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Contributed by G.H. Goldman, R. Geremia, M. Van Montagu, and A. Herrera-Estrella, Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium
Introduction
Virtually all fungal transformation protocols call for the addition of
a high concentration of polyethylene glycol (PEG) following the initial
period of exposure to DNA. However, electroporation could also be useful
for introducing genes into filamentous fungi. Recently, electroporation
has become a valuable technique for transferring nucleic acids into both
eukaryotic and prokaryotic cells (Miller et al., 1988; Fster and Neumann,
1989). In this report, we show the use of high voltage-mediated transformation
as an efficient method for genetic transformation of Trichoderma harzianum
with plasmid DNA.
Materials and Methods
Strains and plasmid. T. harzianum strain IMI206040 was used as the transformation
host. The plasmid used was pAN7-1, a derivative of pUC19 containing the
E. coli hygromycin B phosphotransferase gene as a dominant selectable
marker, and the gpd promoter and trpC terminator signals from Aspergillus
nidulans (Punt et al., 1987). Plasmid DNA was purified from E. coli MC1061
by standard procedures (Maniatis et al., 1982) and dialyzed against distilled
water prior to use.
Electro-competent cells. Osmotically sensitive cells (OSC) were prepared
according to Laurila et al. (1985) with the following modifications: cellophane
sheets placed on Potato Dextrose Agar (PDA) plates were inoculated with
5 x 106 spores/ml and incubated for 21 hours at 28 C; the germinative
tubes from five cellophane sheets were suspended in 15 ml of buffer (1.2
M MgSO4<
/sub>, 10 mM sodium phosphate, pH 5.8) containing Novozyme-234 (5 mg/ml)
in a Petri dish; and, these plates were incubated at 28 C for 30 minutes
with agitation in a rotary shaker at 150 rpm. The OSC generated with this
treatment were then centrifuged in corex tubes at 4,000 x g for 5 minutes
and the pellet was washed twice with 1.2 M sorbitol in water. After the
last wash, the OSC were resuspended in 1.2 M sorbitol at the desired concentration.
Transformation by electroporation. Two micrograms of transforming DNA
were mixed with 400 l of the OSC suspension and kept on ice. High voltage
pulses were delivered to 400 l samples in 0.2 cm electrode gap cuvettes
(Bio-Rad Laboratories) by using a Gene Pulser apparatus with the Pulse
Controller (Bio-Rad Laboratories). Following delivery of the electrical
pulse, OSC were mixed with 1.0 ml of Potato Dextrose Broth (PDB) plus
1.2 M sorbitol (PDBS), incubated for 10 minutes on ice, and then for 2
hours at 28 C. When PEG was used, the following modifications were made:
before adding the transforming DNA, spheroplasts were centrifuged at 4,000
x g for 15 minutes; the pellet was resuspended in 400 l of 1.2 M sorbitol
plus 1.0% PEG 6000 (Fluka); and, after electroporation, the OSC were mixed
with 5.0 ml PDBS. After the incubation period of either treatment, aliquots
were plated using an agar overlay on plates containing PDA plus 1.2M sorbitol
and 100 g/ml of hygromycin B as previously described by Herrera-Estrella
et al. (1990).
DNA preparation. DNA was isolated from T. harzianum mycelia grown in
liquid cultures in PDB medium plus 20 g/ml of hygromycin B (Calbiochem)
according to the method described by Raeder and Broda (1985).
The effect of the parallel resistor (and thus the time constant, τ=RxC)
on electroporation efficiency was also examined (Figure 1B). The best
yield was obtained when the pulse was delivered using a field strength
of 2.0 kV/cm, a parallel resistance of 800 ohms and a capacitance of 25
F. Using 2.0 kV/cm with 25 F, the survival of the OSC decreased sharply
at 100 ohms reaching 20% survival, and continued to decrease slowly from
200 ohms to 800 ohms. At 800 ohms, the survival was about 10.0%. From
these initial experiments, the best electrical conditions found for the
electroporation of T. harzianum were a field strength of 2.8 k
V/cm with
a capacitance of 25 F and a parallel resistance of 800 ohms. T. harzianum
OSC subjected to these conditions in the absence of the pAN7-1 plasmid
did not produce any hygromycin-resistant colonies.
Total cellular DNA of several independent transformants was isolated.
Undigested DNA, as well as DNA digested with EcoRV, BamHI, HindIII, and
EcoRI was subjected to agarose gel electrophoresis and analyzed by Southern
blot using pAN7-1 as a probe. Wild-type T. harzianum contained no sequences
hybridizing to the vector. Figure 2 shows the pattern of hybridization
for three transformants. The pAN7-1 plasmid is about 6.5 kb in size, and
it has no EcoRV sites, one site each for BamHI and HindIII, and two sites
for EcoRI (which yields two fragments of about 3.9 and 2.6 kb). The digestions
showed the restriction patterns expected from the restriction map of pAN7-1.
Each transformant most likely contains tandem repeats of the vector. Hybridization
of undigested DNA occurred only in the high molecular weight genomic band
(UC), suggesting that pAN7-1 integrated into the genome and did not replicate
autonomously.
