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Quantitative Evaluation of Plant Actin Cytoskeletal Organization During Immune Signaling

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1578))

Abstract

High spatial and temporal resolution microscopy-based methods are valuable tools for the precise real-time imaging of changes in cellular organization in response to stimulus perception. Here, we describe a quantitative method for the evaluation of the plant actin cytoskeleton during immune stimulus perception and the activation of defense signaling. As a measure of the biotic stress-induced changes in actin filament organization, we present methods for analyzing changes in actin filament organization following elicitation of pattern-triggered immunity and effector-triggered immunity. Using these methods, it is possible to not only quantitatively evaluate changes in actin cytoskeletal organization following biotic stress perception, but to also use these protocols to assess changes in actin filament organization following perception of a wide range of stimuli, including abiotic and developmental cues. As described herein, we present an example application of this method, designed to evaluate changes in actin cytoskeletal organization following pathogen perception and immune signaling.

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References

  1. Day B, Henty JL, Porter KJ, Staiger CJ (2011) The pathogen-actin connection: a platform for defense signaling in plants. Annu Rev Phytopathol 49:483–506

    Article  CAS  PubMed  Google Scholar 

  2. Henty-Ridilla JL, Shimono M, Li J, Chang JH, Day B, Staiger CJ (2013) The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns. PLoS Pathog 9:e1003290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Porter K, Shimono M, Tian M, Day B (2012) Arabidopsis actin-depolymerizing factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics. PLoS Pathog 8:e1003006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tian M, Chaudhry F, Ruzicka DR, Meagher RB, Staiger CJ, Day B (2009) Arabidopsis actin-depolymerizing factor AtADF4 mediates defense signal transduction triggered by the Pseudomonas syringae effector AvrPphB. Plant Physiol 150:815–824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Shimono M, Higaki T, Kaku H, Shibuya N, Hasezawa S, Day B (2016) Quantitative evaluation of stomatal cytoskeletal patterns during the activation of immune signaling in Arabidopsis thaliana. PLoS One 11:e0159291

    Article  PubMed  PubMed Central  Google Scholar 

  6. Shimono M, Lu YJ, Porter K, Kvitko BH, Henty-Ridilla J, Creason A, He SY, Chang JH, Staiger CJ, Day B (2016) The Pseudomonas syringae type III effector HopG1 induces actin remodeling to promote symptom development and susceptibility during Infection. Plant Physiol 171:2239–2255

    Article  PubMed  PubMed Central  Google Scholar 

  7. Dong B, Yang X, Zhu S, Bassham DC, Fang N (2015) Stochastic optical reconstruction microscopy imaging of microtubule arrays in intact Arabidopsis thaliana seedling roots. Sci Rep 5:15694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S (2010) Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells. Plant J 61:156–165

    Article  CAS  PubMed  Google Scholar 

  9. Shaw SL, Ehrhardt DW (2013) Smaller, faster, brighter: advances in optical imaging of living plant cells. Annu Rev Plant Biol 64:351–375

    Article  CAS  PubMed  Google Scholar 

  10. Dyachok J, Paez-Garcia A, Yoo CM, Palanichelvam K, Blancaflor EB (2016) Fluorescence imaging of the cytoskeleton in plant roots. Methods Mol Biol 1365:139–153

    Article  PubMed  Google Scholar 

  11. Daniels MJ, Barber CE, Turner PC, Sawczyc MK, Byrde RJ, Fielding AH (1984) Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. EMBO J 3:3323–3328

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW (2004) A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol 136:3968–3978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zipfel C (2014) Plant pattern-recognition receptors. Trends Immunol 35:345–351

    Article  CAS  PubMed  Google Scholar 

  14. Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N, Takio K, Minami E, Shibuya N (2006) Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Natl Acad Sci U S A 103:11086–11091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276

    Article  CAS  PubMed  Google Scholar 

  16. Kunze G, Zipfel C, Robatzek S, Niehaus K, Boller T, Felix G (2004) The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16:3496–3507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Special thanks to Amy Baetsen-Young for critical review and comments. Work in the Day Lab is supported by a National Science Foundation grant (IOS-1557437).

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Correspondence to Brad Day .

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Lu, YJ., Day, B. (2017). Quantitative Evaluation of Plant Actin Cytoskeletal Organization During Immune Signaling. In: Shan, L., He, P. (eds) Plant Pattern Recognition Receptors. Methods in Molecular Biology, vol 1578. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6859-6_17

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  • DOI: https://doi.org/10.1007/978-1-4939-6859-6_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6858-9

  • Online ISBN: 978-1-4939-6859-6

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