Agrobacterium tumefaciens mediated transformation of

Leptosphaeria maculans and L. biglobosa

 

based on Bundock et al (1995); Rimmer (2003), Gardiner & Howlett (2004) and Eckert (2005)

 

 

Time scale and protocol summary:

Total time: approx. 4 weeks

 

2 weeks prior to transformation: prepare V8 agar plate with Leptosphaeria isolate to be transformed; grow in conditions favouring sporulation

 

Day 1: transform Agrobacterium with plasmid/revive from storage

           Prepare media required for day 4

 

Day 4: inoculate Agrobacterium into liquid medium

          Prepare media, antibiotics and utensils required for day 5

           

Day 5: Harvest conidia and inoculate into liquid medium; prepare

media, buffer and utensils required for day 6

 

Day 6:  Transformation Day co-cultivate spores with

Agrobacterium

 

Day 9, pm: overlay with selective medium

 

2 weeks after overlay: check plates for transformants, sub-culture onto fresh selective medium

 

 

 

Day 1: Transformation of Agrobacterium tumefaciens with

integrating vector (e.g. pCAMsgfp, pCAMDsRed)

note: the preparation of electro-competent Agrobacterium cells (see Appendix) requires up to 5 days

 

TIME SCALE:

0.5 day for transformation + recovery period, 3 days incubation

                          

MATERIALS:

Equipment:

§    Gene Pulser^® Transfection Apparatus (Bio-Rad Laboratories Ltd., UK) consisting of a Gene Pulser unit connected to a Pulse Controller

§    28°C incubator

§    28°C shaking incubator

§    class I sterile flow hood

§    tabletop centrifuge

 

Consumables:

§    electro-competent Agrobacterium tumefaciens cells (freshly prepared – see Appendix - or thawed on ice from a glycerol stock kept at -80°C)

§    0.2mm electroporation cuvette, chilled to 4°C

§    transforming DNA at a concentration of 25ng/µl, dissolved in water (no salts!)

§    sterile 1.5ml Eppendorf tubes

§    sterile disposable spreaders

§    100ml of YMB (pH 7 @ 22°C: 10g/l mannitol, 400mg/l yeast extract, 100mg/l NaCl, 200mg/l MgSO₄.7H₂O, 500mg/l K₂HPO₄.3H₂O), autoclaved and chilled to 4°C

§    YMB plates (pH 7 @ 22°C: 10g/l mannitol, 400mg/l yeast extract, 100mg/l NaCl, 200mg/l MgSO₄.7H₂O, 500mg/l K₂HPO₄.3H₂O, 1.5% (w/v) bactoagar) containing appropriate antibiotic to select for transforming DNA (e.g. 50µg/ml kanamycin for pCAMBIA based plasmids)

§    ice

 

Method

1. Gently transfer 40µl of electro-competent Agrobacterium cells into a pre-chilled 0.2mm electroporation cuvette, ensuring bacterial suspension makes contact with both sides of the cuvette; place on ice
2. add 25ng of transforming DNA (in a volume of 1µl sterile distilled water)
3. gently tap cuvette to mix and electroporate, conditions:

                   Resistance: 400 Ω

                   Capacitance: 25µF

                   Volts: 2.5kV

(resulting in a field strength of 12.5kV/cm)

 

4. immediately add 1ml of chilled YMB to cuvette and gently mix by pipetting
5. transfer cell suspension to 1.5ml Eppendorf tube
6. incubate shaking at 200rpm in 28°C for 3 hours
7. pellet cells in a tabletop centrifuge, re-suspend in 100µl of YMB medium and plate (using sterile spreader) on YMB agar containing the appropriate antibiotic
8. seal with Parafilm and incubate at 28°C in darkness for 3 days. Colonies of transformed Agrobacterium should then be visible

 

 

 

 

Day 1: preparation of media required for Day 4

 

                          

MATERIALS:

Equipment:

§    autoclave

 

Consumables:

§    20ml of Luria-Bertoni (LB) medium (10g/l Tryptone, 5g/l Yeast extract, 10g/l NaCl)

§    100ml Duran bottle

§    100ml distilled water

§    250ml Duran bottle

§    Kanamycin monosulsphate, 50mg

§    1ml distilled water

§    2ml sterile syringe

§    0.45μm filter

§    sterile 1.5ml Eppendorf tube

 

 

 

 

Method

1. prepare LB medium in 100ml Duran bottle, autoclave
2. fill 250ml Duran bottle with 100ml of distilled water, autoclave
3. dissolve 50mg of kanamycin in 1ml of distilled water, filter sterilise into 1.5ml Eppendorf tube, store at -20°C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Day 4: Inoculation of Agrobacterium into liquid medium, preparation of

media, buffers and utensils

 

MATERIALS:

Equipment:

