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CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

  • Author(s): Trigg, Shelly A
  • Garza, Renee M
  • MacWilliams, Andrew
  • Nery, Joseph R
  • Bartlett, Anna
  • Castanon, Rosa
  • Goubil, Adeline
  • Feeney, Joseph
  • O'Malley, Ronan
  • Huang, Shao-Shan C
  • Zhang, Zhuzhu Z
  • Galli, Mary
  • Ecker, Joseph R
  • et al.
Abstract

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

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