    Stone BA, Evans NA, Bonig I, Clarke AE (1984) The application of Sirofluor, a chemically defined fluorochrome from aniline blue for the histochemical detection of callose. Protoplasma 122(3):191–195CrossRefGoogle Scholar
  17. 17.
    Ham JH, Kim MG, Lee SY, Mackey D (2007) Layered basal defenses underlie non-host resistance of Arabidopsis to Pseudomonas syringae pv. phaseolicola. Plant J 51(4):604–616CrossRefPubMedGoogle Scholar
  18. 18.
    Zhou J, Spallek T, Faulkner C, Robatzek S (2012) CalloseMeasurer: a novel software solution to measure callose deposition and recognise spreading callose patterns. Plant Methods 8(1):49CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Cumbie JS, Pankow RC, Thomas WJ, JH, C (2010) AutoSPOTs: automated image analysis for enumerating callose deposition. In: Akimitsu K et al. (eds) 10th Japan-US Seminar: genome-enabled integration of research in plant pathogen systems, 2010. APS press: St Paul, MNGoogle Scholar
  20. 20.
    Kim MG, da Cunha L, McFall AJ, Belkhadir Y, DebRoy S, Dangl JL, Mackey D (2005) Two Pseudomonas syringae type III effectors inhibit RIN4-regulated basal defense in Arabidopsis. Cell 121(5):749–759CrossRefPubMedGoogle Scholar
  21. 21.
    Daudi A, Cheng Z, O'Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP (2012) The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell 24(1):275–287CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kim MG, Mackey D (2008) Measuring cell-wall-based defenses and their effect on bacterial growth in Arabidopsis. Methods Mol Biol 415:443–452PubMedGoogle Scholar
  23. 23.
    Yuan J, He YH (1996) The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. J Bacteriol 178:6399–6402CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Horticulture and Crop ScienceThe Ohio State UniversityColumbusUSA
  2. 2.Department of Molecular GeneticsThe Ohio State UniversityColumbusUSA

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