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Alain Goossens and Laurens Pauwels (eds.), Jasmonate Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1011, DOI 10.1007/978-1-62703-414-2_16, © Springer Science+Business Media, LLC 2013

Chapter 16

Agroin fi ltration of Nicotiana benthamiana Leaves for Co-localization of Regulatory Proteins Involved in Jasmonate Signaling

Volkan Çevik and Kemal Kazan

Abstract

Protein–protein interactions play important roles in many cellular processes, including the regulation of phytohormone signaling pathways. Identi fi cation of interacting partners of key proteins involved in the cellular signaling control can provide potentially unexpected insights into the molecular events occurring in any signaling pathway. Over the years, various techniques have been developed to examine protein–protein interactions, but, besides certain advantages, most of them have various pitfalls, such as yielding nonspeci fi c interactions. Therefore, additional information obtained through different methods may be needed to substantiate protein–protein interaction data. One of these techniques involves the co-localization of proteins suspected to interact in the same subcellular compartment. In this chapter, we describe a method for co-expression of proteins associated with jasmonate signaling in Nicotiana benthamiana for studies such as co-localization.

Key words Nicotiana benthamiana , Jasmonate signaling , Mediator complex , Yellow fl uorescent protein (YFP) , Cyan fl uorescent protein (CFP) , Red fl uorescent protein (RFP) , Agrobacterium , Agroin fi ltration

Identi fi cation of interacting partner(s) of a protein can potentially reveal new insights into its function. A number of techniques are available to identify protein–protein interactions in plant cells. Some of these techniques, such as yeast two-hybrid (Y2H), tandem af fi nity puri fi cation, mass spectroscopy, and protein microarrays, are among the most commonly used and particularly suitable for high-throughput analyses [ 1, 2 ] . However, a positive protein–protein interaction detected with some of these techniques, particularly those operating in vitro, is only an indication of an interaction between the two proteins. In addition, as some methods are notorious

1 Introduction

200 Volkan Çevik and Kemal Kazan

in producing false positives (such as nonspeci fi c interactions), the data obtained should preferably be supported by independent analyses that employ alternative techniques, such as fl uorescence resonance energy transfer, co-immunoprecipitation, and bimolecular fl uorescence complementation (for a review, see ref. 3 ) . One of the methods that provides supporting evidence for in vitro protein–protein interaction analysis, such as Y2H, is protein co-localization in which the subcellular localizations are investigated of two assumed interacting proteins [ 4 ] . If candidate proteins co-localized in the same subcellular compartment, the reliability would be con fi rmed of the initial interaction detected before the functional studies had been undertaken. In addition, such studies could also provide information about the spatial and temporal distribution of the interacting proteins.

Brie fl y, in protein co-localization studies, proteins are fused to different fl uorescent tags cloned into different expression plasmids and introduced into plant cells through transformation mediated by Agrobacterium tumefaciens or microprojectiles. To monitor the intra-cellular localization of fl uorescently tagged proteins, transformed cells are examined under a confocal microscope. Because different fl uorescent tags display different emission maxima, the localization of these proteins can be detected individually under different wave-lengths. When two proteins are produced in the same subcellular com-partment, the fl uorescent tags should be co-localized as well [ 4, 5 ] .

In this chapter, we provide speci fi c details of this co-localization method by using selected regulatory proteins with relatively well-characterized roles in jasmonate (JA) signaling that regu-lates plant defense and development (for reviews, see refs. 6, 7 ) . The selected proteins are MYC2, ETHYLENE RESPONSE FACTOR 1 (ERF1), and OCTADECANOID-RESPONSIVE ARABIDOPSIS APETALA 2/ERF 59 (ORA59), all of which are key transcription factors regulating the JA signaling pathway [ 8– 13 ] and PHYTOCHROME FLOWERING TIME 1/MEDIATOR 25 (PFT1/MED25), a subunit of the plant Mediator complex [ 14– 16 ] implicated in JA signaling [ 17 ] . Previously, mutant analy-sis had suggested that PFT1/MED25 and the Mediator complex functioned as a bridge between DNA-bound transcription factors and RNA polymerase II transcriptional machinery in the JA signal-ing control [ 17, 18 ] . Recent Y2H and immunoprecipitation exper-iments have revealed physical interactions between the MED25 protein and ERF1, ORA59, and MYC2, suggesting that MED25 is required for the speci fi c functions of these key transcriptional regulators in the JA signaling [ 18 ] .

