Journal of General Microbiology (1989), 135, 601-604.
Printed in Great Britain
60 1
Heat Shock at an Elevated Temperature Improves Transformation
Efficiency of Protoplasts from Podospuva ansevina
By T H I E R R Y B E R G E S * A N D C H R I S T I A N B A R R E A U
Laboratoire de Gknitique, UniversitP de Bordeaux 11, Avenue des Facultks, 33405 Talence Cedex,
France
(Received 26 October 1988; accepted 7 November 1988)
We have developed an improved transformation procedure for the filamentous fungus
Podospora anserina. This procedure is based on the observation that a heat shock at an elevated
temperature (48 "C) improves the competence of P. anserina protoplasts for transformation 5- to
10-fold. This is observable only if the heat shock is applied before the addition of transforming
DNA. An increase in competence is observed immediately after the heat shock, and heatshocked cells are still competent after 20-30 min. The mechanism by which heat shock improves
competence remains unclear. The modified transformation procedure gives as many as 200-500
stable transformants per pg of plasmid DNA containing the P. anserina ura5 gene. This should
allow direct cloning of P. anserina genes from a cosmid library.
INTRODUCTION
Fungi are widely used in biotechnology and represent a potent genetic tool for studying
multicellular eukaryotes. The lack of efficient transformation systems has hampered progress in
the study of fungi. Recently, efficient transformation procedures have been developed in
Neurospora crassa using spheroplasts from germinated conidiospores (Case et al., 1979; Akins &
Lambowitz, 1985) and in Aspergillus nidulans using a vector containing the particular ansl
sequence whose function is still unknown (Ballance & Turner, 1985).An autonomous replicating
vector has also been reported to give a high number of transformants in Mucor circinelloides(Van
Heeswijk, 1986). Successful transformation has been reported in many other fungi (Ballance,
1986; Munoz-Rivas et al., 1986; Sanchez et al., 1987; Rodriguez & Yoder, 1987) but the number
of transformants generally obtained is very low (about 10-50 per pg of plasmid DNA).
Protoplasts from mycelium are easily obtained in large amounts using cell wall cellulases but
they are generally poorly competent. Competence has been reported to vary greatly with the
source of cellulases and with the batch of enzyme used (Akins & Lambowitz, 1985). The
physiological state of the mycelium used to produce the protoplasts is also important. Other
factors that influence transformation have been studied in N . crassa (Buxton & Radford, 1984)
and in A . nidulans (Turner & Ballance, 1985). They include the purity of the plasmid
preparation, and the quality of buffers, CaCl, and polyethylene glycol (PEG). Moreover the best
results are obtained when transformed cells are spread embedded in an overlay of the selective
medium.
Successful transformation of a ura.5 mutant has been previously described in the filamentous
fungus Podospora anserina (Begueret el al., 1984). Efficiency was about 5-20 transformants per
pg of plasmid DNA, which did not permit cloning genes of P. anserina directly by
complementation. Here we report the effect of a heat shock treatment at an elevated
temperature on the competence of P. anserina protoplasts.
METHODS
Podospora anserina and Escherichia coli strains. Wild-type P. anserina was strain s isolated by Bernet (1965). The
isolation and characterization of a ura5 mutant deficient in orotidylic acid pyrophosphorylase (OMPppase) have
0001-5221 0 1989 SGM
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602
T . BERGES AND
c.
BARREAU
been described previously (Razanamparany & Begueret, 1986). Transformation of a Leu-- strain with the su8
suppressor gene has been reported (Brygoo & Debuchy, 1985).
Plasmids were propagated in E. coli strain BJ5183 (endA sbcB recBC galK met StrR thi-1 bioT hsdR).
Plasmids. The plasmid pPAura5-6 used in the transformation of the auxotrophic ura5 mutant is derived from a
tandem insertion into pBR322 of the 1.55 kb EcoRI-EcoRI fragment carrying the ura5 gene of P. anserina
(Begueret et al., 1984). The plasmid pSu8, carrying the su8 tRNA gene that suppresses a Leu- opale mutation of P.
anserina, was kindly provided by Y. Brygoo. The plasmid pBT3, conferring resistance to benomyl upon
transformed protoplasts (Orbach et al., 1986), was generously given by Dr C. Yanofsky. Plasmids were isolated by
two CsCl/ethidium bromide density-gradient centrifugations of cleared lysates (Clewell & Helinski, 1972).
Standard transjormation ofprotoplasts. Protoplasts were prepared from the mycelium of 36-h-old cultures grown
in liquid medium supplemented with uridine. The mycelium was washed in 25 mM-potassium phosphate
pH 58/04l M-sorbitol buffer and cell walls were digested in the same buffer containing 80 mg P-D-glucanase
('Glucanex' ; Novo Ferments, Basel, Switzerland) per g of mycelium. Protoplasts were separated from mycelial
debris by filtration, collected by centrifugation and suspended in ST washing buffer (0.1 M-Tris/HCl pH 7-5,
0.8 M-sorbitol). The protoplasts were pelleted again and resuspended in ST buffer containing 50 mM-CaC1, at a
concentration of lo9 cells ml-'. DNA (1-10 pg in 5 pl H 2 0 )was mixed with 50 p1 of protoplasts ( 5 x lo7 cells) for
10 min at room temperature, and then 500 p1 of a solution containing 60% (w/v) PEG 4000, 0.01 M-Tris/HCI
pH 7.5 and 50 mM-CaC1, was added. After 10 min at room temperature, 450 p1 ST buffer containing 0.01 M-CaCl,
was added. Transformed protoplasts were mixed at 45 "C in melted selective medium containing 0.8 M-SUCrOSe and
2% (w/v) agar and overlaid onto the same medium.
