The Yeast Two-Hybrid Assay in Plants

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The yeast 2-hybrid (Y2H) assay is a very powerful tool for detecting protein-protein or protein-DNA interactions. Since its discovery in the 1980s, this assay has become one of the most popular approaches for probing physical interactions in vivo. The Y2H assay is not only straightforward to perform, but also highly feasible and can be applied in a broad categories of proteins, including those in plants. Here at Lifeasible, we provide well established platforms for Y2H assays with guaranteed high quality results and rapid turnaround time.

The Y2H Assay

The principle of a traditional Y2H assay is to utilize a reporter gene, whose activation relies on its particular transcription factor (1). Specifically, proteins to be tested, X and Y, are fused with the DNA-binding domain (BD), and the activation domain (AD) of the transcription factor, respectively. The BD-X fusion protein binds to the upstream activator sequence (UAS) the reporter gene’s promoter. Upon interaction between X and Y, the AD will be recruited to the promoter and reconstitute a functional transcription factor, which therefore triggers the subsequent transcription of the reporter gene (Figure 1).

../Picture1.pngFigure 1. A schematic cartoon of a classical yeast two hybrid system.

In order to study the interactions of a broad group of proteins, several variants of the Y2H systems have been developed, adapting to the differential subcellular localizations and biochemical characterizations of different proteins. Lifeasible provides service for multiple variations of the Y2H system to accommodate the diverse needs of different projects.

  • Yeast one-hybrid System: for the study of protein-DNA interactions (2) (Figure 2).

Y1H.pngFigure 2. A schematic cartoon of the yeast one hybrid system.

  • Yeast three-hybrid system: for the study of protein interactions that mediated by a third component (3) (Figure 3).

Y3H.pngFigure 3. A schematic cartoon of the yeast three hybrid system.

  • Split-ubiquitin Y2H System: for the study of membrane proteins (4) (Figure 4).

../Picture1.pngFigure 4. A schematic cartoon of the split-ubiquitin Y2H system.

Y2H screening

When investigating the function of an unknown protein, it is essential to identify its interactive partners. Aside from examining interactions between noted proteins/DNAs, the Y2H technique has also been widely used as a screening method for the uncovering of protein-protein interactions. Based on the construction of cDNA libraries, Y2H can be easily applied for high throughput screens of protein interactions on a genome-wide base (5). So far, the Y2H screen has been applied for protein functional identifications in the model plant Arabidopsis and multiple crops, including wheat and maize (5, 6).

Advantages of the Y2H screening are:

  • Feasible for genome-based high throughput screens
  • In vivo assay that avoids artificial influence
  • Straightforward method, easy to perform


  1. Bruckner A, Polge C, Lentze N, Auerbach D, & Schlattner U (2009) Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci 10(6):2763-2788.
  2. Ouwerkerk PB & Meijer AH (2001) Yeast one-hybrid screening for DNA-protein interactions. Curr Protoc Mol Biol Chapter 12:Unit 12 12.
  3. Maruta N, Trusov Y, & Botella JR (2016) Yeast Three-Hybrid System for the Detection of Protein-Protein Interactions. Methods Mol Biol 1363:145-154.
  4. Thaminy S, Miller J, & Stagljar I (2004) The split-ubiquitin membrane-based yeast two-hybrid system. Methods Mol Biol 261:297-312.
  5. 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):287-301.
  6. Matiolli CC & Melotto M (2018) A Comprehensive Arabidopsis Yeast Two-Hybrid Library for Protein-Protein Interaction Studies: A Resource to the Plant Research Community. Mol Plant Microbe Interact:MPMI02180047A.
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