Monitoring Bacterial Infection and Effector Protein Delivery in Plant Cells

Samenvatting

Source: Lee, H. et. al., Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection. J. Vis. Exp. (2018)

This video demonstrates a method for monitoring bacterial infection and effector protein delivery in plant cells using split superfolder green fluorescent protein (sfGFP) complementation combined with confocal microscopy. The approach enables precise visualization of effector translocation into the host cell cytosol.

Protocol

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

1. Preparation of Plant Materials (4 Weeks)

1. Preparation for the Nicotiana 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. Continue to grow plants under the same growth conditions as applied for germination in step 1.1.1. Add 1 L of water per tray every two days.
      NOTE: The growth conditions for plants may vary across labs. Therefore, follow the regular watering protocol to grow the plant in a healthy condition.
   3. In a week, transfer the plants to a new tray and arrange them with adequate space for further growth. Keep growing the plants under the conditions described in step 1.1.1 until they are ready to be infiltrated at 4 weeks of age.
      NOTE: Plant growth may differ depending on the growth conditions across labs. Usually, we find that the 4-week-old N. benthamiana plants bear about six leaves.
2. Preparation for Arabidopsis thaliana transgenic plants
   1. Refer to the Table of Materials and order the transgenic Arabidopsis seeds.
   2. Soak ~50 - 100 transgenic Arabidopsis seeds in 1 mL of distilled water and store them at 4 °C for 3 days in the dark to synchronize the onset of germination.
   3. Sow ~2 - 3 seeds on the soil surface of a plug plant tray and cover the tray with a plastic dome. Allow seeds to germinate at 23 °C, 60% humidity with a 10/14-h light/dark photoperiod cycle.
      NOTE: The seeds should be homozygous. However, we recommend reconfirming the presence of the transgene in the batch. In this case, sterilize the seeds by washing with 70% ethanol for 2 min, 50% bleach (about 2% hypochlorite) containing 0.05% Triton X-100 for 5 min. Followed by washing 5 - 6 times with sterile double-distilled water (ddH₂O). After sterilization, stratify at 4 °C for 3 days and plate them on plant germination media containing 25 µg/L of hygromycin B to select the transgenic plants.
   4. After a week, leave only one plant per plug and continue growing plants under the same growth conditions used for step 1.2.3.
      NOTE: Four-week-old plants were used for the Pseudomonas syringae infection. Water plants every other day to keep plants healthy.

2. Preparation of Pseudomonas Culture (~1 Week)

1. Construction of the plasmid for Pseudomonas transformation
   1. Refer to the Table of Materials and order the desired vector(s) of the type III secretion system (T3SS)-based effector delivery system vector.
   2. Insert the effector gene of interest into the effector delivery vector using site-specific recombination cloning.
      NOTE: When monitoring subcellular localization of full-length effector protein, put the full-length gene into pBK-GW-1-2 or pBG-GW-1-2. It is also possible to choose pBK-GW-1-4 or pBG-GW-1-4 containing 2x sfGFP11 (11th β-strand of sfGFP) for increased fluorescence signal. In the case of a partial effector lacking a signal peptide, use pBK-GW-2-2 or pBK-GW-2-4.
2. Transform the plasmid carrying an effector fused to the sfGFP11 tag to P. syringae pv. Tomato (Pst) CUCPB5500 using standard electroporation.
   NOTE: Other Pseudomonas strains can be used if necessary. The sfGFP11 tag system is constructed for a broad range of vectors, and the gene expression of the effector is regulated by the AvrRpm1 promoter, which is comparable with, e.g., Pseudomonas fluorescens (EthAn).
3. Spread the transformed bacterial cells gently over the surface of the King’s B agar plates containing 100 µg/mL rifampicin and 25 µg/mL kanamycin or 25 µg/mL gentamycin. Incubate at 28 °C for 2 days.
4. Inoculate one colony into King’s B liquid media with antibiotics appropriate for the vector used, and grow the cells overnight at 28 °C with shaking at 200 rpm.
5. Make a glycerol stock. Add autoclaved glycerol to a final concentration of 50% and store at -80 °C.

