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

BackgroundIdentification 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.ResultsHere 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.ConclusionsWe 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.

[1]  S. Somerville,et al.  Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P. Schulze-Lefert,et al.  Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments. , 2009, The Plant journal : for cell and molecular biology.

[3]  G. Coaker,et al.  The type III effector HopF2 Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence , 2010, Proceedings of the National Academy of Sciences.

[4]  D. Guttman,et al.  Evolution of the type III secretion system and its effectors in plant-microbe interactions. , 2007, The New phytologist.

[5]  Z Zhang,et al.  Plasmid stability in recombinant Saccharomyces cerevisiae. , 1996, Biotechnology advances.

[6]  R. Panstruga,et al.  mlo-based powdery mildew immunity: silver bullet or simply non-host resistance? , 2006, Molecular plant pathology.

[7]  N. Chua,et al.  A glucocorticoid-mediated transcriptional induction system in transgenic plants. , 1997, The Plant journal : for cell and molecular biology.

[8]  D. Guttman,et al.  Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein , 2010, PLoS genetics.

[9]  J. Alfano,et al.  Disabling surveillance: bacterial type III secretion system effectors that suppress innate immunity , 2004, Cellular microbiology.

[10]  I. Stagljar,et al.  A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Axtell,et al.  Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease , 2003, Molecular microbiology.

[12]  Sheng Yang He,et al.  A Bacterial Virulence Protein Suppresses Host Innate Immunity to Cause Plant Disease , 2006, Science.

[13]  Anna Block,et al.  Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys? , 2011, Current opinion in microbiology.

[14]  M. Vidal,et al.  High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. , 2001, Methods.

[15]  David Mackey,et al.  RIN4 Interacts with Pseudomonas syringae Type III Effector Molecules and Is Required for RPM1-Mediated Resistance in Arabidopsis , 2002, Cell.

[16]  J. Alfano,et al.  Phytopathogen type III effector weaponry and their plant targets. , 2008, Current opinion in plant biology.

[17]  Jörg Durner,et al.  Conserved requirement for a plant host cell protein in powdery mildew pathogenesis , 2006, Nature Genetics.

[18]  D. Guttman,et al.  The HopZ Family of Pseudomonas syringae Type III Effectors Require Myristoylation for Virulence and Avirulence Functions in Arabidopsis thaliana , 2008, Journal of bacteriology.

[19]  John P. Rathjen,et al.  Plant immunity: towards an integrated view of plant–pathogen interactions , 2010, Nature Reviews Genetics.

[20]  David S Guttman,et al.  Type III Effector Diversification via Both Pathoadaptation and Horizontal Transfer in Response to a Coevolutionary Arms Race , 2006, PLoS genetics.

[21]  X. Puente,et al.  Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors , 2004, Biological chemistry.

[22]  T. Boller,et al.  Innate Immunity in Plants: An Arms Race Between Pattern Recognition Receptors in Plants and Effectors in Microbial Pathogens , 2009, Science.

[23]  Erich Kombrink,et al.  SNARE-protein-mediated disease resistance at the plant cell wall , 2003, Nature.

[24]  Tony Yeung,et al.  Membrane Phosphatidylserine Regulates Surface Charge and Protein Localization , 2008, Science.

[25]  M. Tremblay,et al.  Substrate-trapping techniques in the identification of cellular PTP targets. , 2005, Methods.

[26]  J. Dangl,et al.  The Pseudomonas syringae effector AvrRpt2 cleaves its C-terminally acylated target, RIN4, from Arabidopsis membranes to block RPM1 activation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Kiana Toufighi,et al.  The Botany Array Resource: E-northerns, Expression Angling, and Promoter Analyses , 2022 .

[28]  Paul Schulze-Lefert,et al.  Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[29]  David E Hill,et al.  next-generation sequencing to generate interactome datasets , 2011 .

[30]  Sara K. Hotton,et al.  The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non‐host plants , 2006, Molecular microbiology.

[31]  M. Kriegler.  Gene Transfer and Expression: A Laboratory Manual , 1990 .

[32]  G. Martin,et al.  Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto–Pto‐mediated hypersensitive response , 2000, The EMBO journal.

[33]  D. Guttman,et al.  The targeting of plant cellular systems by injected type III effector proteins. , 2009, Seminars in cell & developmental biology.

[34]  Jonathan D. G. Jones,et al.  Plant pathogens and integrated defence responses to infection , 2001, Nature.

[35]  Jeff H. Chang,et al.  Subterfuge and manipulation: type III effector proteins of phytopathogenic bacteria. , 2006, Annual review of microbiology.

[36]  D. Guttman,et al.  The YopJ superfamily in plant-associated bacteria. , 2011, Molecular plant pathology.

[37]  A. Barabasi,et al.  High-Quality Binary Protein Interaction Map of the Yeast Interactome Network , 2008, Science.

[38]  I. Stagljar,et al.  Utilizing the Split-Ubiquitin Membrane Yeast Two-Hybrid System to Identify Protein-Protein Interactions of Integral Membrane Proteins , 2005, Science's STKE.

[39]  J. Ecker,et al.  Arabidopsis RIN4 Is a Target of the Type III Virulence Effector AvrRpt2 and Modulates RPS2-Mediated Resistance , 2003, Cell.

[40]  A. Barabasi,et al.  An empirical framework for binary interactome mapping , 2008, Nature Methods.

[41]  F. Katagiri,et al.  Eukaryotic Fatty Acylation Drives Plasma Membrane Targeting and Enhances Function of Several Type III Effector Proteins from Pseudomonas syringae , 2000, Cell.

[42]  Heather Youngs,et al.  The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae. , 2004, Molecular biology of the cell.

[43]  F. Katagiri,et al.  The Arabidopsis Thaliana-Pseudomonas Syringae Interaction , 2002, The arabidopsis book.

[44]  D. Guttman,et al.  Allelic variants of the Pseudomonas syringae type III effector HopZ1 are differentially recognized by plant resistance systems. , 2009, Molecular plant-microbe interactions : MPMI.

[45]  M. Wolter,et al.  The Barley Mlo Gene: A Novel Control Element of Plant Pathogen Resistance , 1997, Cell.

[46]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[47]  G. von Heijne,et al.  Topology, Subcellular Localization, and Sequence Diversity of the Mlo Family in Plants* , 1999, The Journal of Biological Chemistry.

[48]  David E Hill,et al.  High-quality binary interactome mapping. , 2010, Methods in enzymology.

[49]  C. Marshall,et al.  A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21 ras to the plasma membrane , 1990, Cell.

[50]  Jonathan D. G. Jones,et al.  The plant immune system , 2006, Nature.