Abstract
Pull-down assays are key tools to test specific protein–protein interactions and have been particularly fruitful in jasmonate signaling research. Here, we describe a standard protocol in which a matrix-bound “bait” protein, expressed in Escherichia coli, pulls down a “prey” protein that is soluble in a protein extract obtained from Arabidopsis thaliana plant tissues. The pulled-down protein can be detected by immunoblot with protein-specific or epitope-specific antibodies. Such a pull-down method was used to reveal interactions among components of the jasmonic acid signaling, including hormone-dependent coreceptor interaction, homodimerization and heterodimerization of JASMONATE ZIM DOMAIN repressors, and interactions among other corepressor components and with transcription factors. Pull-down assays contributed not only to shape this signaling pathway but also to identify the active jasmonate hormone.
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References
Dharmasiri N, Dharmasiri S, Estelle M (2005) The F-box protein TIR1 is an auxin receptor. Nature 435:441–445
Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, Miersch O, Wasternack C, Solano R (2009) (+)-7-iso-Jasmonoyl-l-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol 5:344–350
Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008) COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine. Proc Natl Acad Sci USA 105:7100–7105
Chini A, Fonseca S, Fernández G, Adie B, Chico JM, Lorenzo O, García-Casado G, López-Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448:666–671
Kepinski S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435:446–451
Pauwels L, Fernández Barbero G, Geerinck J, Tilleman S, Grunewald W, Cuéllar Pérez A, Chico JM, Vanden Bossche R, Sewell J, Gil E, García-Casado G, Witters E, Inzé D, Long JA, De Jaeger G, Solano R, Goossens A (2010) NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature 464:788–791
Feng S, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW (2003) The COP9 signalosome interacts physically with SCFCOI1 and modulates jasmonate responses. Plant Cell 15:1083–1094
Chini A, Fonseca S, Chico JM, Fernández-Calvo P, Solano R (2009) The ZIM domain mediates homo- and heteromeric interactions between Arabidopsis JAZ proteins. Plant J 59:77–87
Fernández-Calvo P, Chini A, Fernández-Barbero G, Chico J-M, Gimenez-Ibanez S, Geerinck J, Eeckhout D, Schweizer F, Godoy M, Franco-Zorrilla JM, Pauwels L, Witters E, Puga MI, Paz-Ares J, Goossens A, Reymond P, De Jaeger G, Solano R (2011) The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of JA responses. Plant Cell 23:701–715
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743
Niu Y, Figueroa P, Browse J (2011) Characterization of JAZ-interacting bHLH transcription factors that regulate jasmonate responses in Arabidopsis. J Exp Bot 62:2143–2154
Melotto M, Mecey C, Niu Y, Chung HS, Katsir L, Yao J, Zeng W, Thines B, Staswick P, Browse J, Howe GA, He SY (2008) A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine- and jasmonoyl isoleucine-dependent interactions with the COI1 F-box protein. Plant J 55:979–988
Yan J, Zhang C, Gu M, Bai Z, Zhang W, Qi T, Cheng Z, Peng W, Luo H, Nan F, Wang Z, Xie D (2009) The Arabidopsis CORONATINE INSENSITIVE1 protein is a jasmonate receptor. Plant Cell 21:2220–2236
Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448:661–665
Hammarström M, Hellgren N, Van Den Berg S, Berglund H, Härd T (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci 11:313–321
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Acknowledgments
We thank the members of the laboratory for critical reading of the manuscript. Research in R.S lab was supported by grants from the Ministry of Science and Innovation (BIO2007-66935, BIO2010-21739, CSD2007-00057-B, and EUI2008-03666) to R.S. S.F. was the recipient of a postdoctoral fellowship from the Portuguese Foundation for Science and Technology and a JAE-Doc contract from the Consejo Superior de Investigaciones Científicas.
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Fonseca, S., Solano, R. (2013). Pull-Down Analysis of Interactions Among Jasmonic Acid Core Signaling Proteins. In: Goossens, A., Pauwels, L. (eds) Jasmonate Signaling. Methods in Molecular Biology, vol 1011. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-414-2_13
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DOI: https://doi.org/10.1007/978-1-62703-414-2_13
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