Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2.
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.
Department of Cell and Systems Biology, University of Toronto, Toronto, ON, M5S 3B2, Canada.
Jennifer D
JD
Lewis
Janet
J
Wan
Rachel
R
Ford
Yunchen
Y
Gong
Pauline
P
Fung
Hardeep
H
Nahal
Pauline W
PW
Wang
Darrell
D
Desveaux
David S
DS
Guttman
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D057166:High-Throughput Screening Assays; D054884:Host-Pathogen Interactions; D008565:Membrane Proteins / Q000235:genetics*; D011485:Protein Binding; D021381:Protein Transport; D044224:Pseudomonas syringae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D020798:Two-Hybrid System Techniques; D014774:Virulence / Q000235:genetics
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; C573497:MLO2 protein, Arabidopsis; D008565:Membrane Proteins