In vivo analysis of plant intron splicing



Summary:

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throughout the text: all the µ in µl (u in ul) got lost, the final version should be a pdf to avoid this problem, I did not mark this individually

Title: In vivo analysis of plant intron splicing.

Craig G. Simpson1, Michele Liney1, Diane Davidson1, Dominika Lewandowska1, Maria Kalyna3, Sean Chapman4, Andrea Barta3 and John WS Brown1,2

1Genetics Programme, Scottish Crop Research Institute, Dundee DD2 5DA, Scotland, UK; 2Division of Plant Sciences, University of Dundee at SCRI, Dundee DD2 5DA, Scotland, UK; 3Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna, Austria; 4Plant Pathology Programme, Crop Research Institute, Dundee DD2 5DA, Scotland

Address correspondence to: Craig G. Simpson, Genetics Programme, Scottish Crop Research Institute, Dundee DD2 5DA, Scotland, UK. E-mail: craig.simpson@scri.ac.uk.

1. Abstract

The characterisation of plant intron signals required for efficient splicing has relied on in vivo splicing analyses due to the lack of a plant in vitro splicing extract. Different in vivo systems and a small number of particular introns have been utilised to determine the contribution that intron splicing signals make to efficient plant splicing and to allow comparisons both among the main plant families and to other organisms such as human and yeast. In addition, in vivo studies have addressed intron enhancement of expression, the roles of UA-rich sequences in plant introns and the functions of trans-acting factors. We describe protocols for analysing splicing behaviour using either plant protoplast or agroinfiltration systems.

Keywords: splicing reporter, protoplasts, transfection, transient splicing assays, agroinfiltration, RNA binding proteins

2. Theoretical background

2.1 Plant splicing analysis in vivo

Tremendous progress in our understanding of eukaryotic splicing has been made through the use of human nuclear or yeast whole cell extracts that are splicing competent (see chapter 26 for in vitro protocol and chapter 5 for a theoretical introduction). Such an in vitro splicing system has not been available for plants despite valiant numerous? attempts by different labs. Rem: people also tried mammalian tissue-specific splicing, which never worked, on can add a brain extract to hela extract and it sort of splices, do you do the same in plants?) In the absence of plant nuclear or whole cell extracts that support in vitro splicing, detailed analysis of plant splicing has progressed through the development of in vivo splicing analysis systems. Transcriptional assays in plants have commonly used transfection of plant protoplasts (plant cells stripped of their cell walls) as a rapid tool for promoter analysis. For analysing plant intron splicing, protoplast transfection of different intron constructs has been invaluable in defining intron splicing signals and features which determine the accuracy and efficiency of plant splicing (Goodall and Filipowicz, 1989, 1991; Simpson et al., 1996, 2000, 2002; Waigmann and Barta, 1992).

2.2 Splicing of plant and animal introns in reciprocal systems

Experiments to examine whether animal introns are spliced in plants and plant introns in animal splicing extracts gave variable results (Brown et al., 1986; Hartmuth and Barta, 1986; van Santen and Spritz, 1987). Naturally-occurring plant introns from a variety of plant species (wheat, oat, pea and soybean) and synthetic intron constructs have been accurately and efficiently spliced in HeLa cell in vitro splicing extracts, while other plant introns were spliced inefficiently or not at all in this system. The variation in splicing is likely to reflect the degree of similarity of the plant intron sequences to the requirements of the animal system (e.g. polypyrimidine tract sequence found near the 3’ splice site). On the other hand, with very few exceptions, transcripts containing animal introns have not been spliced when introduced into plant cells (Barta et al., 1986), again most likely reflecting the requirements of the plant splicing machinery for high UA content (>59%) in introns. In addition to plant/animal splicing differences, there are also differences in splicing efficiency between the two main branches of flowering plants (angiosperms): monocotyledonous (single seed leaf) and dicotyledonous (two seed leaves). Monocotyledons have a lower requirement for UA content of introns than dicotyledons and are therefore dicots are more restrictive in the introns which they can splice – for example, some monocotyledonous introns have been poorly spliced in dicotyledonous cells. Thus despite the many similarities in splicing signals and splicing factors found in plants and animals, there are clearly differences in splicing and in vivo splicing systems have been an essential development. We describe the construction of exemplar splicing reporters and protocols for two plant protoplast systems and a system based on agroinfiltration.

