Apr 30, 2026
Genetic transformation of Aureobasidium pullulans via Agrobacterium tumefaciens-mediated transformation
- Julian Liber1,
- Claudia Petrucco1
- 1Duke University Department of Biology
- Duke University
Protocol Citation: Julian Liber, Claudia Petrucco 2026. Genetic transformation of Aureobasidium pullulans via Agrobacterium tumefaciens-mediated transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5y5wjl1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 314477
Keywords: genetic transformation of aureobasidium pullulan, mediated transformation aureobasidium pullulan, transformation aureobasidium pullulan, heterologous expression mutants in this fungus, aureobasidium pullulan, protocol adapts agrobacterium tumefacien, agrobacterium tumefacien, ubiquitous extremotolerant yeast, generating heterologous expression mutant, biosynthesi, biosynthesis of useful biomaterial, fungus, genetic transformation, transformation to this species, resistance markers for competition assay, label proteins of interest, label protein, plant disease, approach to rescue mutant, antibiotic
Funders Acknowledgements:
Julian Liber
Grant ID: Duke Microbiome Center
Julian Liber
Grant ID: Triangle Center for Evolutionary Medicine
Abstract
Aureobasidium pullulans is a ubiquitous extremotolerant yeast, researched for its unusually cell division, ability to prevent plant diseases, extremotolerance of pH, salinity, osmolarity, and radiation, and its biosynthesis of useful biomaterials such as polymers and antibiotics. This protocol adapts Agrobacterium tumefaciens-mediated transformation to this species, using an efficient and low-cost method for generating heterologous expression mutants in this fungus. The authors have used this approach to rescue mutants, fluorescently label proteins of interest, and express resistance markers for competition assays.
Materials
- LM medium (10 g tryptone, 6 g yeast extract, 1.5 g K2HPO4, 0.6 g NaCl, and 0.4 g MgSO4*7H2O per 1L)
- Add 15-20 µg/mL rifampicin and appropriate selection antibiotic
- YPD (1% YE, 2% peptone, 2% dextrose)
- YPD agar with antibiotics, in square plates
- 200-500 µg/mL hygromycin B, or 100 µg/mL nourseothricin, or 100 µg/mL G418
- 50 µg/mL tetracycline HCl
- 34 µg/mL chloramphenicol
- IM plates, 1 per 4 transformations, see Recipe
- IM liquid, 1x, ~5 mL per plasmid, see Recipe
- Round bottom glass test tube, at least 15 mm in diameter
- Flame or other heat sterilization process
- 50 mL conical tubes, can be bleached, rinsed, and autoclaved, then reused
- 1 cm polystyrene cuvettes
- Visible light spectrophotometer
- 1.5 – 2 mL microcentrifuge tubes
- Sterile water
- PCR strips or microcentrifuge tubes, for mixing yeast agro
- Sterilized wood streakers, or cell scrapers
- Bundock’s Induction Media (IM) for *Agrobacterium* transformation of *Aureobasidium pullulans*:
- Per 500 mL 2x or 1L 1x, add in order, to ~400 mL water:
- 500 mg MgSO4 * 7H2O
- 150 mg NaCl
- 100 mg CaCl2 * 6H2O
- 500 mg (NH4)2SO4
- 2.5 mg Fe(II)SO4 * 7H2O (made a 10 mg/mL solution, then added 250 µL)
- 0.05 g K2HPO4
- 1.45 g KH2PO4
- 7.81 g MES hydrate
- 3.6 g D-glucose
- 5 mL or 6 g glycerol
- 250 µL 20 mg/mL bromocresol purple in 100% EtOH (optional, but a good sanity check for pH of the medium)
- 2 mL 100 mM acetosyringone (3’,5’-dimethoxy-4’-hydroxyacetophenone) in DMSO
- Adjust pH to 5.2 with HCl (will be yellowish green with bromocresol purple)
- Fill to 500 mL for 2x
- Filter sterilize with a 0.22 µm filter
- Make a 2x agar solution containing 4% w/v agar. After autoclaving, keep warm. Mix 1:1 agar and 2x IM, then pour into 100x15mm round plates.
Procedure
Transform binary plasmids capable of replicating in A. tumefaciens and expressing in A. pullulans into A. tumefaciens AGL1 via electroporation. This vector on Addgene is a good starting point. See this protocol: https://dx.doi.org/10.17504/protocols.io.g8ubzww
Modified from the cited protocol: without diluting, plate 20 µL of the resuspended cells on appropriate selective medium. Tilt the plate to create a drip, then incubate for 48:00:00 at 28 °C .
Day 3
Inoculate 5 mL of LM+ 15 ug/mL Rif with the appropriate antibiotic in a 50 mL tube with a streak of colonies taken from the electroporation-transformed cells.
Inoculate5 mL of YPD in a 50 mL tube with the yeast line intended for transformation.
Grow cells at 230 rpm, 28°C overnight.
Day 4
Pellet Agro cultures in tubes, by centrifuging at 3000 x g for 00:10:00 .
Pour off or aspirate the supernatant, and replace with 5 mL of 1x IM. Vortex to resuspend.
Replace into the incubator at 230 rpm, 28°C , for 4-6 hr.
When Agrobacterium cells are nearly ready, centrifuge 1.5-2 mL of yeast culture in a microcentrifuge tube at 15000 x g, 00:00:30 . Discard supernatant, resuspend in 1 mL water, centrifuge again. Discard supernatant and suspend in 300 µL water (you will need 50 µL of 0.25 OD600 suspension per transformation).
Measure the OD600 of the yeast suspensions. Prepare dilutions at OD600 = 0.25 in water.
Flame the glass test tube, and allow to cool in a sterile environment. Use it to press 4 divots in the IM plates, distributed in quadrants, as below.
Mix 50 µL of yeast with 50 µL of induced Agrobacterium culture, then transfer all of the mixture to a divot in the plate.
Allow plates to dry overnight on the bench with lids on, then transfer to a 24C incubator in a bag or wrapped in parafilm. Incubate for 3+ days (ideally 5) until a lawn of colonies is present in the divot.
Day 8-9
Using a wood streaker, collect as much of the co-culture in the divot as possible. Transfer to one quadrant of a square YPD plate with antibiotics as follows: Make a line at one edge of the area with all the cells. Streak back and forth over the area, moving gradually away from the line, to create a dilution from one side to the other, as below.
Incubate the plate at 28 °C until colonies are visible and can be selected by fluorescence. Transfer the colonies to a new plate before genotyping by PCR because holdover bacteria can create false positives.
Protocol references
Petrucco, Claudia A., Alex W. Crocker, Alec D’Alessandro, et al. “Tools for Live-Cell Imaging of Cytoskeletal and Nuclear Behavior in the Unconventional Yeast, Aureobasidium pullulans.” Molecular Biology of the Cell 35, no. 4 (2024): br10. https://doi.org/10.1091/mbc.E23-10-0388.
Bundock, P., A. den Dulk-Ras, A. Beijersbergen, and P.j. Hooykaas. “Trans-Kingdom T-DNA Transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae.” The EMBO Journal 14, no. 13 (1995): 3206–14. https://doi.org/10.1002/j.1460-2075.1995.tb07323.x.