Here, we describe a method for co-expression in N. benthami-ana of proteins fused to fl uorescent tags for studying co-localiza-tion. We discuss (1) production of binary vectors for fusions to red fl uorescent protein (RFP) and cyan fl uorescent protein (CFP), (2) introduction into leaf epidermal cells of tobacco ( N. benthamiana )

201Agroinfi ltration of N. benthamiana

via Agrobacterium tumefaciens -mediated transformation, and (3) confocal microscopy of the transformed cells.

1. Laminar fl ow. 2. Shaker. 3. Sterile Petri plates. 4. 1-mL syringe without needle. 5. Laser scanning microscope (LSM710; Carl Zeiss, Jena, Germany)

and image analysis software, such as the ZEN software (Carl Zeiss) and ImageJ ( http://rsbweb.nih.gov/ij/ ) [ 19 ] .

1. Tobacco ( Nicotiana benthamiana ) seeds. 2. Gateway-compatible binary vectors pEarlygate102 [ 20 ] and

pVGWmRFP (V. Çevik, unpublished). 3. 35S:mRFP and 35S:CFP control vectors (V. Çevik,

unpublished).

1. 150 mM acetosyringone stock solution: 0.59 g of 4 ¢ -hydroxy-3 ¢ ,5 ¢ -dimethoxyacetophenone (acetosyringone; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 20 mL dimethyl sulfoxide (DMSO). Aliquot into 1.5-mL Eppendorf tubes and store at −20 °C.

2. Kanamycin (100 mg/mL) and gentamicin (25 mg/mL) stock solutions. Dissolve 1 g kanamycin sulfate (Sigma-Aldrich) or 0.25 g gentamicin sulfate (Sigma-Aldrich) in 10 mL sterile deionized water and fi lter-sterilize. Store in 1 mL aliquots at −20 °C.

3. Rifampicin stock solution (25 mg/mL). Dissolve 0.25 g rifam-picin (Sigma-Aldrich) in DMSO ( see Note 1 ). Store in 1 mL aliquots at −20 °C.

4. Agrobacterium liquid and solid growth medium: Dissolve 5 g yeast extract, 10 g NaCl, and 10 g peptone in a fi nal volume of 1 L deionized water and adjust pH to 7.2. Store the ster-ile liquid medium at room temperature, and add sterile anti-biotics (e.g., for Agrobacterium strain GV3101 (pMP90) add rifampicin, kanamycin ( see Note 2 ), and gentamicin to give a fi nal concentration of 50, 50, and 25 m g/mL, respec-tively). For solid growth medium, add 10 g of agar per liter of liquid medium and sterilize by autoclaving at 121 °C for 15 min. Store at room temperature and before use, melt the medium in a microwave oven, cool it to ~50–60°C, and add sterile antibiotics. Brie fl y and gently shake the media and

2 Materials

2.1 Equipment

2.2 Plant Material and Vectors

2.3 Media, Buffers, and Solutions

202 Volkan Çevik and Kemal Kazan

dispense (approximately 25 mL) into sterile Petri plates under sterile conditions (e.g., in a laminar fl ow hood) ( see Note 3).

5. YEB medium: 5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose, and 2 mM MgSO 4 .

6. In fi ltration medium: Filter-sterilized 10 mM of 2-( N -morpholino) ethanesulfonic acid (MES) (pH 5.6), 10 mM MgCl 2 , and 150 m M acetosyringone. The in fi ltration medium without acetosyringone can be stored at RT for sev-eral months. Add acetosyringone just before use.

1. Use seeds that are stored under suitable conditions for uniform germination.

2. Store dried seeds in a sealed container at low humidly at 4 °C. 3. Grow N. benthamiana plants from seeds in pots containing

compost or any other medium that supports plant growth in a greenhouse or a plant growth chamber under 16-h light pho-toperiod at 25–28 °C.

4. Keep the plants free from pests and pathogens by following hygienic growth conditions.

1. Grow E. coli and A. tumefaciens carrying binary vector plas-mids in liquid media by shaking at 37 °C overnight for E. coli and at 28 °C for 2 days (or until the culture becomes cloudy) for A. tumefaciens .

2. Add up to 50 % (w/v) glycerol and store at −80 °C. 3. When required, start a new culture by streaking the bacteria

onto a solid medium containing appropriate antibiotic(s). 4. Incubate the plates at 28 °C for A. tumefaciens and 37 °C for

E. coli cultures. 5. Pick up a single colony using a sterile toothpick and start a

liquid culture as described above.

For Agrobacterium -mediated transient expression of proteins in tobacco epidermal cells, fl uorescently tagged proteins should be cloned into binary expression vectors, such as those reported [ 20 ] , according to a molecular biology laboratory manual using stan-dard cloning procedures [ 21 ] .