2.3 Plant splicing reporter constructs

A basic splicing reporter construct (pDH515) to study intron splicing was made by cloning an intronless zein storage protein gene behind the Cauliflower Mosaic Virus (CaMV) 35S RNA promoter and followed by the CaMV terminator sequence (Figure 1A). A restriction site was introduced into the zein gene to allow intron sequences (with short flanking exons) to be introduced. The advantage of using the zein coding region was that as a maize storage protein gene it was highly unlikely that a similar sequence would be present in the dicotyledonous cell systems used for protoplast production. Thus, primers to zein sequences flanking the inserted intron(s) could be used to specifically amplify pre-mRNAs and spliced mRNAs from the intron construct.

A sensitive splicing reporter for detailed studies of splicing signal sequences was based on a potato invertase mini-exon sequence. Mini-exons have requirements for strong or additional splicing signals to ensure that they are recognised and spliced into an mRNA. The GF invertase gene from potato (Acc #: AJ133765) consists of 6 exons and 5 introns. Exon 2 is a short 9 nt exon that is spliced by default into the final message (Simpson et el., 1996). Part of the GF invertase gene consisting of 50 nt of exon 1, intron 1 (219 nt), the 9nt mini-exon 2, intron 2 (108 nt) and 70nt of exon 3 was inserted into the unique BamHI site in the zein gene of expression vector pDH515 (Simpson et al., 1996, Simpson et al., 2000) (Figure 1B). This construct (inv1) has been used to generate a series of mutations in the splicing signals (Simpson et al., 2000, 2002) allowing it to be used to report on both splicing activation and repression.

Finally, the mini-exon system has also been used to generate a GFP-based splicing reporter to visualise changes in splicing behaviour. The first 9 nt of the 3’ end of exon 1, intron 1 (219 nt), the 9 nt mini-exon 2, intron 2 (108 nt) and 9nt of the 5’ end of exon 3 were fused to the 5’ end of the mGFP5 gene (Siemering et al., 1996). The exon 1 sequence was modified to include a translation initiation codon and the mini-exon 2 was modified to include an in-frame stop codon (Figure 1C). Skipping of the mini-exon would produce mGFP5 protein with an N-terminal extension of seven amino acids, while inclusion of the mini-exon would result in premature termination and expression of a five amino acid peptide or to degradation by the nonsense-mediated decay pathway. Can you give this construct a name?

Craig, John can you please insert the blue marked constructs into the reagent database:



user: superadmin

pw:golgi

 

the idea behind this is to collect reagents for Eurasnet, but also later make this reagents available for users (distributed by Dundee Cell products). Any user will be able to export data he wants to make available to a site there. However, at least internally, we should be able to see and exchange all the reagents.

2.4 Expression of trans-acting factors

The Arabidopsis genome encodes more than 200 proteins that contain recognised RNA binding domains. About half of these are highly conserved factors known to be involved in RNA processing events, in other eukaryotes, but the other half are plant-specific and most are of unknown function (Lorković, 2009). For example, plants contain many more SR protein genes than humans – some are orthologues of the human proteins while others are plant-specific (Kalyna and Barta, 2004). Similarly, Arabidopsis contains three genes with similarity to the human polypyrimidine tract binding protein (PTB), a negative regulator of splicing. One of the Arabidopsis PTB genes contains four RNA binding domains (RRM) and has the highest identity to human PTB while the remaining two are unique to plants. In vivo splicing reporter systems can be used to examine whether particular RNA-binding proteins influence splicing. This is achieved by over-expression of genes or cDNAs of RNA binding proteins from plant expression vectors usually driven by the CaMV 35S RNA promoter. These constructs are co-transfected or co-inoculated with splicing reporter constructs into plant cells. The inclusion of epitope tags allows monitoring of the expression of the RNA-binding protein or splicing factor from the plasmids.

3. Protocols

Transfection of plasmid DNA into plant protoplasts

Plant cells, unlike metazoan cells, have a large vacuole that makes up 80-90% of the cell volume and has an important role in cell shape maintenance (Oda et al., 2009). During protoplast preparation, the plant cell wall is removed and cells lose their shape and form spherical protoplasts that are susceptible to disruption by osmosis. Complex plant media that contain different salts and the plasmolysing agent mannitol are essential to maintain protoplasts and allow them to continue to function. We describe protocols for use of protoplasts from different sources of plant material: 1) tobacco leaves and 2) Arabidopsis cell cultures. When would you use tabacco vs Arabidopsis?

3.1 Protocol 1 Transfection of tobacco leaf protoplasts

The protocol described in detail below is based on that of Guerineau et al. (1988).

3.1.1 Solutions

To- (minus) = Solution To lacking sucrose, FeSO4 and Na2EDTA.

To+ (plus) = Solution To containing 0.02% Tween 20 or 80.

