Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Summary

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Abstract

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.

Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFP^OPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11^th β-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10^OPT (1-10^th β-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Introduction

Plants are sessile organisms that encounter numerous invading pathogens including bacteria, fungi, viruses, insects, and nematodes throughout their life cycle. Among the phytopathogens, the gram-negative bacterial pathogens such as Pseudomonas spp. and Ralstonia spp., infect their host plants by entering through wounds or natural openings, such as the stomata and hydathode¹. To successfully colonize host plants, bacterial pathogens have evolved to develop a variety of virulence factors². When bacteria invade a host plant, they inject a series of virulence proteins — known as effectors — dire....

Protocol

Note: All steps are performed at room temperature, unless stated otherwise.

1. Preparation of Plant Materials (4 Weeks)

1. Preparation for the N. benthamiana plants
   1. Sow 2 seeds of N. benthamiana on the soil surface of each pot, cover the tray with a plastic dome, and allow seeds to germinate in a 25 °C, 60% humidity growth chamber with a 16/8-h light/dark photoperiod cycle.
   2. After two weeks, pick out and discard the smallest seedling in each pot and continue to grow plants under the same growth conditions as applied for the germination in step 1.1.1. Add 1 L of water per tray every two d....

Results

The β-barrel structure of GFP is composed of eleven β strands and can be divided into two fragments, the 1 - 10^th strand (GFP1-10^OPT) and the 11^th (GFP11) strand. Although neither of two fragments fluorescent by themselves, self-assembled sfGFP can emit the fluorescence when the two fragments exist in close proximity (Figure 1A). In this system, sfGFP1-10^OPT-expressing Arabidopsis or N. benthamiana.......

Discussion

The protocol described here is used for monitoring the accurate localization of the effector proteins injected by the bacterial T3SS into the host plant cell upon infection. Previously, the split GFP system was used as a tool to study the subcellular localization of mammalian proteins^23,36, Salmonella T3E localization, and Agrobacterium VirE2 delivery through the T4SS into the plant cells³⁷. To apply this system in the pl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Arabidopsis transgenic lines			Park, E., Lee, H. Y., Woo, J., Choi, D. & Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)
CYTO-sfGFP1-10	ABRC	CS69831
NU-sfGFP1-10	ABRC	CS69832
PT-sfGFP1-10	ABRC	CS69833
MT-sfGFP1-10	ABRC	CS69834
PX-sfGFP1-10	ABRC	CS69835
ER-sfGFP1-10	ABRC	CS69836
GO-sfGFP1-10	ABRC	CS69837
PM-sfGFP1-10	ABRC	CS69838
Organelle-targeted sfGFP1-10OPT plasmid			Park, E., Lee, H. Y., Woo, J., Choi, D. & Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)
CYTO-sfGFP1-10	Addgene	97387
NU-sfGFP1-10	Addgene	97388
PT-sfGFP1-10	Addgene	97389
MT-sfGFP1-10	Addgene	97390
PX-sfGFP1-10	Addgene	97391
ER-sfGFP1-10	Addgene	97392
GO-sfGFP1-10	Addgene	97393
PM-sfGFP1-10	Addgene	97394
ER-sfCherry1-10	Addgene	97403
ER-sfYFP1-10	Addgene	97404
CYTO-sfCFP1-10	Addgene	97405
sfGFP11-tagged Gateway compatible vector for T3SS-based effector delivery system	Park, E., Lee, H. Y., Woo, J., Choi, D. & Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)
pBK-GW-1-2	Addgene	98250	pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)
pBK-GW-1-4	Addgene	98251	pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)
pBK-GW-2-2	Addgene	98252	pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)
pBK-GW-2-4	Addgene	98253	pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)
pBG-GW-1-2	Addgene	98254	pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)
pBG-GW-1-4	Addgene	98255	pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)
pBG-GW-2-2	Addgene	98256	pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)
pBG-GW-2-4	Addgene	98257	pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)
Bacterial strains
Agrobacterium tumefaciens GV3101			Csaba Koncz and Jeff Schell, The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet. 204,383-396 (1986); Resistant to gentamycin (50 ug/ml) and rifampicin (50 ug/ml)
Pseudomonas syringae pv. Tomato CUCPB5500			Kvitko, B. H. et al. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5 (4) (2009).; Resistant to rifampicin (100 ug/ml)
Media components
Plant germination media			Add 2.165g/L Murashige & Skoog powder, 10 g/L sucrose to water. Adjust to pH 5.8 and add 2.2 g/L phytagel. Autocalve.
Murashige & Skoog medium including vitamins	Duchefa Biochemie	M0222	Store at 4 °C.
Sucrose	Duchefa Biochemie	S0809
Phytagel	Sigma-Aldrich	P8169
LB media			Add 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl to water. For solid media, add 15 g/L micro agar. Autoclave.  Allow solution to cool to 55 °C, and add antibiotic if needed.
Tryptone	BD Bioscience	211705
Yeast extract	BD Bioscience	212750
NaCl	Duchefa Biochemie	S0520
Micro agar	Duchefa Biochemie	M1002
King's B media			10 g/L protease peptone #2, 1.5 g/L anhydrous K2HPO4, 15 g/L of agar to water. Autoclave. Cool down to 55 °C and add sterile 15 ml/L glycerol, 5 ml/L MgSO4 to the medium. Add antibiotics if needed.
Proteose peptone	BD Bioscience	212120
Anhydrous K2HPO4	Sigma-Aldrich	1551128 USP
Glycerol	Duchefa Biochemie	G1345
MgSO4	Sigma-Aldrich	M7506
Bacto Agar	BD Bioscience	214010
Mannitol-Glutamate (MG) liquid media			Add 10 g/L of mannitol, 2 g/L of L-glutamic acid, 0.5 g/L of KH2PO4, 0.2 g/L of NaCl, and 0.2 g/L of MgSO4 to water. Adjust to pH 7
Mannitol	Duchefa Biochemie	M0803
L-glutamic acid	Duchefa Biochemie	G0707
KH2PO4	Sigma-Aldrich	NIST200B
Infiltration buffer			10 mM MES (2-(N-morpholino)-ethane sulfonic acid), 10 mM MgCl2, 150 µM acetosyringone. pH 5.6; Prepare a fresh buffer before use.
MES	Duchefa Biochemie	M1503	Prepare 100 mM (pH 5.6) stock in water. Filter sterilize.
MgCl2	Sigma-Aldrich	M8266	Prepare 100 mM stock in water. Autoclave.
Acetosyringone	Sigma-Aldrich	D134406	Prepare 150 mM stock in DMSO.
Confocal microscope equipments/materials
710 laser scanning confocal system	Carl Zeiss
Axio observer Z1 inverted microscope	Carl Zeiss
Propidium iodide	ThermoFisher	P1304MP