In the experiments including a heat shock, protoplasts were treated at the desired temperature for 5 min, cooled
on ice for 30 s and incubated at room temperature for 5 min before the addition of DNA.
R E S U L T S AND DISCUSSION
Protoplasts from mycelium of P. anserina can be obtained repeatedly in large amounts using
Glucanex, which is a fungal cellulase preparation used to eliminate glucans in some wine
preparations. Our standard transformation protocol includes incubation of protoplasts with
DNA in the presence of CaCI2, treatment of the cell suspension with PEG 4000 and spreading
of the transformed cells on a selective medium. We have found that optimal concentrations are
50 mM for CaC12 and 60% (w/v) for PEG. Spreading of the transformed cells embedded in an
overlay of selective medium containing 2% (w/v) agar maintained melted at 45 "C significantly
improves the transformation yield. The quality of the plasmid DNA preparation is crucial:
plasmids prepared by two CsCl centrifugation steps were 5-10 times more efficient in
transformation than those obtained after one such step.
A new protocol including a heat shock treatment was tried to improve the transformation
efficiency, as is done with some bacteria and plants. Protoplasts were heat shocked for 5 min at
the temperatures indicated in Fig. 1. Plasmid DNA was added 10 min later and the cells were
treated with 60% PEG 4000 as described in Methods. Survival of the cells was checked by
plating appropriate dilutions of the transformation mixture onto non-selective medium. Fig. 1
shows the results of a typical experiment. Both the number of transformants and the proportion
of surviving cells without heat shock may vary by up to threefold between different such
experiments, depending on the protoplast preparation. However, the overall response to heat
shock is always the same, with transformation efficiency best around 48 "C, and the survival of
the protoplasts beginning to decrease at about 46 "C. Transformation efficiencies after heat
shock treatment at 48 "C vary from about 200 to 1000 transformants per pg DNA, depending on
the protoplast preparation and on the plasmid used. This represents an increase of about 5-10fold in the yield of transformants, and about 0.5-2-5% of the viable cells are competent for
trans format ion.
In order to determine the delay of onset of competence we transformed the protoplasts at
different times before and after heat shock treatment at 48 "C (Table 1). Heat shock had no
effect if it was applied after the addition of the DNA. Improvement of competence was
observed immediately after the heat shock and there was a further slight increase in the yield of
transformants after 10 min. Heat-shocked cells were still competent after 20-30 min.
The efficiency of transformation after heat shock is significantly increased only if the
treatment is applied before the addition of DNA. The optimal effect is obtained when survival
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603
Improued transjormation of P . anserina
I
1
I
I
I
I
Temperature ("C)
Fig. 1. Effect of treatment at various temperatures before transformation on the number of
transformants. Protoplasts were prepared as described in Methods, divided into 50 p1 portions
(5 x lo7 cells) in 1.5 ml microcentrifuge tubes and treated for 5 min at the temperatures indicated. The
tubes were briefly cooled, and 1 pg DNA was added to each tube after 10 min incubation at room
temperature. Then the samples were treated as for standard transformation. Viability of the cells after
transformation was estimated by plating various dilutions onto non-selective medium. -,
Number of
transformants; ---, plating efficiency.
Table 1. Efiect of'the time of addition of DNA to the heat-shocked protopjasts on the number of
transjormants
No. of
transformants
per pg DNA
Time of addition of DNA
Expt 1
Expt 2
DNA without heat shock
DNA added:
10 rnin before heat shock
Just before heat shock
Just after heat shock
10 min after heat shock
20 rnin after heat shock
30 rnin after heat shock
First heat shock at 44"C,
30 rnin at 20 "C and DNA
added 30 min after a second
heat shock at 48 "C
40
80
10
20
348
418
406
-
-
346
-
116
-, Not tested.
of the cells begins to be altered. These results may explain why heat shock has not been used in
fungal transformations. First, workers attempted the heat shock treatment at temperatures too
low to be effective, and secondly the heat shock was applied after the addition of DNA. Our
protocol can be attempted with other fungi (the optimal temperature may of course differ for
different organisms).
The nature of the mechanism responsible for the improvement of competence after the heat
shock treatment is unknown. As the results in Table 2 demonstrate, the improvement in
transformation yield after heat shock does not depend on the selectable marker used for
transformation, indicating that there is no relation with the expression of the transforming gene.
The effect may be rather at the level of the physiological state of the protoplasts. Treated cells
may have increased their capability to take up or (and) to integrate the transforming DNA.