To- and To+ are made up from a number of different stock solutions:

Solution 1 10X

10.3 mM NH4NO3 8.25 g

9.4 mM KNO3 9.5 g

1.5 mM CaCl2.2H2O 2.2 g

0.75 mM MgSO4.7H2O 1.85 g

0.62 mM KH2PO4 0.85 g

Make up 200 ml

Solution 2 (for To+ only) 10X

100 μM FeSO4 0.278 g

100 μM Na2EDTA 0.372 g

Make up 200 ml

Solution 3 200X

16 μM H3BO3 200 mg

0.6 μM MnSO4 200 mg

3.5 μM ZnSO4.7H2O 20 mg

0.12 μM CuSO4.5H2O 6 mg

0.22 μM AlCl3 6 mg

0.13 μM NiCl2.6H2O 6 mg

0.06 μM KI 1 mg

Make up 200 ml

Solution 4 10X

555 μM myo-Inositol 1000 mg

3 μM Thiamine 10 mg

5 μM Pyridoxine 10 mg

8 μM Nicotinic acid (Niacin) 10 mg

2 μM Calcium Pantothenate 10 mg

0.04 μM Biotin 0.1 mg

(Make separate stock of biotin at 10 mg/ml dissolving first in 2 drops of 1 M NaOH. Add 100 μl)

Make up 10 ml

Other stock solutions

16.1 μM NAA

Make up 10 ml of a 3 mg/ml solution dissolving in 50% ethanol.

4.4 μM BAP

Make up 10 ml of a 1 mg/ml solution. Dissolve first in 2 drops 1M NaOH.

Solution To-

To prepare 200 ml of To- solution add the following stock solutions:

4 ml Solution 1

0.2 ml Solution 3

0.2 ml Solution 4

0.2 ml NAA (3 mg/ml)

0.2 ml BAP (1 mg/ml)

16 g Mannitol

pH 5.5 with NaOH

Make up to 200 ml with sterile, distilled water

Filter sterilise

Solution To+

4 ml Solution 1

4 ml Solution 2

0.2 ml Solution 3

0.2 ml Solution 4

0.2 ml NAA (3 mg/ml)

0.2 ml BAP (1 mg/ml)

16 g Mannitol

4 g Sucrose

40 μl Tween 20

pH 5.5 with NaOH

Make up to 200 ml with sterile, distilled water

Filter sterilise

Protoplasting enzyme solution for 100 ml

1 mg/ml Cellulase 100 mg

0.5 mg/ml Driselase 50 mg

0.2 mg/ml Macerozyme 20 mg

Suspend in To-

Filter sterilise

16% (w/v) Sucrose

16 g sucrose in 100 ml water.

Autoclave.

PEG Solution for 10 ml

25% PEG 8000 2.5 g

0.1 M Ca(NO3)2 0.24 g

0.45 M Mannitol 0.82 g

10 mM MES 1 ml of 0.1 M MES pH6

pH 6

Filter sterilise

Calcium Nitrate Solution for 100 ml

0.275 M Ca(NO3)2 6.5 g

10 mM MES 10 ml 0.1 M MES pH 6

Autoclave

3.1.2 Preparation of tobacco leaf protoplasts

1. Select young, fully expanded leaves of tobacco (Nicotiana tabacum var. Xanthii) from plants grown in a controlled environment chamber at 20ºC in a 16h light/8h dark regime.

2. Avoid damaged or infested leaves; harvest 2 leaves for 4 transfection assays.

3. Sterilise leaves by soaking in 7% Domestos for 10 min at room temperature (RT).

4. Remove bleach and wash 4X in sterile water (~400 ml/wash).

5. Dry leaves by blotting gently with absorbent paper.

6. Peel the lower epidermis of the leaf off with a pair of fine forceps and place the leaves with exposed areas downwards onto 15 ml of enzyme solution in a 9 cm petri dish. Fill two dishes with peeled leaf material.

7. Cover in aluminium foil and incubate overnight at 25ºC.

8. Released protoplasts fall to the bottom of the petri dish; pipette protoplasts onto a sterile 100 µm sieve (use sterile disposable pipettes).

9. Transfer filtered protoplasts into 2x sterile 10 ml plastic round bottom tubes (round bottom tubes used throughout the protocol) and centrifuged at low speed (32xg) for 5 min at RT.

10. Remove supernatant and resuspend protoplasts in 10 ml To- and divide into 2x10 ml tubes.

11. Prepare 4x10 ml tubes with 2.5 ml 16% sucrose and gently layer 5 ml of protoplasts onto the sucrose solution.

12. Centrifuge samples at 130xg for 5 min at RT.

13. Protoplasts band at the sucrose/protoplast media interface and are collected with a glass pipette and transferred to two new 10 ml tubes and suspended in 10 ml of To-.