1. Amplify the full-length coding sequences of your genes of interest from cDNA without stop codon, attaching Gateway-compatible sites according to the manufacturer.

3 Methods

3.1 Plant Culture

3.2 Agrobacterium and E. coli Cultures

3.3 Vector Construction

203Agroinfi ltration of N. benthamiana

2. Subclone into the binary vector, e.g., pEarleyGate102 [ 22 ] for C-terminal fusions to CFP or, e.g., pVGWmFR for C-terminal fusions to mRFP ( see Note 4 ).

Agrobacterium can be transformed by a variety of methods as described [ 23 ] .

1. Dilute an overnight culture of Agrobacterium in YEB medium containing the appropriate antibiotics.

2. Grow Agrobacterium at 28 °C by shaking until the culture reaches an OD 550 value of 0.5–0.8.

3. Pellet the cells by centrifugation at 3,000 × g for 20 min at room temperature.

4. Wash the pellet with sterile distilled water and resuspend it in a smaller volume of YEB (1/10th of the initial volume of YEB used to grow the bacteria should be suf fi cient for this purpose).

5. Freeze 0.2-mL aliquots in liquid nitrogen. 6. Store at −80 °C. 7. For transformation, thaw the cells on ice. 8. Add the puri fi ed vector DNA through miniprep. 9. Incubate on ice for 5 min. 10. Transfer the mixture into liquid nitrogen for 5 min. 11. Incubate at 37 °C for 5 min. 12. Add 1 mL of liquid growth medium into the tube. 13. Shake at 28 °C for 2–4 h. 14. Working in a laminar fl ow, spread small aliquots of the culture

into Petri plates containing the appropriate antibiotics. 15. Incubate the plates in a laminar fl ow until they are completely

dry. 16. Incubate in the dark at 28 °C for 2 days or until distinct colo-

nies emerge. 17. Grow a small culture from one of the colonies. 18. Con fi rm the presence of the vector either by polymerase chain

reaction or restriction digest of miniprep DNA. 19. Prepare the glycerol stock of the con fi rmed culture. 20. Keep at −80 °C, until further use.

1. Grow each Agrobacterium strain harboring a binary vector in YEB and liquid growth media by shaking (220 rpm) at 28 °C for 24 h. Grow next to the mRFP- and CFP-tagged proteins of interest also Agrobacteria containing the 35S:mRFP and 35S:CFP control vectors.

3.4 Transformation of Agrobacterium

3.5 Transient Expression in N. benthamiana Epidermal Cells

204 Volkan Çevik and Kemal Kazan

2. Harvest the cells by centrifugation at 3,000 × g for 20 min at 20 °C.

3. Resuspend the bacterial pellet in the in fi ltration medium. 4. Adjust the bacterial density to give a fi nal OD 550 of 0.1–0.2. 5. Mix equal volumes of Agrobacterium cultures containing

mRFP and CFP fusions. 6. Prepare control mixes containing the 35S:CFP or 35S:mRFP

in addition to mRFP or CFP fusions, respectively. 7. In fi ltrate into leaves of approximately 4-week-old N. bentha-

miana plants with a needless syringe (e.g., 1 mL). 8. Carefully and slowly inject the bacteria into the abaxial side

(backside) of the leaf ( see Note 5 ). 9. Label in fi ltrated leaf with mixture composition. Multiple leaves

can be in fi ltrated with different mixtures on one plant.

1. Prepare samples 2–3 days after in fi ltration with Agrobacterium . Cut approximately 0.25 cm 2 leaf sample from the in fi ltrated area avoiding the veins and place the leaf sample onto a micro-scope slide. Add a drop of distilled water onto the leaf sample and then place the cover slide. Make sure that the abaxial side of the leaf sample faces the microscope objective.

2. Microscope settings are dependent on the laser scanning con-focal microscopy being used. Laser power, gain, and pinhole sizes should be adjusted. Generally, 40× or 60× objective lens is used to resolve individual cell nuclei. Lasers at 458 nm and 543 nm are used to excite CFP and mRFP, respectively.

3. Images from multiple epidermal cells coexpressing both pro-teins should be examined. In addition, the proportion of the cells showing co-localization pattern should be reported ( see Note 6 ).

In many cases, co-localization alone is not a suf fi cient evidence for protein–protein interaction and therefore one should be cautious about the interpretation of results from these analyses alone. In addi-tion, potential pitfalls associated with image acquisition techniques have been identi fi ed. We therefore recommend various guidelines published elsewhere (e.g., 24, 25 ) be consulted to fi nd out what these pitfalls are and how they can affect the interpretation of the data.