The experiments described above were performed using 1 pg DNA (20 pg ml-l) in each
transformation assay. In other experiments, the number of transformants obtained increased
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604
T. B E R G E S A N D
c.
BARREAU
Table 2. Eflect of the heat shock on transjormation eflciency with dierent selectable markers
No. of transformants per pg DNA*
Transforming vector
Recipient strain
f
No heat shock
With heat shock
pPAura5-6 (ura5 gene)
ura5
95
40
426
178
pSu8 (opale suppressor)
leui
14
16
84
72
W ild-type
0
1
10
14
pBT3 (benomyl resistance gene)
* The results of
two independent experiments are reported for each vector.
proportionally with the quantity of added DNA in the range 10-100 pg ml-I. Addition of pUC8
DNA (100 pg ml-l) as carrier DNA increased the transformation efficiency by three- to fourfold, but only at low concentrations of the transforming vector. With or without carrier DNA the
system appears to be saturated at the same level, suggesting that for high DNA concentration
the limiting factor is the number of competent cells. Nevertheless protoplasts can be obtained in
large amounts, and using 150-250 pg DNA ml-l in each transformation experiment, thousands
of transformants can be obtained. This is sufficient to perform the direct isolation of genes from
a P. anserina genomic library. Such a library has been realized in the cosmid pHC79 containing
the ura5 gene. Cloning of some genomic sequences has now been undertaken.
This work was supported by the French Centre National de la Recherche Scientifique and the Universitk de
Bordeaux 11.
REFERENCES
AKINS,R. A. & LAMBOWITZ,
A. M. (1985). General
method for cloning Neurospora crassa nuclear genes
by complementation of mutants. Molecular and
Cellular Biology 5 , 2272-2278.
BALLANCE,
D. J . (1986). Sequences important for gene
expression in filamentous fungi. Yeast 2, 229-236.
BALLANCE,
D. J . & TURNER,G. (1985). Development
of a high-frequency transforming vector for Aspergillus nidulans. Gene 36, 321-331.
J., RAZANAMPARANY,
V., PERROT,M. &
BEGUERET,
BARREAU,
C. (1984). Cloning gene ura5 for the
orotydylic acid pyrophosphorylase of the filamentous fungus Podospora anserina : transformation of
protoplasts. Gene 32, 487-492.
BERNET,
J. (1965). Mode d’action des genes de barrage
et relation entre I’incompatibilitk cellulaire et I’incompatibilitk sexuelle chez le Podospora anserina.
Annales des Sciences Naturelles, Botaniques, Paris 12,
61 1-768.
BRYGOO,
Y. & DEBUCHY,
R. (1985). Transformation by
integration in Podospora anserina. I. Methodology
and phenomenology. Molecular and General Genetics
200, 128-131.
BUXTON,
F. P. & RADFORD,
A. (1984). The transformation of mycelial spheroplasts of Neurospora crassa
and the attempted isolation of an autonomous
replicator. Molecular and General Genetics 196, 339344.
CASE,M. E., SCHEWEITZER,
M., KUSHNER,S. R. &
GILES,N. H. (1979). Efficient transformation of
Neurospora crassa by utilizing hybrid plasmid DNA.
Proceedings of the National Academy of Sciences of the
United States of America 76, 5259-5263.
D. B. & HELINSKI,
D. R. (1972). Effect of
CLEWELL,
growth on the formation of the relaxation complex of
supercoiled ColEl , deoxyribonucleic acid and protein in Escherichia coli. Journal of Bacteriology 110,
1135-1 146.
MUNOZ-RIVAS,
A., SPECHT,C. A., DRUMMOND,
B. J.,
FROELIGER,
E., NOVOTRY,
C. P. & ULLRICH,R. C .
(1 986). Transformation of the basidiomycete Schizophyllum commune. Molecular and General Genetics
205, 103-106.
ORBACH,
M.J., PORRO,E. B. & YANOFSKY,
C. (1986).
Cloning and characterization of the gene for betatubuline from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable
marker. Molecular and Cellular Biology 6,2452-2461.
RAZANAMPARANY,
V. & BEGUERET,
J. (1986). Positive
screening of ura5 mutants in the fungus Podospora
anserina : characterization of the transformants.
Current Genetics 10, 8 1 1-8 17.
RODRIGUEZ,
R. J. & YODER,0. C. (1987). Selectable
genes for transformation of the fungal pathogen
Glomerella cingulata fsp. phaseoli (Colletotrichum
lindemuthianum). Gene 54, 73-81.
F., LOZANO,M., RUBIO,V. & PENALVA,
A.
SANCHEZ,
(1987). Transformation in Penicillium chrysogenum.
Gene 51, 97-102.
D. J. (1985). Cloning and
TURNER,
G. & BALLANCE,
transformation in Aspergillus. In Gene Manipulation
in Fungi, pp. 280-282. Edited by J. W. Bennett &
L. L. Lasure. New York: Academic Press.
VANHEESWIJK,
R. (1986). Autonomous replication of
plasmids in Mucor transformants. Carlsberg Research Communication 51, 433-443.
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