14. The number of protoplasts is determined by haemocytometer.

15. Centrifuge protoplast suspension at 32xg for 5 min at RT and the supernatant removed.

16. Resuspend protoplasts in To- to a concentration of approximately 1x106 protoplasts/ml. (This should give a dark green colour in a volume of around 2 ml).

3.1.3 PEG-mediated transfection of tobacco protoplasts

1. Precipitate up to 30 μg plasmid DNA with 1/16 volume 5M NaCl and 2.5 volumes of ethanol. For co-transfection of two plasmids (e.g. splicing reporter and protein factor), 30 µg of each plasmid (60 µg total) are precipitated.

2. Resuspend plasmid DNA pellet in 20 μl water.

3. Aliquot 200 μl protoplasts (approximately 200,000 protoplasts) per assay into a 10 ml round bottom tubes (each experiment usually consisted of 8 assays).

4. Add plasmid DNA and mix gently.

5. Add 200 μl PEG solution drop-wise, mixing gently while adding and leave to stand for 20 min.

(Note: There is enough time to do 8 assays comfortably in a 20 min period – time each assay accurately).

6. Add 4x 200 μl Calcium Nitrate solution very slowly and mix carefully.

7. Finally, add 4 ml Calcium Nitrate solution (giving a final volume of 5 ml) and leave to stand for 20 min.

(Note: 4 assays can be done in a 20 min period so a second set of assays can be done while the first is left to stand).

8. Centrifuge assays at 32xg for 3 min at RT.

9. Remove supernatant and resuspend protoplasts in 5 ml To+ by gentle shaking.

10. Pour the protoplasts gently into a small petri dish (3 cm diameter) and seal with Nescofilm.

11. Incubate at 25ºC for 24 hrs under light.

12. Collect protoplasts by centrifugation at 32xg for 5 min at RT.

13. Remove supernatant and transfer protoplasts to microfuge tube with a glass pipette and centrifuge at 110xg for 1 min.

14. Remove as much supernatant as possible and flash freeze protoplasts in liquid nitrogen.

15. Store at -80ºC until RNA extraction.

3.2 Protocol 2 Transfection of Arabidopsis cell suspension protoplasts

3.2.1 Solutions

Growth medium for Arabidopsis cell suspension for 1 liter

Murashige and Skoog medium including Gamborg B5 vitamins (Duchefa) 4.4 g

87 mM Sucrose 30 g

4.5 µM 2,4-D (10 mg/ml) 100 µl

pH 5.8 with KOH

Filter sterilise.

Store at 4ºC

Make separate stock of 2.4-Dichlorophenoxyacetic acid (2.4-D) at 10 mg/ml dissolving in dimethyl sulfoxide. Store aliquotes of 100 µl at -20ºC.

B5-0.28 M Sucrose Solution for 1 liter

Gamborg B5 medium including vitamins (Duchefa) 3.18 g

0.28 M Sucrose 95.76 g

pH 5.5 with KOH

Filter sterilise

Store at 4ºC

B5-0.34 M Glucose Mannitol (GM) Solution for 1 liter

Gamborg B5 medium including vitamins (Duchefa) 3.18 g

0.17 M Glucose 30.5 g

0.17 M Mannitol 30.5 g

4.5 µM 2.4-D (10 mg/ml) 100 µl

pH to 5.5 with KOH

Filter sterilize

Store at 4ºC

Protoplasting Enzyme Solution for 100 ml

1% Cellulase (Duchefa) 1 g

0.2% Macerozyme (Duchefa) 0.2 g

Make up to 100 ml with B5-0.34 M GM Solution

Stir slowly for 30 min. Filter through Whatmann paper.

Filter sterilise

Store aliquots of 25 ml at -20ºC

PEG Solution for 10 ml

30% PEG 6000 3 g

0.1 M Ca(NO3)2. 4H2O 0.24 g

0.45 M Mannitol 0.82 g

pH 9.0 with KOH (adjust pH carefully, may take several hours)

Filter sterilise

Store at -20ºC

Calcium Nitrate Solution for 100 ml

0.275 M Ca(NO3)2. 4H2O 6.5 g

Filter sterilize

Store at room temperature

3.2.2 Preparation of protoplasts from Arabidopsis cell cultures

1. Grow Arabidopsis cell suspension cultures in 40 ml of the growth medium in 250 ml flasks at 23°C in the dark with shaking at 200 rpm (Model G25 Incubator Shaker - New Brunswick Scientific Co. Inc). Dilute the cells every 7 days into 3 parts of growth medium.

