A user-friendly platform for yeast two-hybrid library screening using next generation sequencing

PLoS One. 2018 Dec 21;13(12):e0201270. doi: 10.1371/journal.pone.0201270. eCollection 2018.


Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism's ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics*
  • Arabidopsis Proteins / genetics*
  • Gene Library*
  • High-Throughput Nucleotide Sequencing*
  • Open Reading Frames
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Two-Hybrid System Techniques*


  • Arabidopsis Proteins

Grant support

This research was supported by the Research Foundation Flanders by a research project grant to A.G. (G004515N) and for postdoctoral fellowships to J.P. and L.P., the Program Ciências Sem Fronteiras for a predoctoral fellowship to B.R. (Grant 201135/2014-0), the BEC.AR program for overseas training of Argentine professionals in the fields of science, technology and productive innovation for a scholarship to M.P. and the Special Research Fund from Ghent University (project O1J14813). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.