Quantitative Proteomics Reveals Dynamic Changes in the Plasma Membrane During Arabidopsis Immune Signaling*

The plant plasma membrane is a crucial mediator of the interaction between plants and microbes. Understanding how the plasma membrane proteome responds to diverse immune signaling events will lead to a greater understanding of plant immunity and uncover novel targets for crop improvement. Here we report the results from a large scale quantitative proteomics study of plasma membrane-enriched fractions upon activation of the Arabidopsis thaliana immune receptor RPS2. More than 2300 proteins were identified in total, with 1353 proteins reproducibly identified across multiple replications. Label-free spectral counting was employed to quantify the relative protein abundance between different treatment samples. Over 20% of up-regulated proteins have known roles in plant immune responses. Significantly changing proteins include those involved in calcium and lipid signaling, membrane transport, primary and secondary metabolism, protein phosphorylation, redox homeostasis, and vesicle trafficking. A subset of differentially regulated proteins was independently validated during bacterial infection. This study presents the largest quantitative proteomics data set of plant immunity to date and provides a framework for understanding global plasma membrane proteome dynamics during plant immune responses.

[1]  M. Grant,et al.  Arabidopsis Auxin Mutants Are Compromised in Systemic Acquired Resistance and Exhibit Aberrant Accumulation of Various Indolic Compounds1[W][OA] , 2010, Plant Physiology.

[2]  S. Bak,et al.  The involvement of two p450 enzymes, CYP83B1 and CYP83A1, in auxin homeostasis and glucosinolate biosynthesis. , 2001, Plant physiology.

[3]  G. Jürgens,et al.  Co-option of a default secretory pathway for plant immune responses , 2008, Nature.

[4]  P. Khaitovich,et al.  BMC Genomics BioMed Central Methodology article Estimating accuracy of RNA-Seq and microarrays with proteomics , 2022 .

[5]  Hyungwon Choi,et al.  Significance Analysis of Spectral Count Data in Label-free Shotgun Proteomics*S , 2008, Molecular & Cellular Proteomics.

[6]  Albert J R Heck,et al.  Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis*S , 2007, Molecular & Cellular Proteomics.

[7]  J. Dangl,et al.  Phospholipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana. , 2006, The Plant journal : for cell and molecular biology.

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

[9]  Joshua L. Heazlewood,et al.  SUBA: the Arabidopsis Subcellular Database , 2006, Nucleic Acids Res..

[10]  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.

[11]  M. Washburn,et al.  Refinements to label free proteome quantitation: how to deal with peptides shared by multiple proteins. , 2010, Analytical chemistry.

[12]  S. Somerville,et al.  Phytoalexin Accumulation in Arabidopsis thaliana during the Hypersensitive Reaction to Pseudomonas syringae pv syringae. , 1992, Plant physiology.

[13]  Adrian D Hegeman,et al.  A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling * S , 2006, Molecular & Cellular Proteomics.

[14]  Michael J. Axtell,et al.  Initiation of RPS2-Specified Disease Resistance in Arabidopsis Is Coupled to the AvrRpt2-Directed Elimination of RIN4 , 2003, Cell.

[15]  Qun Zhang,et al.  Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis[C][W][OA] , 2009, The Plant Cell Online.

[16]  D. Ren,et al.  Ethylene signaling is required for the acceleration of cell death induced by the activation of AtMEK5 in Arabidopsis , 2008, Cell Research.

[17]  T. Heitz,et al.  A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis. , 2005, The Plant journal : for cell and molecular biology.

[18]  Alexey I Nesvizhskii,et al.  Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. , 2002, Analytical chemistry.

[19]  J. Schultz,et al.  Overexpression of CRK13, an Arabidopsis cysteine-rich receptor-like kinase, results in enhanced resistance to Pseudomonas syringae. , 2007, The Plant journal : for cell and molecular biology.

[20]  P. He,et al.  Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology , 2007, Proceedings of the National Academy of Sciences.

[21]  J. Yates,et al.  A model for random sampling and estimation of relative protein abundance in shotgun proteomics. , 2004, Analytical chemistry.

[22]  T. Boller,et al.  A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. , 2009, Annual review of plant biology.