2. Transfer 40 ml of a 5 days post-subculture Arabidopsis cell suspension into a sterile 50 ml centrifuge tube and spin at 1500 rpm (Heraeus Megafuge 1.OR) for 5 min at RT.

3. Remove supernatant, add 25 ml of enzyme solution and fill up to 50 ml (total volume) with B5-0.34 M GM solution.

4. Resuspend the cells and transfer 25 ml to each of two large petri dishes (15 cm diameter).

5. Incubate the plates in an incubator shaker at 50 rpm for 3-4 h at 25°C. Check the cells every hour during incubation.

6. Filter the cells through a sterile 100 µm sieve, collecting the filtrate into 50 ml centrifuge tubes.

7. Spin at 1500 rpm for 5 min and discard the supernatant.

8. Resuspend the pellets in 25 ml of B5-0.28 M Sucrose solution and transfer each into two 14 ml round-bottom tubes.

9. Spin at 800 rpm for 7 min. The protoplasts should float on the top of the solution - if not, increase the centrifugation time.

10. Collect the floating protoplasts into a new 14 ml tube, add B5-0.28 M Sucrose solution to fill the tubes.

11. Spin at 1000 rpm for 6 min.

12. Collect all protoplasts into one 14 ml tube, count the protoplast density using a haemocytometer.

13. Resuspend protoplasts in B5-0.28 M Sucrose solution to a density of 1 x 106 protoplasts/ml.

3.2.3 PEG-mediated transfection of Arabidopsis protoplasts

1. Put 5 µl (up to a maximum of 15 µg) of plasmid DNA in 15 µl of water per assay in a microfuge tube.

2. Add 2 x 105 protoplasts in 50 µl per tube, mix protoplasts and DNA gently.

3. Immediately add 150 µl of PEG solution. Mix well.

4. Incubate 15 min at RT in the dark.

5. Wash PEG solution by adding Calcium Nitrate solution in two steps of 0.5 ml.

6. Mix by inverting.

7. Spin 7 min 800 rpm.

8. Remove supernatant.

9. Resuspend in 0.5 ml of B5-0.34 M GM.

10. Incubate protoplasts in tubes laid on the side in the dark at 25°C for 12-24 h.

11. Collect protoplasts by centrifugation for 5 min at 1500 rpm.

12. Remove all supernatant and freeze protoplasts in liquid nitrogen.

13. Store at -80°C until RNA extraction.

3.3 Protocol 3 – Agrobacterium mediated infiltration of Nicotiana benthamiana leaves

Infiltration of Agrobacterium containing expression plasmids into leaves of N. benthamiana provides a second rapid method of expression of splicing reporters and trans-acting factors for splicing analysis. The Agrobacterium-mediated infiltration method is widely used in RNA silencing assays and the method described here is adapted from Voinnet et al. (2003). The splicing reporter is based on the invertase mini-exon construct fused to GFP (Figure 1C) such that splicing can be detected on the basis of fluorescence and by RT-PCR of RNA extracted from the infiltrated region. For expression of proteins, coding sequences are PCR amplified with gene-specific primers that also introduce a 5’ AscI site and a 3’ NotI site. Amplification products were cloned in to pGEM-T Easy (Invitrogen) and then recovered by digestion with AscI and NotI prior to cloning into pGRAB, a derivative of the binary not clear to me what binary means in this context pGreen II 0229 (Hellens et al., 2000). The plasmid pGRAB contains the 35S promoter and terminator cassette from a derivative of pRTL2 (Carrington and Freed, 1990) with a modified multiple cloning site, containing AscI and NotI sites, inserted between the TEV leader sequence and 35S terminator sequence.

3.3.1 Solutions

LBG medium.

10g/l SELECT Peptone 140 (Invitrogen)

5g/l SELECT Yeast Extract

5g/l NaCl

1g/l glucose

Make up in distilled water, adjust pH to 7.5 with 5M NaOH and autoclave to sterilize.

Rifampicin 10mg/ml stock.

Dissolve 250mg of rifampicin in 25ml of methanol. Store at -20°C.

Kanamycin 50mg/ml stock.

Dissolve 1g of kanamycin sulphate in 20ml of distilled water, filter sterilize and store at 4°C.

10% glycerol.

Dilute 100ml of glycerol to 1l with distilled water and autoclave to sterilize.

LB agar.

