Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries

  • Rocío Sánchez-Montesino
  • Luis Oñate-Sánchez
Part of the Methods in Molecular Biology book series (MIMB, volume 1629)


Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA–protein (Y1H) and protein–protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic “test tube” and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).

Key words

Arrayed libraries DNA–protein interaction High-throughput One-hybrid system Open reading frame Protein–protein interaction Transcription factors Two-hybrid system Yeast 



The work in L.O.-S. lab is supported by MINECO grants BIO2013-46076-R and BIO2016-77840-R. We thank all people that contributed to the development of the Arabidopsis TF library [22] as well as all the labs that have used this resource helping this way to ascertain its potential. Dr. Begoña Benito is also thanked for critical reading. We apologize to those publications not cited here due to space limitations.


  1. 1.
    Brent R, Ptashne M (1985) A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43:729–736CrossRefPubMedGoogle Scholar
  2. 2.
    Ma J, Ptashne M (1998) Converting a eukaryotic transcriptional inhibitor into an activator. Cell 55:443–446CrossRefGoogle Scholar
  3. 3.
    Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246CrossRefPubMedGoogle Scholar
  4. 4.
    Wilson TE, Fahrner TJ, Johnston M, Milbrandt J (1991) Identification of the DNA binding site for NGFIB by genetic selection in yeast. Science 252:1296–1300CrossRefPubMedGoogle Scholar
  5. 5.
    Li JJ, Herskowitz I (1993) Isolation of the ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262:1870–1874CrossRefPubMedGoogle Scholar
  6. 6.
    Wang MM, Reed RR (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364:121–126CrossRefPubMedGoogle Scholar
  7. 7.
    Dowell SJ, Romanowski P, Diffley JF (1994) Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. Science 265:1243–1246CrossRefPubMedGoogle Scholar
  8. 8.
    Inouye C, Remondelli P, Karin M, Elledge S (1994) Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system. DNA Cell Biol 13:731–742CrossRefPubMedGoogle Scholar
  9. 9.
    Rezwan M, Auerbach D (2012) Yeast “N”-hybrid systems for protein-protein and drug-protein interaction discovery. Methods 57(4):423–429CrossRefPubMedGoogle Scholar
  10. 10.
    Ferro E, Trabalzini L (2013) The yeast two-hybrid and related methods as powerful tools to study plant cell signalling. Plant Mol Biol 83(4–5):287–301CrossRefPubMedGoogle Scholar
  11. 11.
    Ji X, Wang L, Nie X, He L, Zang D, Liu Y, Zhang B, Wang Y (2014) A novel method to identify the DNA motifs recognized by a defined transcription factor. Plant Mol Biol 86:367–380CrossRefPubMedGoogle Scholar
  12. 12.
    Ota K, Feng SY, Ito T (2014) Detecting protein-DNA interactions using a modified yeast one-hybrid system. Methods Mol Biol 1164:39–50CrossRefPubMedGoogle Scholar
  13. 13.
    Mallick J, Jansen G, Wu C, Whiteway M (2016) SRYTH: a new yeast two-hybrid method. Methods Mol Biol 1356:31–41CrossRefPubMedGoogle Scholar
  14. 14.
    Snider J, Stagljar I (2016) Membrane Yeast Two-Hybrid (MYTH) mapping of full-length membrane protein interactions. Cold Spring Harb Protoc. doi: 10.1101/pdb.top077560 Google Scholar
  15. 15.
    Reece-Hoyes JS, Walhout AJ (2012) Yeast one-hybrid assays: a historical and technical perspective. Methods 57(4):441–447CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mehla J, Caufield JH, Uetz P (2015) The yeast two-hybrid system: a tool for mapping protein-protein interactions. Cold Spring Harb Protoc 5:425–430Google Scholar
  17. 17.
    Paz-Ares J (2002) REGIA, an EU project on functional genomics of transcription factors from Arabidopsis thaliana. Comp Funct Genomics 3:102–108CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gong W, Shen YP, Ma LG, Pan Y, Du YL, Wang DH, Yang JY, Hu LD, Liu XF, Dong CX, Ma L, Chen YH, Yang XY, Gao Y, Zhu D, Tan X, Mu JY, Zhang DB, Liu YL, Dinesh-Kumar SP, Li Y, Wang XP, Gu HY, Qu LJ, Bai SN, Lu YT, Li JY, Zhao JD, Zuo J, Huang H, Deng XW, Zhu YX (2004) Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol 135:773–782CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mitsuda N, Ikeda M, Takada S, Takiguchi Y, Kondou Y, Yoshizumi T, Fujita M, Shinozaki K, Matsui M, Ohme-Takagi M (2010) Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana. Plant Cell Physiol 51:2145–2151CrossRefPubMedGoogle Scholar
  20. 20.
    Arabidopsis Interactome Mapping Consortium (2011) Evidence for network evolution in an Arabidopsis interactome map. Science 333(6042):601–607CrossRefPubMedCentralGoogle Scholar
  21. 21.
