Nucleic Acids Research, Vol. 18, No. 22 6737
%.) 1990 Oxford University Press
Transformation of filamentous fungi by electroporation B.N.Chakraborty and M.Kapoor* Cellular, Molecular and Microbial Biology Division, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada Submitted October 3, 1990 Transformation systems have been reported for a number of filamentous fungi (1) based on the procedure perfected for Neurospora crassa by Case et al. employing sphaeroplasts (2). While efficient transformation is achieved with sphaeroplasts, their preparation demands a careful monitoring of the individual steps, along with optimization of conditions for each batch of cell wall-degrading enzymes. Therefore, a new efficient transformation system was developed involving direct electroporation of germinating conidia of N. crassa. Initially we employed a recombinant plasmid harboring the qa-2 + gene, encoding the catabolic dehydroquinase (2) in conjunction with a double mutant (qa-2 arom-9) recipient strain, devoid of both the biosynthetic and catabolic dehydroquinase activities. Fifteen-
of a wild type strain of N. crassa (74A) with the plasmid pCSN44 (4)-carrying the bacterial hygromycin phosphotransferase gene-yielded transformation frequencies ranging from 1,100 to 1,750 per sg DNA. Following a slight modification of this procedure, conidia of another filamentous fungus, Penicillium urticae, were transformed to hygromycin resistance at an efficiency of 2.8 x 103 per isg DNA. We have described a rapid and effective means of transformation, without prior sphaeroplast formnation, that will be readily applicable to other conidial as well as mycelial fungi.
day-old conidia were grown in 0.5 xFries' medium (3) with
We are grateful to Dr. Mary E. Case for permission to use the qa-2+ gene in these experiments. A culture of P. urticae was provided by Dr. G.M.Gaucher. This work was supported by an operating grant from the Natural Sciences and Engineering Research Council to M.K. and a Government of India, Biotechnology Department Fellowship to B.N.C.
aromatic amino acid supplements, for 4 h at 300C, while shaking. Germinated conidia were washed and suspended in 1 mM HEPES buffer (pH 7.0)-50 mM mannitol and 100-jl (6 x 106) samples were mixed with 2 to 7 ,ug of plasmid DNA and subjected to electroporation using the Bio-Rad Gene Pulser apparatus. After electroporation, 1 ml Vogel's minimal medium, Vm (3) was added and incubation resumed at 30°C for 3 h. Transformants were selected by plating on Vm-1 .0% sorbose-0. 1% glucose-0.1% fructose-1.5% agar. Virtually no transformants were recovered when field strength ranging from 3.0 to 9 KV/cm was employed, with capacitance values of 1 and 3 tsF. On increasing the field strength to 12.5 KV/cm, at 25 ltF capacitance and 5 msec pulse length, 2 to 3 stable transformants per ,sg DNA were obtained. Raising the pulse length to 25 msec resulted in suppression of transformation efficiency. Assuming that the cell wall impedes the uptake of DNA, we added 1 mg/ml ,-glucuronidase (Sigma: from Helix pomatia) to the growth medium for the final 2 h of germination. With this treatment a dramatic improvement in the efficiency of transformation was achieved: yields averaging 21 stable transformants per jtg DNA, comparable to those reported for other filamentous fungi (1) were observed routinely. Southern blot analysis of restriction endonuclease-digested DNA from a random sample of transformants demonstrated the integration of the plasmid in the genome. Hybridization with 32P-labelled probes showed only the resident qa-2 genes in the untransformed recipient strain (Figure IA, B: lane 1). In contrast, the DNA of transformants revealed ectopically integrated copies of qa-2 + gene (Figure IA: lanes 2-5) as well as vector sequences (Figure IB: lanes 2-5), a majority of the integration events being attributable to non-homologous recombination. Electroporation of (3-glucuronidase-treated germinating conidia *
To whom
correspondence
should be addressed
ACKNOWLEDGEMENTS
REFERENCES 1. 2. 3. 4.
