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A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

André Alcântara, Denise Seitner, Fernando Navarrete, Armin Djamei
doi: https://doi.org/10.1101/825190
André Alcântara
1Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
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Denise Seitner
1Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
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Fernando Navarrete
1Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
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Armin Djamei
1Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
2Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, Germany
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  • For correspondence: djamei@ipk-gatersleben.de
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Abstract

Background The unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.

Results We developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.

Conclusions We have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

  • List of abbreviations

    BiP
    luminol binding protein;
    bZIP
    basic leucin zipper;
    DTT
    dithiothreitol;
    ER
    endoplasmic reticulum;
    ERSE
    ER stress elements;
    HY5
    Elongated Hypocotyl 5;
    IRE1
    Inositol-requiring enzyme 1;
    LB
    Luria broth cell culture medium;
    mCh
    mCherry;
    MES
    2-(N-morpholino)ethanesulfonic acid;
    OD600 nm
    optical density at 600 nm;
    p35S
    CaMV 35S promoter;
    PCD
    programmed cell death;
    qRT-PCR
    quantitative real time polymerase chain reaction;
    S1P
    site 1 protease;
    S2P
    site 2 protease;
    SA
    Salicylic acid;
    Skp1
    S-phase kinase-associated protein 1;
    Tm
    tunicamycin;
    UPR
    unfolded protein response;
    UPRE
    UPR responsive elements;
    eYFP
    enhanced yellow fluorescent protein.
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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    Posted October 31, 2019.
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    A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants
    André Alcântara, Denise Seitner, Fernando Navarrete, Armin Djamei
    bioRxiv 825190; doi: https://doi.org/10.1101/825190
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    A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants
    André Alcântara, Denise Seitner, Fernando Navarrete, Armin Djamei
    bioRxiv 825190; doi: https://doi.org/10.1101/825190

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