    Brady SM, Zhang L, Megraw M, Martinez NJ, Jiang E, Yi CS, Liu W, Zeng A, Taylor-Teeples M, Kim D, Ahnert S, Ohler U, Ware D, Walhout AJ, Benfey PN (2011) A stele-enriched gene regulatory network in the Arabidopsis root. Mol Syst Biol 7:459CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Castrillo G, Turck F, Leveugle M, Lecharny A, Carbonero P, Coupland G, Paz-Ares J, Oñate-Sánchez L (2011) Speeding cis-trans regulation discovery by phylogenomic analyses coupled with screenings of an arrayed library of Arabidopsis transcription factors. PLoS One 6:e21524CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gaudinier A, Zhang L, Reece-Hoyes JS, Taylor-Teeples M, Pu L, Liu Z, Breton G, Pruneda-Paz JL, Kim D, Kay SA, Walhout AJ, Ware D, Brady SM (2011) Enhanced Y1H assays for Arabidopsis. Nat Methods 8(12):1053–1055CrossRefPubMedGoogle Scholar
  24. 24.
    Ou B, Yin KQ, Liu SN, Yang Y, Gu T, Wing Hui JM, Zhang L, Miao J, Kondou Y, Matsui M, Gu HY, Qu LJ (2011) A high-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation. Mol Plant 4:546–555CrossRefPubMedGoogle Scholar
  25. 25.
    Burdo B, Gray J, Goetting-Minesky MP, Wittler B, Hunt M, Li T, Velliquette D, Thomas J, Gentzel I, dos Santos Brito M, Mejía-Guerra MK, Connolly LN, Qaisi D, Li W, Casas MI, Doseff AI, Grotewold E (2014) The Maize TFome—development of a transcription factor open reading frame collection for functional genomics. Plant J 80:356–366CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pruneda-Paz JL, Breton G, Nagel DH, Kang SE, Bonaldi K, Doherty CJ, Ravelo S, Galli M, Ecker JR, Kay SA (2014) A genome-scale resource for the functional characterization of Arabidopsis transcription factors. Cell Rep 8:622–632CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Taylor-Teeples M, Lin L, de Lucas M, Turco G, Toal TW, Gaudinier A, Young NF, Trabucco GM, Veling MT, Lamothe R, Handakumbura PP, Xiong G, Wang C, Corwin J, Tsoukalas A, Zhang L, Ware D, Pauly M, Kliebenstein DJ, Dehesh K, Tagkopoulos I, Breton G, Pruneda-Paz JL, Ahnert SE, Kay SA, Hazen SP, Brady SM (2015) An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517(7536):571–575CrossRefPubMedGoogle Scholar
  28. 28.
    Rueda-Romero P, Barrero-Sicilia C, Gómez-Cadenas A, Carbonero P, Oñate-Sánchez L (2012) Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14. J Exp Bot 63:1937–1949CrossRefPubMedGoogle Scholar
  29. 29.
    Iglesias-Fernández R, Barrero-Sicilia C, Carrillo-Barral N, Oñate-Sánchez L, Carbonero P (2013) Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase encoding gene AtMAN7. Plant J 74:767–780CrossRefPubMedGoogle Scholar
  30. 30.
    Iglesias-Fernández R, Wozny D, Iriondo-de Hond M, Oñate-Sánchez L, Carbonero P, Barrero-Sicilia C (2014) The AtCathB3 gene, encoding a cathepsin B-like protease, is expressed during germination of Arabidopsis thaliana and transcriptionally repressed by the basic leucine zipperP protein GBF1. J Exp Bot 65:2009–2021CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Marín-de la Rosa N, Sotillo B, Mizckolczi P, Gibbs DJ, Vicente J, Carbonero P, Oñate-Sánchez L, Holdsworth MJ, Bhalerao R, Alabadí D, Blázquez MA (2014) Large-scale identification of gibberellin-related transcription factors defines Group VII ERFs as functional DELLA partners. Plant Physiol 166:1022–1032CrossRefPubMedGoogle Scholar
  32. 32.
    Ballester P, Navarrete-Gomez M, Carbonero P, Oñate-Sánchez L, Ferrándiz C (2015) Leaf expansion in Arabidopsis is controlled by a TCP-NGA regulatory module likely conserved in distantly related species. Physiol Plant 155:21–32CrossRefPubMedGoogle Scholar
  33. 33.
    Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816CrossRefPubMedGoogle Scholar
  34. 34.
    James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436PubMedPubMedCentralGoogle Scholar
  35. 35.
    Liu J, Wilson TE, Milbrandt J, Johnston M (1993) Identifying DNA-binding sites and analyzing DNA-binding domains using a yeast selection system. Methods 5:125–137CrossRefGoogle Scholar
  36. 36.
    Elble R (1992) A simple and efficient procedure for transformation of yeasts. BioTechniques 13(1):18–20PubMedGoogle Scholar
  37. 37.
    Dobi KC, Winston F (2007) Analysis of transcriptional activation at a distance in Saccharomyces cerevisiae. Mol Cell Biol 27(15):5575–5586CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Aronheim A, Zandi E, Hennemann H, Elledge SJ, Karin M (1997) Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol 17:3094–3102CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Stagljar I, Korostensky C, Johnsson N, te Heesen S (1998) A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95:5187–5192CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Smirnov MN, Smirnov VN, Budowsky EI, Inge-Vechtomov SG, Serebrjakov NG (1967) Red pigment of adenine-deficient yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 27(3):299–304CrossRefPubMedGoogle Scholar
  41. 41.
    Weisman LS, Bacallao R, Wickner W (1987) Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J Cell Biol 105(4):1539–1547CrossRefPubMedGoogle Scholar
  42. 42.
    Rajagopala SV, Hughes KT, Uetz P (2009) Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins. Proteomics 9:5296–5302CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Rocío Sánchez-Montesino
    • 1
  • Luis Oñate-Sánchez
    • 1
  1. 1.Centro de Biotecnología y Genómica de Plantas (UPM-INIA)Universidad Politécnica de MadridMadridSpain

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