The effect of the number of electro-competent cells on the transformation
efficiency has already been observed in bacteria, mammalian cells, and
plant protoplasts (Fromm et al., 1985; Dower et al., 1988; Miller et al.,
1988; Shigekawa and Dower, 1988). We investigated the effect of the number
of electro-competent T. harzianum cells on the transformation efficiency.
By increasing the concentration of OSC to about 1.0 x 109/ml, we were
able to increase the transformation efficiency 2.3-fold. When a concentration
of 2.5 x 109 OSC/ml was used, a sharp decrease in the number
of transformants/g
of DNA occurred. This low efficiency correlated with a decrease in the
time constant probably caused by the high concentrations of OSC providing
a higher resistance (data not shown). A sharp decrease in the transformation
efficiency was observed when concentrations of OSC lower than 2.0 x 108/ml
were used. The effect of plasmid DNA concentration on the number of transformants
obtained by the electroporation of identical quantities of cells was also
examined. The transformation efficiency increased with increasing concentrations
(up to 40 g/ml) of plasmid DNA (data not shown).
To date, all protocols known for the chemical transformation of filamentous
fungi are based on the utilization of PEG. Figure 3 shows the influence
of different concentrations of PEG on the transformation efficiency. The
use of 1.0% PEG in the electroporation medium made it possible to obtain
an efficiency of 435 transformants/g of DNA. This efficiency is about
four times greater than electroporation without PEG. A control pulse using
only PEG in the same concentration did not yield any transformants. Recently,
efficient transformation of Rhodococcus fascians (Desomer et al., 1990)
and Bacillus thuringiensis (Mahillon et al., 1989) has been obtained by
combining PEG and electroporation.
The decreased efficiency of transformation obtained by increasing the
concentration of PEG above 1% did not correlate to the survival of the
OSC (data not shown). Unexpectedly, the number of transformants was lower
when concentrations of PEG below 1% were used than in the control treatment
without PEG. This behavior was found to be reproducible in at least three
independent experiments. Volumes smaller than
400 l produced lower numbers
of transformants. This result could be expected since reducing the cross-sectional
area of the solution at the electrode surface increases the resistance
(Shigekawa and Dower, 1988).
The results presented here show that OSC from T. harzianum can be efficiently
transformed by electroporation. Electroporation is rapid, easy to perform,
and requires minimal sample preparation. Therefore, it may prove to be
a general method useful for introducing DNA into many fungal species in
addition to T. harzianum, A. nidulans (Richey et al., 1989), F. solani
(Richey et al., 1989), S. cerevisiae (Delorme, 1989; Hashimoto et al.,
1985; Hill, 1989; Karube et al., 1985; Meilhoc et al., 1990; Rech et al.,
1990; Weaver et al., 1988), S. pombe (Hood and Stachow, 1990; Weaver et al., 1988), and D. discoideum (Dynes and Firtel, 1989; Egelhoff et al.,
1989; Howard et al., 1988). This report presents possibilities to improve
transformation systems that have already been described, or to transform
other filamentous fungi where
PEG-mediated transformation has not been
achieved.
Desomer, J., Dhaese, P., and Van Montagu, M., Appl. Environ. Microbiol.,
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86, 7966 (1989).
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Cell. Biol., 9, 1965 (1989).
Fster, W. and Neumann, E., Gene Transfer by Electroporation. A Practical
Guide. In Neumann, E., Sowers, E. A., and Jordan, C. A., (eds) Electroporation
and Electrofusion in Cell Biology, Plenum Press, New York (1989).
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82, 5824 (1985).
Hashimoto, H., Morikawa, H., Yamada, Y., and Kimura, A., Appl. Microbiol.
Biotechnol., 21, 336 (1985).
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4, 839 (1990).
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USA, 85, 856 (1988).
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Results
Electroporation of germinative tubes or mycelia from T. harzianum did
not yield transformants, so osmotically sensitive cells were tested. The
effect of incubation time of the germinative tubes with the cell wall-degrading
enzymes on the electro-transformation efficiency was tested. Intervals
of 15, 30, and 45 minutes were used. An incubation period shorter than
30 minutes did not yield any transformants while a 45 minute incubation
yielded 50% less transformants than did a 30 minute incubation time (data
not shown). To optimize conditions, different combinations of voltages
and capacitances were chosen, while holding the parallel resistor at 200
ohms and holding the concentration of OSC at 2.0 x 108 per ml (data not
shown). The electroporation medium consisted of distilled water with 1.2
M sorbitol as an osmotic protectant. These preliminary results indicated
that transformation could be obtained at 2.0 kV/cm with a capacitance
of 25 F. Figure 1A shows that this was a good approximation since the
maximum yield of transformants was found at 2.8 kV/cm.
Conclusions
There are two major advantages of electroporation over the traditional
chemical method of transformation. The first is simplicity: the OSC do
not need to be purified by sorbitol gradients, and it is possible to perform
many electro-transformations at one time. The second advantage we found
is that electroporation is more reproducible than PEG-mediated transformation.
An important difference we observed between the two methods was the stability
of transformants from T. harzianum when transformed with the plasmid pAN7-1.
A possible explanation for this effect could be the activation of the
repair system caused by the high-voltage electric pulse.
References
Delorme, E., Appl. Environ. Microbiol., 55, 2242 (1989).
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