§    28°C shaking incubator

§    class I sterile flow hood

§    autoclave

§    drying oven

§    shaker

§    pH meter

 

Consumables:

§    plate containing actively growing Agrobacterium carrying integrating vector (Day 1)

§    7.5ml LB in a 50ml Falcon tube

§    kanamycin @ 50mg/ml (Day 1)

§    sterile bacterial loop

§    40ml Potato dextrose broth (PDB, Sigma P-6685): 24g/l PDB

§    250ml conical flask

§    5x10cm of Miracloth

§    small glass funnel

§    aluminium foil

§    autoclave tape

§    glass spreader

§    Hygromycin B, 50mg (for 1ml)

§    Cefatoxamine, 500mg (for 1ml)

§    KOH, 280.4mg (for 1ml)

§    Acetosyringone, 19.6mg (for 10ml)

§    MES, 2-(N-Morpholino)ethanesulfonic acid, 3.9g (for 20ml)

§    Glucose, 0.9g (for 10ml)

§    5 x 2ml sterile syringe

§    5 x 0.45μm filter

§    2 x 50ml Falcon tube

§    3 x sterile 1.5ml Eppendorf tube

§    5 x sterile 15ml centrifuge tube

§    100ml conical flask + bung

 

 

Method

1. working in the Class I sterile flow hood, add 7.5µl of kanamycin [50mg/ml] to 7.5ml LB in a 50ml Falcon tube
2. working in the Class I sterile flow hood, pick one single Agrobacterium colony and transfer into 7.5ml LB^{+Kan 50µg/ml}
3. incubate in 28°C shaking incubator, 200rpm for two nights and one day
4. prepare 40ml of PDB in 250ml conical flask, seal with aluminium foil, autoclave
5. fold Miracloth once to produce a 5x5cm square with two layers; line the inside of a small glass funnel with the Miracloth; wrap in aluminium foil, tape shut with autoclaving tape, autoclave and dry in drying oven for 24 hours
6. wrap glass spreader in aluminium foil, tape with autoclaving tape, autoclave, dry in drying oven for 24 hours
7. dissolve 50mg of hygromycin B in 1ml distilled water, filter sterilize using 0.45μm filter into 1.5ml Eppendorf tube, store at -20°C
8. dissolve 500mg of cefatoxamine in 1ml distilled water, filter sterilize using 0.45μm filter into 1.5ml Eppendorf tube, store at -20°C
9. dissolve 280.4mg KOH in 1ml distilled water (= 5M KOH)
10.  in a 50ml Falcon tube, dissolve 19.6mg of acetosyringone in 10ml distilled water, on shaker (this will take about 1 hour); pH with 5M KOH to pH 8 (not much, one drop); filter sterilize using 0.45μm filter into sterile 15ml centrifuge tube, store at 4°C
11.  in a 50ml Falcon tube, dissolve 3.9g of MES in 18ml distilled water; pH with 5M KOH to pH 5.3, make up volume to 20ml with distilled water; filter sterilize using 0.45μm filter into sterile 15ml centrifuge tube, store at 4°C
12.  dissolve 0.9g of glucose in 10ml distilled water, filter sterilize using 0.45μm filter into sterile 15ml centrifuge tube, store at 4°C
13.  place bung in neck of 100ml conical flask, cover with aluminium foil, autoclave and dry in drying oven for 24 hours

 

Day 5: Harvesting of conidia and inoculation into liquid medium,

preparation of media, buffers and utensils

 

MATERIALS:

Equipment:

§    28°C shaking incubator

§    class I sterile flow hood

§    centrifuge for 15ml tubes

§    haemocytometer + cover slip

§    stereo light microscope

§    balance

§    magnetic stirrer + flea (sterile)

§    autoclave

 

Consumables:

§    plate containing actively growing Leptosphaeria, sporulating

§    3ml sterile distilled water

§    glass spreader, sterile (Day 4)

§    small glass funnel lined with 2 layers of Miracloth, sterile (Day 4)

§    sterile 15ml centrifuge tube

§    40ml Potato dextrose broth in 250ml conical flask, sterile (Day 4)

 

§    2.5x stock solution of minimal media salts for Induction Media (IM):

o        KH₂PO₄          3.625g

o        K₂HPO₄          5.125g

o        NaCl                       0.375g

o        MgSO₄.7H₂O             1.250g

o        CaCl₂.2H₂O               0.165g

o        FeSO₄.7H₂O             0.0062g

o        (NH₄).2SO₄              1.250g

§    1l sterile distilled water

 

§    Induction Media (IM), 200ml, part I:

o        80ml of 2.5x minimal media salt stock

o        1ml glycerol

o        103ml distilled water

§    500ml Duran bottle

 