10g/l SELECT Peptone 140 (Invitrogen)

5g/l SELECT Yeast Extract

10g/l NaCl

15g/l SELECT Agar

Make up in distilled water, adjust pH to 7.5 with 5M NaOH and autoclave to sterilize. Cool molten media to 60°C prior to addition of antibiotics. Pour about 25ml aliquots in to sterile, 90mm, Petri dishes. When plates are set, store at 4°C prior to use.

Infiltration medium.

0.5ml 1M MgCl2 (from a filter sterilized stock)

0.5ml 1M MES buffer (from a stock adjusted to pH 5.6 with KOH and filter sterilized)

0.75ml 10mM acetosyringone (3’, 5’-Dimethoxy-4’-hydroxy-acetophenone) (from a stock dissolved in ethanol and stored at -20°C)

48.75ml sterile distilled water

Prepare on day of use and protect from light.

3.3.2 Preparation and transformation of Agrobacterium electro-competent cells

1. Pick a fresh colony of Agrobacterium, e.g. strain AGL1, into 5ml of LBG medium supplemented with rifampicin (50µg/ml) and grow up at 28°C in a shaking incubator until the culture attains an OD600 of more than 1.0.

2. Inoculate the 5ml culture in to 500ml of LBG medium and grow up at 28°C in a shaking incubator until the culture reaches mid-log phase with an OD600 of between 0.5 and 1.0.

3. Chill the bacterial culture on ice for 30min, prior to collecting the cells through centrifugation at 4°C for 15min at 4000xg.

4. Gently resuspend the bacterial pellet in 240ml of ice-cold 10% glycerol before collecting the cells again through centrifugation.

5. Repeat the resuspension in 10% glycerol three times more, progressively reducing the resuspension volume, i.e. resuspend in 120ml, 60ml and then 30ml.

6. Finally collect the cells by centrifugation and resuspend in a small volume, about 0.5ml, of 10% glycerol to produce a bacterial slurry. Use the cells immediately or snap-freeze in liquid nitrogen and store at -80°C, thawing on ice before use.

7. Add 1µl of plasmid (100μg/ml) to 50µl cells and incubate on ice for 1min.

8. Transfer the cells to pre-chilled electroporation cuvettes with a 2mm inter-electrode gap and electroporate at 2800V in an EC100 electroporator (E-C Apparatus Corporation) or similar.

9. Immediately add 1ml of LBG medium, pipette up and down to mix and transfer to a 2ml micro-centrifuge tube.

10. Incubate at 28°C for 1h in a shaking incubator.

11. Plate 100µl and 10µl aliquots of the transformations on to LB agar plates supplemented with rifampicin (50µg/ml) and kanamycin (50µg/ml) to select for binary-containing Agrobacterium.

12. Seal plates with Nesco film ( what is necsco film) and incubate at 28°C for 2 days until colonies appear.

13. Confirm binary not clear to me what binary means in this context transformation through colony PCR.

3.3.3 Preparation of cultures for infiltration

1. Pick fresh colonies of transformed Agrobacterium into 5ml of LBG medium supplemented with rifampicin (50µg/ml) and kanamycin (50µg/ml). Grow up at 28°C in a shaking incubator for 24h to 48h until cultures reach stationary phase.

2. Collect bacterial cells by centrifugation of the cultures at room temperature for fifteen minutes at 1800g.

3. Resuspend the cells in 5ml of infiltration medium and incubate at RT in the dark for 2h.

4. After 2h, make a 1 in 10 dilution of each resuspension and measure the OD600 using infiltration media as a blank.

5. Calculate the OD600 of the undiluted resuspensions. Prepare mixtures of different resuspensions, e.g. Agrobacterium transformed with empty binary, binaries for expression of RNA binding proteins, the splicing reporter construct or GFP, with infiltration buffer so that the final OD600 of individual components is 0.1. not clear to me what binary means in this context, do you mean duplicate experiments?

3.3.4 Agroinfiltration

1. Nicotiana benthamiana plants were grown from seed with a 16h light-period at 26°C and a 8h dark-period at 22°C. The inocula were delivered to young, expanded leaves of circa six-week-old plants beyond nine-leaf stage.

2. Make a small wound on the underside of a leaf using a 23G hypodermic needle to facilitate entry of the bacterial suspension.

3. Fill a 1ml syringe with one of the infiltration mixtures and infiltrate an area of about 1cm2 by placing the end of the syringe (without a needle attached!) over the puncture site, applying gentle finger pressure on the upper side of the leaf above the puncture site and slowly infiltrating the liquid in to air spaces within the leaf.