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

[24]  O. Maudoux,et al.  Single Point Mutations Distributed in 10 Soluble and Membrane Regions of the Nicotiana plumbaginifolia Plasma Membrane PMA2 H+-ATPase Activate the Enzyme and Modify the Structure of the C-terminal Region* , 1998, The Journal of Biological Chemistry.

[25]  M. G. Kim,et al.  Two Pseudomonas syringae Type III Effectors Inhibit RIN4-Regulated Basal Defense in Arabidopsis , 2005, Cell.

[26]  M. Bolton Primary metabolism and plant defense--fuel for the fire. , 2009, Molecular plant-microbe interactions : MPMI.

[27]  K. Sjolander,et al.  Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Synan F. AbuQamar,et al.  The Membrane-Anchored BOTRYTIS-INDUCED KINASE1 Plays Distinct Roles in Arabidopsis Resistance to Necrotrophic and Biotrophic Pathogens[W] , 2005, The Plant Cell Online.

[29]  N. Chua,et al.  Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death. , 1998, The Plant journal : for cell and molecular biology.

[30]  M. B. Mudgett,et al.  SOBER1 phospholipase activity suppresses phosphatidic acid accumulation and plant immunity in response to bacterial effector AvrBsT , 2009, Proceedings of the National Academy of Sciences.

[31]  C. Larsson,et al.  Isolation of highly purified plant plasma membranes and separation of inside-out and right-side-out vesicles , 1994 .

[32]  William Stafford Noble,et al.  Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. , 2008, Journal of proteome research.

[33]  S. Dinesh-Kumar,et al.  Induced ER chaperones regulate a receptor-like kinase to mediate antiviral innate immune response in plants. , 2009, Cell host & microbe.

[34]  P. Naur,et al.  CYP83A1 and CYP83B1, Two Nonredundant Cytochrome P450 Enzymes Metabolizing Oximes in the Biosynthesis of Glucosinolates in Arabidopsis1 , 2003, Plant Physiology.

[35]  M. Nishimura,et al.  A novel membrane fusion-mediated plant immunity against bacterial pathogens. , 2009, Genes & development.

[36]  P. Schulze-Lefert,et al.  Not a peripheral issue: secretion in plant-microbe interactions. , 2010, Current opinion in plant biology.

[37]  F. Katagiri,et al.  Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity. , 2010, Current opinion in plant biology.

[38]  L. Du,et al.  Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from Arabidopsis , 2004, Plant Molecular Biology.

[39]  Robertson Craig,et al.  TANDEM: matching proteins with tandem mass spectra. , 2004, Bioinformatics.

[40]  Linfeng Wu,et al.  Role of spectral counting in quantitative proteomics , 2010, Expert review of proteomics.

[41]  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.

[42]  She Chen,et al.  Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. , 2009, Cell host & microbe.

[43]  G. Fink,et al.  The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis. , 2006, The Plant journal : for cell and molecular biology.

[44]  P. He,et al.  Direct Ubiquitination of Pattern Recognition Receptor FLS2 Attenuates Plant Innate Immunity , 2011, Science.

[45]  A. Macone,et al.  A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides , 2010, Proceedings of the National Academy of Sciences.

[46]  J. Dangl,et al.  Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense , 2009, Proteomics.

[47]  Alexandra M. E. Jones,et al.  Specific changes in the Arabidopsis proteome in response to bacterial challenge: differentiating basal and R-gene mediated resistance. , 2004, Phytochemistry.

[48]  D. Roby,et al.  Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants? , 2003, Trends in plant science.

[49]  P. Schulze-Lefert,et al.  Activity Determinants and Functional Specialization of Arabidopsis PEN1 Syntaxin in Innate Immunity* , 2008, Journal of Biological Chemistry.

[50]  T. McNellis,et al.  A Humidity-Sensitive Arabidopsis Copine Mutant Exhibits Precocious Cell Death and Increased Disease Resistance , 2001, The Plant Cell Online.

[51]  Xuemin Wang Lipid signaling. , 2004, Current opinion in plant biology.