§    Induction Media Agar (IMA), 200ml, part I:

o        80ml of 2.5x minimal media salt stock

o        1ml glycerol

o        103ml distilled water

o        3g Agar, technical no. 3

§    500ml conical flask

§    200ml of PDA: 7.8g/l, autoclaved in 500ml conical flask

 

Method

1. working in the Class I sterile flow hood, place 3ml of sterile water on top of actively sporulating fungal culture; ensure plate is covered by gently tapping plate
2. leave at room temperature for 30 minutes
3. gently scrape the surface of the culture using a sterile glass spreader
4. filter spore suspension through miracloth and collect in sterile 15ml tube
5. pellet spores by centrifugation at 4500rpm for 5 min
6. wash pellet twice in 5ml of sterile water, pelleting spores in between as in step 5.
7. count spores using a haemocytometer
8. inoculate 10⁸ spores into 40ml of sterile PDB in a 250ml conical flask, incubate at 28°C shaking @ 200rpm for 24 hours.

 

9. 2.5x stock of minimal media salts: place bottle containing 1l sterile distilled water on magnetic stirrer, add flea
10.  weigh out one chemical, add to stirred water; when completely dissolved, add next chemical etc. until all dissolved. Store at room temperature and do not autoclave

 

11.  Induction media, part I: combine minimal media salts, glycerol and water in 500ml Duran bottle, autoclave

 

12.  Induction medium agar, part I: combine minimal media salts, glycerol, water and agar in 500ml flask, autoclave

 

Day 6:                                     TRANSFORMATION DAY

 

TIME SCALE: Induction of Agrobacterium needs to be done first thing in the morning and the transformation is set up last thing in the evening, allowing 8 – 10 hours of incubation

 

MATERIALS:

Equipment:

§    spectrophotometer, reading at 600nm

§    28°C shaking incubator

§    class I sterile flow hood

§    haemocytometer + cover slip

§    stereo light microscope

§    centrifuge for 50ml tubes

§    tabletop centrifuge

§    22°C incubator

 

Consumables:

§    plastic cuvettes, 1ml

§    Induction Media (IM), 200ml, part II:

o        IM (part I) prepared Day 5, autoclaved and cooled to room temperature

o        8ml 1M MES (Day 4)

o        4ml 500mM glucose (Day 4)

o        4ml AS (Day 4)

§    100ml conical flask + bung, sterile (Day 4)

§    Induction Media Agar (IMA), 200ml, part II:

o        IMA (part I) prepared Day 5, autoclaved and cooled to room temperature

o        8ml 1M MES (Day 4)

o        4ml 500mM glucose (Day 4)

o        4ml AS (Day 4)

§    9cm Petri dishes

§    50ml Falcon tube, sterile

§    1.5ml Eppendorf tubes, sterile

§    sterile glass spreaders

§    Parafilm

 

Method

1. finish preparing the IM by adding MES, glucose and AS to the autoclaved and cooled media prepared on Day 5
2. transfer 15ml of IM into a 100ml sterile conical flask
3. take 1ml from LB Agrobacterium culture (Day 4) and read optical density (OD) at 600nm
4. add appropriate amount of Agrobacterium to the 15ml IM to obtain an OD₆₀₀ = 0.15 and adjust volume to 20ml (ie. 1:20 dilution)
5. seal flask with sterile bung and incubate in 28°C shaking incubator, 200rpm for 8-10 hours,  until OD₆₀₀ = 0.6
6. finish preparing the IMA by adding MES, glucose and AS to the autoclaved and cooled media prepared on Day 5
7. working in class I flow hood, prepare IMA plates by poring 10ml (exactly) into 9cm Petri dishes, leave to set and store in 4°C
8. check Leptosphaeria culture for germination by placing 10μl on an haemocytometer slide; the germination rate should be >50%.
9. Transfer Leptosphaeria culture to 50ml sterile Falcon tube and pellet germlings at 4500rpm, 10min.
10.  wash twice in 10ml ml of IM, pelleting spores in between as in step 9; re-suspend spores in 10ml of IM
11.  aliquot 200μl of spores into sterile 1.5ml Eppendorf tubes, place on ice
12.  After 8 hours of incubating the Agrobacterium culture in IM, take 1ml of the culture and read OD₆₀₀ (if close to this value, continue to step 9., otherwise return to incubator)
13.  add 200μl of the log-phase Agrobacterium culture to the prepared fungal spores.
14.  TRANSFER ALL WORK TO CERTIFIED CLASS II LAB, CONTAINING CLASS II BIOSAFETY CABINET
15.  vortex, pellet bacteria and spores by centrifugation in a tabletop centrifuge at 14 000rpm for 3 min and decant supernatant by poring
16.  re-suspend bacteria and spores in liquid left in tube (~ 50μl) and place in

the middle of a prepared IMA plate

17.  using a sterile glass spreader, spread bacteria/spore mix on plate (careful not to over-spread), briefly leave to dry (careful not to over-dry) and seal with Parafilm
18.  incubate for 3 days in 22°C and darkness

