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

Abstract

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 (sfGFP OPT ) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th b-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 b-strand (sfGFP1-10 OPT ), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10 OPT 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 sfGFP OPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.

Plant–microbe interactions: organelles and the cytoskeleton in action

“…The N-termini of HopK1 and AvrRPS4 are required for virulence and have been shown to localize to chloroplasts in transgenic Arabidopsis plants overexpressing HopK1, but not when transiently expressed in N. benthamiana . In addition, when AvrRPS4 is directly delivered from Pst in a split fluorescent protein assay, it does not localize to the chloroplast (Park et al, 2017). HopU1 is a mono-ADP-ribosyltransferase from Pst DC3000 that targets three chloroplast-localized, RNA-binding proteins to suppress innate immunity in plants (Fu et al, 2007).…”

“…Therefore, studies that monitor the dynamics of effectors that are delivered into host organelles directly by the pathogen are needed to understand the precise localization and role of these effectors in virulence. Recently, a split fluorescent protein-based assay has been described to detect the subcellular localization of effectors delivered directly by bacterial pathogens into host cells (Henry et al, 2017;Park et al, 2017). Using this assay, Pseudomonas effectors are fused to the 11 th b-strand of super-folder GFP (sfGFP11) and, when delivered into plant cells expressing the sfGFP 1-10 b-strand (sfGFP1-10), sfGFP is reconstituted in the cytosol or in target organelles and can be visualized by confocal microscopy (Park et al, 2017).…”

“…Recently, a split fluorescent protein-based assay has been described to detect the subcellular localization of effectors delivered directly by bacterial pathogens into host cells (Henry et al, 2017;Park et al, 2017). Using this assay, Pseudomonas effectors are fused to the 11 th b-strand of super-folder GFP (sfGFP11) and, when delivered into plant cells expressing the sfGFP 1-10 b-strand (sfGFP1-10), sfGFP is reconstituted in the cytosol or in target organelles and can be visualized by confocal microscopy (Park et al, 2017). This approach is not only useful for detecting the bona fide localization of bacterial effectors secreted through the T3SS, but also for monitoring the dynamics of fungal and other pathogen effectors.…”

A human pathogenic bacterium Shigella proliferates in plants through adoption of type III effectors for shigellosis

“…In this system, the nonfluorescent GFP protein (GFP1–10) harbouring β‐strands 1–10 is expressed in the host cell, and the bacterial effector is linked to the 11th β‐strand (GFP11). Only when the effector fused to GFP11 is introduced into plant cells via bacterial T3SS is a fluorescent signal induced through reconstitution of GFP (Park et al, ; Figure a). First, to select a host plant expressing GFP1–10 capable of exploring the delivery of OspF and OspG, we observed subcellular localization of OspF:GFP and OspG:GFP by expressing them in Nicotiana benthamiana leaves using the Agrobacterium system (Figure S8).…”

Tracking the Bacterial Type III Secretion System: Visualization of Effector Delivery Using Split Fluorescent Proteins

“…In a complementary approach, a Breakthrough Report by Park et al (2017) examined effectors in various subcellular compartments. The authors generated a set of transgenic Arabidopsis plants expressing GFP 1-10 in the nucleus, Golgi, endoplasmic reticulum, plasma membrane, mitochondria, plastids, peroxisome, and cytoplasm.…”