LeEIX2 Interactors’ Analysis and EIX-Mediated Responses Measurement

  • Meirav Leibman-Markus
  • Silvia Schuster
  • Adi AvniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)


Plant-pathogen interactions involve a large number of wide regulatory systems, necessary for plant defense responses against pathogen attack. The fungal protein ethylene-inducing xylanase (EIX) elicits defense responses in specific cultivars of tobacco and tomato. The response to EIX is controlled by a single locus encoding for LeEIX2, a leucine-rich-repeat receptor-like-protein (LRR-RLP). As an RLP, LeEIX2 does not possess an obvious cytoplasmic signaling moiety such as a kinase domain. To study LeEIX2 mode of action, it is essential to identify the potential interactors involved after EIX perception. Here, we describe the in vivo co-IP methodology used for protein interaction verification and ethylene and ROS (reactive oxygen species) measurements used for physiological effects assessment.

Key words

LRR-RLP Ethylene-inducing xylanase MAMP Defense responses Co-IP ROS 



This work was partly supported by the Israel Science Foundation administered by the Israel Academy of Science and Humanities no. 388/12 and Research Grant Award no. IS-4842-15R from BARD, the United States–Israel Binational Agriculture Research and Development Fund and by the United States-Israel Binational Science Foundation no. 2013227.


  1. 1.
    Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124(4):803–814CrossRefPubMedGoogle Scholar
  2. 2.
    Bohm H, Albert I, Fan L, Reinhard A, Nurnberger T (2014) Immune receptor complexes at the plant cell surface. Curr Opin Plant Biol 20(1):47–54CrossRefPubMedGoogle Scholar
  3. 3.
    Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11(6):725–732CrossRefPubMedGoogle Scholar
  4. 4.
    De Smet I, Voss U, Jurgens G, Beeckman T (2009) Receptor-like kinases shape the plant. Nat Cell Biol 11(10):1166–1173CrossRefPubMedGoogle Scholar
  5. 5.
    Wang G, Fiers M (2010) CLE peptide signaling during plant development. Protoplasma 240(1–4):33–43CrossRefPubMedGoogle Scholar
  6. 6.
    Sakamoto T, Deguchi M, Brustolini OJ, Santos AA, Silva FF, Fontes EP (2012) The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense. BMC Plant Biol 12:229CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Fritz-Laylin LK, Krishnamurthy N, Tor M, Sjolander KV, Jones JD (2005) Phylogenomic analysis of the receptor-like proteins of rice and Arabidopsis. Plant Physiol 138(2):611–623CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jones DA, Thomas CM, Hammond-Kosack KE, Balint-Kurti PJ, Jones JD (1994) Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging. Science 266(5186):789–793CrossRefPubMedGoogle Scholar
  9. 9.
    Thomma BP, VANEsse HP, Crous PW, DE Wit PJ (2005) Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae. Mol Plant Pathol 6(4):379–393CrossRefPubMedGoogle Scholar
  10. 10.
    Fradin EF, Zhang Z, Juarez Ayala JC, Castroverde CD, Nazar RN, Robb J, Liu CM, Thomma BP (2009) Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol 150(1):320–332CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ron M, Avni A (2004) The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato. Plant Cell 16(6):1604–1615CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hammond-Kosack KE, Jones JD (1996) Resistance gene-dependent plant defense responses. Plant Cell 8(10):1773–1791CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tenhaken R, Levine A, Brisson LF, Dixon RA, Lamb C (1995) Function of the oxidative burst in hypersensitive disease resistance. Proc Natl Acad Sci U S A 92(10):4158–4163CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Felix G, Grosskopf DG, Regenass M, Basse CW, Boller T (1991) Elicitor-induced ethylene biosynthesis in tomato cells: characterization and use as a bioassay for elicitor action. Plant Physiol 97(1):19–25CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Meirav Leibman-Markus
    • 1
  • Silvia Schuster
    • 1
  • Adi Avni
    • 1
    Email author
  1. 1.Department of Molecular Biology and Ecology of PlantsTel-Aviv UniversityTel-AvivIsrael

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