[52]  Yingzhong Li,et al.  Stability of plant immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box (SCF)-mediated protein degradation , 2011, Proceedings of the National Academy of Sciences.

[53]  B. Halkier,et al.  CYP71B15 (PAD3) Catalyzes the Final Step in Camalexin Biosynthesis1 , 2006, Plant Physiology.

[54]  S. Grün,et al.  Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem1[W] , 2009, Plant Physiology.

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

[56]  U. Grossniklaus,et al.  PAMP (Pathogen-associated Molecular Pattern)-induced Changes in Plasma Membrane Compartmentalization Reveal Novel Components of Plant Immunity* , 2010, The Journal of Biological Chemistry.

[57]  C. Larsson,et al.  [52] Preparation of high-purity plasma membranes , 1987 .

[58]  Johannes Vogel,et al.  Quantifying Western blots: Pitfalls of densitometry , 2009, Electrophoresis.

[59]  M. Palmgren,et al.  RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack , 2009, PLoS biology.

[60]  B. Phinney,et al.  Proteomic characterization of human milk whey proteins during a twelve‐month lactation period , 2011, Journal of proteome research.

[61]  Kentaro Inoue,et al.  Two chloroplastic protein translocation components, Tic110 and Toc75, are conserved in different plastid types from multiple plant species , 2004, Plant Molecular Biology.

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

[63]  B. Phinney,et al.  A comparative study of in-gel digestions using microwave and pressure-accelerated technologies. , 2010, Journal of biomolecular techniques : JBT.

[64]  J. Braam,et al.  Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis. , 1995, The Plant journal : for cell and molecular biology.

[65]  M. Mann,et al.  In-gel digestion for mass spectrometric characterization of proteins and proteomes , 2006, Nature Protocols.

[66]  Jun Liu,et al.  A receptor-like cytoplasmic kinase phosphorylates the host target RIN4, leading to the activation of a plant innate immune receptor. , 2011, Cell host & microbe.

[67]  B. Bartel,et al.  Peroxisome-associated matrix protein degradation in Arabidopsis , 2009, Proceedings of the National Academy of Sciences.

[68]  A. Falick,et al.  Activation of a Phytopathogenic Bacterial Effector Protein by a Eukaryotic Cyclophilin , 2005, Science.

[69]  T. Boller,et al.  A Plasma Membrane Syntaxin Is Phosphorylated in Response to the Bacterial Elicitor Flagellin* , 2003, Journal of Biological Chemistry.

[70]  D. Wessel,et al.  A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. , 1984, Analytical biochemistry.

[71]  J. Dangl,et al.  Pathogen-induced, NADPH oxidase–derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana , 2005, Nature Genetics.

[72]  A. Lloyd,et al.  The hypersensitive response; the centenary is upon us but how much do we know? , 2008, Journal of experimental botany.

[73]  C. Larsson,et al.  Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles. , 1995, The Plant journal : for cell and molecular biology.

[74]  A. Bent,et al.  RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2. , 1993, The Plant cell.

[75]  S. Roje S-Adenosyl-L-methionine: beyond the universal methyl group donor. , 2006, Phytochemistry.

[76]  J. Dangl,et al.  Specific threonine phosphorylation of a host target by two unrelated type III effectors activates a host innate immune receptor in plants. , 2011, Cell host & microbe.

[77]  J. Glazebrook,et al.  Arabidopsis Cytochrome P450 Monooxygenase 71A13 Catalyzes the Conversion of Indole-3-Acetaldoxime in Camalexin Synthesis[W] , 2007, The Plant Cell Online.

[78]  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.

[79]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[80]  T. Mengiste,et al.  Biochemical and Genetic Requirements for Function of the Immune Response Regulator BOTRYTIS-INDUCED KINASE1 in Plant Growth, Ethylene Signaling, and PAMP-Triggered Immunity in Arabidopsis , 2011 .

[81]  Alexandra ME Jones,et al.  Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses , 2007, The Plant journal : for cell and molecular biology.

[82]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[83]  Yuan Li,et al.  Glutathione-Indole-3-Acetonitrile Is Required for Camalexin Biosynthesis in Arabidopsis thaliana[W][OA] , 2011, Plant Cell.