4. Highlight the infiltrated area gently with a black marker pen.

5. Leave for 3 - 4 days in glasshouse before viewing infiltrated area for GFP fluorescence.

6. Excise the infiltrated area for RNA extraction and RT-PCR analysis of splicing.

3.4 Analysing the results of in vivo splicing analysis.

Below are short summaries of the methods used to analyse in vivo splicing. These methods are commonly used or are described in detail elsewhere in this manual.

3.4.1 RNA extraction

1. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen Cat No. 74904) following the manufacturer’s instructions (see Qiagen RNeasy manual). To aid RNA extraction from the agroinfiltrated Nicotiana benthamiana leaf discs, a little acid purified sand was added to improve grinding of the plant material. (how much is a little?)

3.4.2 RT-PCR analysis

1. RT-PCR analysis was performed essentially as described in the chapter “Monitoring changes in plant alternative splicing events 18b” with the exception that a single pair of primers was used for all splicing analysis.

2. Primers for analysis of the zein-based splicing reporters (Figure 1A and 1B) were designed to zein flanking sequences and were: O8F 5'- CCCAATTGTTCAACCCTAC labelled with the 5' fluorescent phosphoramidite 6-FAM and O9, 5' - GGTAAGATGCCTGTTGCGATTGC. O8F corresponded to the zein sequence 5' to the site of intron construct insertion and O9 was complementary to the zein sequence 3' to the site of intron construct insertion in pDH515 (Simpson et al., 1996). Primers for the agroinoculation mini-exon reporter (Figure 1C) were O786F, 5'- GCGCCAAAAATGGGATCAATG labelled with 6-FAM and an unmodified primer complementary to GFP coding sequence 162bp downstream of the mini-exon splicing reporter insert, O787, 5' - GTGACAAGTGTTGGCCATGG.

3. RT-PCR fragments were separated on an ABI3730 DNA Analyzer (Applied Biosystems) set up for fragment analysis.

4. The size and peak area data were analysed by GeneMapper v3.5.(Applied Biosystems).

5. The percentage inclusion or exclusion of the splicing reporter mini-exon from the peak areas for each processed transcript was determined. Each transient assay was repeated at least three times and standard errors determined.

3.4.3 Western analysis

1. To confirm expression of the tagged proteins, protein was extracted from protoplasts or agroinfiltrated disks by boiling in 50 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 1 mM EDTA, 1% (w/v) SDS and 15% β-mercaptoethanol and bromophenol blue.

2. Proteins were separated on 10% SDS-PAGE gels and blotted onto Immobilon transfer membranes (Millipore).

3. Immunoblots were performed using rabbit anti-HA (Millipore Antibodies) or mouse anti-myc (Abcam) depending on the protein tag.

4. Detection was as described using Amersham Enhanced Chemiluminescence Western blotting detection reagents using horse-radish peroxidise linked secondary antibodies (GE Healthcare). Light produced by the chemiluminescent reaction was detected using Amersham Hyperfilm ECL (GE Healthcare) after exposure for 30 seconds.

3.4.4 Visualising GFP expression

1. Whole leaves were illuminated by black ray long-wave (365 nm) ultraviolet lamp (model B, 100 Amps, UV products, Upland, CA, USA).

2. Protoplasts were mounted on glass microscope under a glass cover and imaged for GFP using a Leica TCS SP (Leica Microsystems, Heidelberg, Germany) spectral confocal laser scanning microscope.

4. Experimental example.

4.1 Splicing of U12 and U2 introns using tobacco protoplast transfection.

The sixth intron (of 11 introns) in the Glutathione synthetase 2 gene (GSH2 - AT5G27380) is a U12 intron, which is spliced by the minor spliceosome (see chapter 4 and 5). To examine the splicing of plant U12 introns and in particular, the influence of flanking normal (U2) introns, different constructs were produced. Firstly, the single U12 intron and part of its flanking exon sequences was inserted into pDH515 (Figure 1A) and then introduced into tobacco protoplasts where it was inefficiently spliced (Lewandowska et al., 2004). Exon bridging interactions influence the efficiency by which adjacent introns are spliced (Robberson et al., 1990) and to examine whether the flanking U2 introns could improve splicing of the U12 intron, a construct was prepared that contained all three introns and associated exons inserted into pDH515 (Figure 2A). RT-PCR of RNA isolated from tobacco protoplasts transfected with this construct using the PEG mediated protocol (3.1) showed four different RT-PCR products which represented different spliced transcripts (Figure 2B).