Day 9: Selection

 

MATERIALS:

Equipment:

§    microwave

§    waterbath, set to 47°C

§    class II biosafety cabinet

§    22°C incubator

 

Consumables:

§    200ml PDA, autoclaved (Day 5)

§    200μl cefatoxamine [500mg/ml], sterile (Day 4)

§    400μl hygromycin [50mg/ml]

§    Parafilm

 

Method

1. melt PDA in microwave, cool in waterbath until 47°C
2. add cefatoxamine and hygromycin
3. in Class II biosafety cabinet, overlay Agrobacterium-fungal spore co-culture plates (Day 6) with 10ml (exactly) of PDA^{+Cef +Hyg}
4. leave to set completely, seal with Parafilm
5. incubate in 22°C and darkness for 2 weeks (L. maculans) or 4 weeks (L. biglobosa), when first transformants should grow through selective media

 

 

 

APPENDIX:

 

Preparation of electro-competent Agrobacterium tumefaciens

Amounts stated will give 25 aliquots of electro-competent Agrobacterium

 

TIME SCALE:

2-3 days incubation + 1.5 days incubation + 0.5 days preparation

 

MATERIALS:

Equipment:

§    CO8000 Cell Density Meter (WPA Biowave, UK) or equivalent, to measure optical density at 600nm

§    28°C incubator

§    28°C shaking incubator

§    class I sterile flow hood

§    centrifuge capable to take 50ml falcon tubes, spin at 12 000rpm and chill to 4°C

§    tabletop centrifuge

 

Consumables:

§    1ml plastic disposable cuvettes

§    Agrobacterium tumefaciens strain (e.g. LBA4404, Agl1) on a plate or as glycerol stock kept at -80°C

§    Sterile LB plate containing appropriate antibiotic for Agrobacterium strain used (e.g. for LBA4404 rifampicin at 50µg/ml and streptomycin at 30µg/ml)

§    Sterile bacterial loop

§    100ml of YT medium (10g/l yeast extract, 16g/l tryptone, 5g/l NaCl), autoclaved in 500ml conical flask, cooled to room temperature

§    ice

§    50ml Falcon tubes

§    200ml of 10% glycerol, autoclaved and chilled to 4°C

§    1.5ml sterile Eppendorf tubes

 

 

 

Method

Day 1:

1. Working in a class I sterile laminar flow cabinet and using a sterile bacterial loop, streak Agrobacterium tumefaciens (from old plate or from storage at -80°C) onto a fresh LB plate containing the appropriate antibiotic

2. Seal the plate with Parafilm and incubate in darkness at 28°C for 2-3 days, until individual colonies are visible

 

Day 3:

3. Working in a class I sterile laminar flow cabinet and using a sterile bacterial loop, select one individual colony and transfer into 100ml of YT medium

4. Seal flask with sterile bung and aluminium foil and incubate in 28°C and shaking at 200rpm for 24 – 30h

 

Day 4:

5. measure the optical density of the bacterial culture at 600nm; the OD₆₀₀  should be 0.5-0.7 (if not, return to incubator)

6. transfer culture to ice and from now on always work on ice or in equipment pre-chilled to 4°C

7. transfer 4x25ml of the culture into 4x50ml sterile Falcon tubes

8. Pellet bacteria by centrifugation at 12 000rpm, 4°C for 2 min

9. remove the supernatant and gently re-suspend (pipetting!) bacteria in 25ml (per tube) of 10% glycerol

10. repeat steps 8 and 9 successively re-suspending the bacteria  in 12.5ml, 5ml and 0.5ml (per tube) of 10% glycerol

11. combine the cell suspensions in two 1.5ml Eppendorf tubes (2x1ml) and pellet in tabletop centrifuge, 12 000rpm 2 min

12. remove supernatant and re-suspend in 2x500µl of 10% glycerol, store on ice and use immediately or aliquot into 40µl portions and store at -80°C

 

 

References:

 

Bundock P, Dendulkras A, Beijersbergen A, Hooykaas PJJ, 1995. Transkingdom T-

DNA Transfer from Agrobacterium tumefaciens to Saccharomyces

cerevisiae. Embo Journal 14, 3206-3214.

 

Eckert M, Maguire K, Urban M, Foster S, Fitt BDL, Lucas JA, Hammond-Kosack K, 2005. Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp FEMS Microbiology Letters 253:67-74.

 

Gardiner DM, Howlett BJ, 2004. Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans. Current Genetics 45, 249-255.

 

Rimmer R, 2003. Agrobacterium tumefaciens mediated transformation of Leptosphaeria maculans. In: Proceedings of the ICPP 2003. Christchurch, NZ