Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2

BMC Genomics. 2012 Jan 9;13:8. doi: 10.1186/1471-2164-13-8.

Abstract

Background: Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system.

Results: Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence.

Conclusions: We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics*
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Gene Library
  • High-Throughput Screening Assays*
  • Host-Pathogen Interactions
  • Membrane Proteins / genetics*
  • Protein Binding
  • Protein Transport
  • Pseudomonas syringae / genetics*
  • Pseudomonas syringae / metabolism
  • Pseudomonas syringae / pathogenicity
  • Two-Hybrid System Techniques
  • Virulence / genetics

Substances

  • Arabidopsis Proteins
  • Bacterial Proteins
  • MLO2 protein, Arabidopsis
  • Membrane Proteins