3. Transient Expression of Organelle-targeted sfGFP1-10^OPT (1- 10^th β-strand of sfGFP) in N. benthamiana (4 Days)

1. Preparation of Agrobacterium culture
   1. Order the desired vector(s) of organelle-targeted sfGFP1-10^OPT plasmid(s) (refer to the Table of Materials).
   2. Transform the plasmid(s) into Agrobacterium tumefaciens strain GV3101 cells. Grow the cells on Luria-Bertani (LB) agar medium supplemented with 50 µg/mL kanamycin and 50 µg/mL rifampicin at 28 °C for 2 days.
   3. From a single colony on the LB agar medium, inoculate the cells into 5 mL of liquid LB media supplemented with 50 µg/mL kanamycin and 50 µg/mL rifampicin. Grow the cells overnight at 28 °C with shaking at 200 rpm.
   4. Harvest the cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant media and resuspend the pellet in 1 mL of freshly made infiltration buffer.
   5. Measure the quantity of Agrobacterium by obtaining the optical density (OD) value at an absorbance of 600 nm (Abs 600 nm). Adjust the OD₆₀₀ of the bacteria to 0.5 with infiltration buffer.
      NOTE: 1 mL of suspension is enough to infiltrate on two spots.
   6. Leave the culture at room temperature on a gentle rocker for 1 - 5 h before infiltration.
   7. Poke a hole into the center of the leaves to be infiltrated with a 10 µL tip. Use a 1-mL needleless syringe to infiltrate the Agrobacterium suspensions. Carefully and slowly inject about 500 µL of the suspensions prepared from step 3.1.5 into the leaf adaxial side via the syringe. Repeat the infiltration on at least three different plants for experimental replicates.
      NOTE: For health and safety reasons, eye protection should be worn during infiltration.
   8. Wipe off the remaining bacterial suspension on the leaves and mark the boundary of the infiltrated region.
   9. Keep the infiltrated plants under the same growth conditions used for step 1.1.1 for 2 days.

4. Inoculation of Pseudomonas (4 Days)

1. Streak the transformed Pseudomonas strain from the glycerol stock in step 2.5 on King’s B agar media with the appropriate antibiotics at 28 °C for 2 days.
   NOTE: The health of Pseudomonas is very critical. If colonies do not form well, streak the cells again or propagate the cells in the King’s liquid media prior to proceeding.
2. Inoculate a loopful of Pseudomonas cells in Mannitol-Glutamate (MG) liquid media at 28 °C with shaking at 200 rpm for overnight.
3. Harvest the cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant media, resuspend the pellet in 10 mM MgCl₂, and adjust the OD₆₀₀ to 0.02 (1 x 10⁷ cfu/mL) for N. benthamiana leaves and to 0.002 (1 x 10⁶ cfu/mL) for Arabidopsis leaves.
4. For the N. benthamiana, infiltrate the Pseudomonas suspension into the area of the leaves where the Agrobacterium carrying sfGFP1-10^OPT construct was infiltrated 2 days previously (as in step 3.1.7). For the sfGFP1-10^OPT transgenic Arabidopsis, infiltrate the Pseudomonas suspension into two 4-week-old short day-grown leaves.
   NOTE: At least three plants are needed for experimental replicates.

5. Observation of sfGFP Signal via Confocal Microscopy (1 Day)

1. Cut out the leaf disc from the Pseudomonas-inoculated leaves. At specific time points after infiltration of Pseudomonas, image two 2-cm² leaf discs from the single plant using a laser scanning confocal system with 40X/1.2 numerical aperture (NA) C-Apochromat water immersion objective or 63X/0.8 NA C-Apochromat oil immersion objective. To avoid dead cells killed by wounding, observe the cells away from the infiltration hole.
2. Start the observation at a low power setting of the 488-nm argon laser. Increase the laser power to detect sfGFP.
   NOTE: We usually use 2 - 15% of the laser intensity to detect the fluorescence signal. However, the laser power and detection settings should be adjusted based on the user’s microscopy system. Here, the emission filters were set to 520 - 550 nm. The dead cells often emit auto-fluorescence under the 488 nm laser excitation. Therefore, as a negative control, the same effector without the sfGFP11 tag should be infiltrated and observed under the same observation conditions. In addition, high laser excitation can induce chlorophyll autofluorescence. Therefore, adjust the laser intensity using the control plant cells so as not to induce chlorophyll autofluorescence.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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