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

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          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.

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          Most cited references36

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          Plant pathogens and integrated defence responses to infection.

          Plants cannot move to escape environmental challenges. Biotic stresses result from a battery of potential pathogens: fungi, bacteria, nematodes and insects intercept the photosynthate produced by plants, and viruses use replication machinery at the host's expense. Plants, in turn, have evolved sophisticated mechanisms to perceive such attacks, and to translate that perception into an adaptive response. Here, we review the current knowledge of recognition-dependent disease resistance in plants. We include a few crucial concepts to compare and contrast plant innate immunity with that more commonly associated with animals. There are appreciable differences, but also surprising parallels.
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            Membrane phosphatidylserine regulates surface charge and protein localization.

            Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.
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              Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens.

              For many years, research on a suite of plant defense responses that begin when plants are exposed to general microbial elicitors was underappreciated, for a good reason: There has been no critical experimental demonstration of their importance in mediating plant resistance during pathogen infection. Today, these microbial elicitors are named pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the plant responses are known as PAMP-triggered immunity (PTI). Recent studies provide an elegant explanation for the difficulty of demonstrating the role of PTI in plant disease resistance. It turns out that the important contribution of PTI to disease resistance is masked by pathogen virulence effectors that have evolved to suppress it.
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                Author and article information

                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2012
                9 January 2012
                : 13
                : 8
                Affiliations
                [1 ]Department of Cell and Systems Biology, University of Toronto, Toronto, ON, M5S 3B2, Canada
                [2 ]Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, ON, M5S 3B2, Canada
                [3 ]Plant Gene Expression Center, USDA, 800 Buchanan St., Albany, CA, 94710, USA
                Article
                1471-2164-13-8
                10.1186/1471-2164-13-8
                3320541
                22230763
                dba2e3ea-1108-4692-b139-7992942d736d
                Copyright ©2012 Lewis et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 16 September 2011
                : 9 January 2012
                Categories
                Research Article

                Genetics
                mlo2,type iii effector,high-throughput screening,arabidopsis,hopz,next-generation sequencing,pseudomonas syringae,yeast two-hybrid

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