2–3 days after in fi ltration, images (Fig. 1 ) are obtained with a laser scanning microscope and processed with the ZEN software and ImageJ [ 19 ] . MED25 co-localizes to the nucleus of N. ben-thamiana cells, when co-expressed with the transcription factors ERF1, MYC2, and ORA59 (Fig. 1 , see Note 7 ). In control exper-iments, co-expression of MED25-RFP with CFP or MYC2-CFP or ERF1-CFP with RFP did not induce speckle formation (Fig. 2 ), suggesting that speckle formation requires co-expression of MED25 and the interacting transcription factors.

3.6 Visualization of Co-localization with Confocal Microscopy

205Agroinfi ltration of N. benthamiana

MergedMED25-RFP

MED25-RFP Merged

MED25-RFP Merged

ERF1-CFP

MYC2-CFP

ORA59-CFP

MED25-RFP Bright field MergedNu

Cy

a

b

c

d

Fig. 1 Co-localization of MED25-RFP to the nucleus and cytoplasm of N. benthamiana cells, forming nuclear speckles when co-expressed with ERF1-CFP , ORA59-CFP , and MYC2-CFP . Confocal images were taken 48 h after Agrobacterium -mediated transient expression in the epidermal cells of N. benthamiana leaves. The expression of MED25-RFP was seen both in the nucleus and in the cytoplasm when transformed on its own ( a ). Nuclear speckles were observed when N. benthamiana leaves were co-transformed with MYC2-CFP with MED25-RFP ( b ), ERF1-CFP with MED25-RFP ( c ), and ORA59-CFP with MED25-RFP ( d ). Cy, cytoplasm; Nu, nucleus. Bars = 5 m m

206 Volkan Çevik and Kemal Kazan

1. Filter sterilization is not required when antibiotics (for instance, rifampicin) are dissolved in solvents, such as EtOH or DMSO, before use.

2. For example, for the Agrobacterium strain GV3101 (pMP90), add rifampicin, kanamycin, and gentamicin to give a fi nal con-centration of 50, 50, and 25 m g/mL, respectively. Here, kana-mycin was used in the selection, because the binary vectors confer kanamycin resistance.

3. Plates can be stored at 4 °C, wrapped in aluminum foil for up to 2 weeks.

4. The fl uorescent tags can be fused to either the N-terminus or the C-terminus of the protein of which the co-localization is to be examined. Here, MED25 and the transcription factor

4 Notes

CFP

mRFPMYC2-CFP

ERF1-CFP

MergedMED25-RFP

Merged

mRFP Merged

a

b

c

Fig. 2 Confocal images of control co-expression experiments of MED25-RFP with CFP ( a ) and of MYC2-CFP ( b ) or ERF1-CFP ( c ) with RFP. Bars = 5 m m

207Agroinfi ltration of N. benthamiana

proteins were C-terminally tagged with the fl uorescent proteins, but similar co-localization patterns are observed when they are N-terminally tagged. The decision on which terminus to fuse the candidate protein with a fl uorescent tag depends on whether the fusion could affect the stability and/or activity of the candidate protein; for instance, if the stability of the protein is affected by the C-terminus fusion, then the N-terminus fusion or vice versa can be tested. It is also possible that a functional fusion protein cannot be pro-duced for some proteins.

5. Although tobacco cells are larger and the transient expression is more ef fi cient, Arabidopsis epidermal cells from cotyledons and root cells have also been reported as a suitable alternative [ 5, 26 ] . In addition, some Arabidopsis fusion proteins are seemingly not expressed in N. benthamiana and, therefore, the use of Arabidopsis cells is required. Epidermal cells of onion ( Allium cepa ) and carrot ( Daucus carota ) could be utilized as material for transient co-localization studies as well.

6. In some instances, MED25-RFP protein was also found in the cytoplasm (Fig. 1 ). Transient over-expression and/or tagging of proteins may sometimes alter their correct subcellular local-ization. Weaker promoters may be tested in transient expres-sion studies if artifacts are observed. Again, fl uorescent tags fused to the N-terminus or the C-terminus of the protein may be tested in order to eliminate potential artifacts.

7. Nuclear speckles are known to serve as hubs of enhanced mRNA metabolic activity and/or involved in active transcrip-tion sites ( see ref. 27 for a review).

Acknowledgment

We thank Brendan Kidd for useful discussions.

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