Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by alteri...

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Veröffentlicht in:The Plant cell 2017-07, Vol.29 (7), p.1571-1584
Hauptverfasser: Park, Eunsook, Lee, Hye-Young, Woo, Jongchan, Choi, Doil, Dinesh-Kumar, Savithramma P.
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container_end_page 1584
container_issue 7
container_start_page 1571
container_title The Plant cell
container_volume 29
creator Park, Eunsook
Lee, Hye-Young
Woo, Jongchan
Choi, Doil
Dinesh-Kumar, Savithramma P.
description Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.
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These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. 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source Jstor Complete Legacy; Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals
subjects Animal diseases
Arabidopsis - genetics
Arabidopsis - microbiology
Bacteria
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Biological activity
BREAKTHROUGH REPORT
Breakthrough Reports
Compartments
Effector cells
Effectors
Fluorescence
Gram-negative bacteria
Green fluorescent protein
Green Fluorescent Proteins - analysis
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Host-Pathogen Interactions
Immune response
Localization
Molecular Imaging - methods
Monitoring systems
Nicotiana - genetics
Nicotiana - microbiology
Pathogens
Peptide Fragments - analysis
Peptide Fragments - genetics
Peptide Fragments - metabolism
Plant cells
Plant Diseases - microbiology
Plant immunity
Plants, Genetically Modified
Protein Folding
Proteins
Pseudomonas
Pseudomonas syringae
Pseudomonas syringae - metabolism
Pseudomonas syringae - pathogenicity
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Transgenic plants
title Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments
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