Characterizing the roles of an Arabidopsis Epsin n-terminal homology domain protein in plant innate immunity
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The plasma membrane (PM) serves as a crucial point of contact between a plant cell and its environment. Localized at this critical interface are proteins involved in the recognition of external stimuli, including Receptor-Like Kinases (RLKs) and Pattern Recognition Receptors (PRRs). PRRs perceive a potential pathogen via conserved microbial features or Pathogen-Associate Molecular Patterns (PAMPs), including molecules from bacteria. PAMP binding by a PRR protein initiates intracellular responses to inhibit the growth of a bacterial pathogen and subsequently, plants that lack some PRR proteins are more susceptible to bacterial infection. For future biochemical analysis of PM-localized proteins, including PRRs, we provide a procedure for the simplified enrichment of PM proteins from intact Arabidopsis seedlings. For this PM enrichment method, differential ultra-centrifugation is first utilized to isolate microsomes that are subsequently treated with a non-ionic detergent Brij-58 to enrich for PM proteins by removing potentially contaminating organellar proteins, such as those derived from internal membrane-bound compartments. Advantages of this PM enrichment procedure are that it is relatively quick and highly reproducible. In addition, it requires minimal optimization when working with different tissue types and species. Combining this protocol with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM. In the model plant Arabidopsis thaliana, the PM-localized Flagellin Sensing 2 (FLS2), recognizes bacterial flagellin or the peptide flg22 to initiate PAMP responses. FLS2 abundance at the PM must be tightly controlled so that it can alert the cell to the presence of the pathogen. A complex and dynamic network of internal vesicular trafficking pathways maintains the correct pool of proteins at the PM. However, few vesicular trafficking components are known with roles in transporting FLS2 to and from the PM. Here, we used a phosphoproteomic screen to identify an Arabidopsis Epsin NTerminal Homology (ENTH) domain-containing protein, Epsin1, as being phosphorylated in response to flg22, which potentially places it in the FLS2-flg22 response pathway. In plants, ENTH-domain proteins are thought to function in clathrin-mediated vesicle formation at the trans-Golgi Network (TGN), which functions in multiple trafficking pathways throughout the cell. Two independent epsin1 mutant alleles showed defects in all investigated flg22-responses and were more susceptible to infection with the Pto DC3000 and Pto DC3000 hrcC- strains of bacteria. Using both a biochemical simplified PM-enrichment and live-cell imaging approach, we correlated impaired flg22-signaling to reduced FLS2 protein levels at the PM. Our data identified Epsin1 as a novel positive regulator of innate immunity against Pto DC3000 and Pto DC3000 hrcC-, with roles in regulating correct FLS2 abundance at the PM. Elongation Factor-Tu Receptor (EFR) and Arabidopsis Plant Elicitor Peptide Receptor 1 (AtPEPR1) are PRRs that recognize the peptides Elongation Factor-Tu (elf26) and Arabidopsis Plant Elicitor Peptide 1 (AtPep1), respectively. We demonstrated that both epsin1 mutant alleles displayed phenotypic defects in all investigated elf26 and AtPep1 signaling events. These results indicate that Epsin1 may be a positive regulator of elf26- and AtPep1-induced responses as well. Using both immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) a subset of proteins were decreased in an enriched PM fraction of an epsin1-2 mutant, including the RLKs Brassinosteriod-Insensitive 1 (BRI1) and BRI1-Associated Kinase 1 (BAK1). Together, these findings expand the role of Epsin1 to include a broad role as a positive regulator of immune-related signaling and the PM accumulation of some PRRs at the PM. Previous preliminary results from the Heese lab indicate that the ADPRibosylation Factor-Guanine Exchange Factor (ARF-GEF) named BFA-visualized endocytic trafficking defective 1/HopM1-Interacting 7/BFA-Inhibited Guanine nucleotide-exchange factor 5 (BEN1/MIN7/BIG5), or MIN7, is found in a complex with Epsin1 in the presence of flg22. Analysis of epsin1-2 min7 double mutants reveals that Epsin1 and MIN7 may function together genetically for proper root growth and leaf development, but perhaps not for the induction of flg22-induced reactive oxygen species production (ROS). We also establish tools for the future study of the biochemical relationship between Epsin1 and MIN7. Taken together, these findings illustrate the importance of a functional vesicular trafficking network necessary for the initiation of plant innate immune responses.
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