To examine whether different RNA-binding proteins could influence splicing of the U12 intron, the splicing reporter construct was co-transfected with plasmids containing the RNA-binding proteins, UBP1 and RBP45. (are there mammalian orthologs for this?) UBP1 strongly enhances the splicing of otherwise inefficiently processed introns (Lambermon et al., 2000) while a similar RNA binding protein RBP45 was unable to stimulate splicing and accumulation when transiently over-expressed in protoplasts (Lorković et al., 2000). Both UBP1 and RBP45 were tagged with the HA epitope to demonstrate expression of protein from the plasmids. Over-expression of UBP1 had little effect on splicing pattern or efficiency with the three intron construct. However, over-expression of RBP45 completely abolished the normal splicing pattern and showed an exon skipping event which removed all three introns and the two intervening exons (Figure 2B). Expression of the HA-tagged proteins in tobacco protoplast was confirmed by western analysis (Figure 2C).

5. Troubleshooting

|Problem |Reason and solution |

| |Use healthy, young fully expanded tobacco leaves. Poor quality |

| |leaves usually results in a poor return of protoplast numbers. |

| |Protoplasts that show a dull green/brown colouration after overnight|

| |incubation are usually the result of acidification of the media by |

| |bacterial contamination and should not be used. All media should be |

| |sterile and the assays should be performed in a flow cabinet to |

| |maintain sterility. |

| |When the majority of protoplasts (>75%) have collapsed then the |

| |osmoticum of the media is incorrect and should be made again. |

| | |

1. Use healthy, young fully expanded tobacco leaves. Poor quality leaves usually results in a poor return of protoplast numbers.

2. Protoplasts that show a dull green/brown colouration after overnight incubation are usually the result of acidification of the media by bacterial contamination and should not be used. All media should be sterile and the assays should be performed in a flow cabinet to maintain sterility.

3. When the majority of protoplasts (>75%) have collapsed then the osmoticum of the media is incorrect and should be made again.

Figure Legends

Figure 1. Splicing reporter vectors

A. An intronless zein gene from maize was cloned between the CaMV promoter and terminator sequences (shaded boxes), a unique BamHI restriction site was introduced into the coding sequence (white box) to produce the plasmid, pDH515. Introns of interest and surrounding exon sequence (solid line separating dark boxes) were introduced in frame into the BamHI site. Lines indicate the intron removed by splicing. B. The potato invertase mini-exon was introduced into pDH515. The mini-exon is 9 nt long and requires strong splicing signals in the upstream intron to be efficiently spliced. This construct formed the basis of a series of intron signal mutations. C. For a visible assay of splicing, the invertase mini-exon system was modified to be expressed as a fusion protein to GFP. Introduction of a stop codon into the mini-exon produced GFP fluorescence only when the mini-exon was skipped. This construct and derivatives of it have been used in agroinfiltration experiments along with protein factors.

Figure 2. Splicing of U12 and U2 introns using tobacco protoplast transfection.

A. Construct pGSH9 consisting of GSH2 U12 intron with both upstream and downstream U2 introns inserted within the coding sequence of the maize zein gene. Lines indicate intron sequences and boxes indicate exons. The zein gene coding sequence is labeled and all possible splicing events are indicated by the lines across the introns. Arrows indicate the primer positions used in the RT-PCR analysis. B. Genescan analysis of splicing of pGSH9 construct alone and with over-expressed RBP45 and UBP1 proteins in tobacco protoplasts. M indicates a DNA size marker in base pairs. Splicing of pGSH9 gave three different RT-PCR products in addition to unspliced transcripts where either the downstream U2 intron was removed (D) or where both U2 introns were removed (UD) and the fully spliced product (FS). Over expression of UBP1 did not alter the splicing pattern of pGSH9 but over-expression of RBP45 caused exon skipping (SK). C. Protein gel blot analysis with anti-HA antibody to HA-tagged RBP45 and UBP1 proteins over-expressed in tobacco protoplasts. Protein bands are indicated by asterisks. C is control untransfected protoplasts.

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Abbreviations

BAP - 6-Benzylaminopurine

CaMV – Cauliflower Mosaic Virus

EDTA - Ethylenediaminetetraacetic acid

GFP - Green Fluorescent Protein

MES - 2[N-Morpholino]ethanesulfonic acid

NAA - 1-Naphthaleneacetic acid

PEG - Polyethylene glycol

RRM - RNA recognition motif

RT - Room temperature

Acknowledgements:

This work was funded by the EU FP6 Programme Network of Excellence on Alternative Splicing (EURASNET) [LSHG-CT-2005-518238]; the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD) [WP114]; the Austrian Science Foundation (FWF: SFB-F017/10/11; DK W1207, RNA Biology).

Figure 1. Splicing reporter constructs

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Figure 2. Splicing of U12 and U2 introns using tobacco protoplast transfection.

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