Fincham,J.R.S. (1989) Microbiol. Revs. 53, 148-170. Case,M.E. et al. (1979) Proc. Natl. Acad. Sci. USA 76, 5259-5263. Davis,R.H. and de Serres,F.J. (1970) Methods Enzymol. 17, 79-143. Staben,C. et al. (1989) Fungal Genet. Newslett. 36, 79-81.
A
1 2 3 4 5
B
1
23 45 -_.
..
_
-.
23 1 94
6-E
2.3 2.0
Figure 1. Southern blot hybridization of genomic DNA of N. crassa transformants digested with EcoRI. The blot was first hybridized with a 2.2 kb BamHI fragment encoding the qa-2+ gene (A) and (after removal of the probe) with Bluescript vector DNA (B). In panels A and B, the lanes contain DNA (5 ug) as follows: 1, untransformed recipient strain; 2 to 5, DNA from transformants E-17, E-20, E-21 and E-22, respectively. Numbers on the left show position of size markers in kb.
%.) 1990 Oxford University Press
Transformation of filamentous fungi by electroporation B.N.Chakraborty and M.Kapoor* Cellular, Molecular and Microbial Biology Division, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada Submitted October 3, 1990 Transformation systems have been reported for a number of filamentous fungi (1) based on the procedure perfected for Neurospora crassa by Case et al. employing sphaeroplasts (2). While efficient transformation is achieved with sphaeroplasts, their preparation demands a careful monitoring of the individual steps, along with optimization of conditions for each batch of cell wall-degrading enzymes. Therefore, a new efficient transformation system was developed involving direct electroporation of germinating conidia of N. crassa. Initially we employed a recombinant plasmid harboring the qa-2 + gene, encoding the catabolic dehydroquinase (2) in conjunction with a double mutant (qa-2 arom-9) recipient strain, devoid of both the biosynthetic and catabolic dehydroquinase activities. Fifteen-
of a wild type strain of N. crassa (74A) with the plasmid pCSN44 (4)-carrying the bacterial hygromycin phosphotransferase gene-yielded transformation frequencies ranging from 1,100 to 1,750 per sg DNA. Following a slight modification of this procedure, conidia of another filamentous fungus, Penicillium urticae, were transformed to hygromycin resistance at an efficiency of 2.8 x 103 per isg DNA. We have described a rapid and effective means of transformation, without prior sphaeroplast formnation, that will be readily applicable to other conidial as well as mycelial fungi.
day-old conidia were grown in 0.5 xFries' medium (3) with
We are grateful to Dr. Mary E. Case for permission to use the qa-2+ gene in these experiments. A culture of P. urticae was provided by Dr. G.M.Gaucher. This work was supported by an operating grant from the Natural Sciences and Engineering Research Council to M.K. and a Government of India, Biotechnology Department Fellowship to B.N.C.
aromatic amino acid supplements, for 4 h at 300C, while shaking. Germinated conidia were washed and suspended in 1 mM HEPES buffer (pH 7.0)-50 mM mannitol and 100-jl (6 x 106) samples were mixed with 2 to 7 ,ug of plasmid DNA and subjected to electroporation using the Bio-Rad Gene Pulser apparatus. After electroporation, 1 ml Vogel's minimal medium, Vm (3) was added and incubation resumed at 30°C for 3 h. Transformants were selected by plating on Vm-1 .0% sorbose-0. 1% glucose-0.1% fructose-1.5% agar. Virtually no transformants were recovered when field strength ranging from 3.0 to 9 KV/cm was employed, with capacitance values of 1 and 3 tsF. On increasing the field strength to 12.5 KV/cm, at 25 ltF capacitance and 5 msec pulse length, 2 to 3 stable transformants per ,sg DNA were obtained. Raising the pulse length to 25 msec resulted in suppression of transformation efficiency. Assuming that the cell wall impedes the uptake of DNA, we added 1 mg/ml ,-glucuronidase (Sigma: from Helix pomatia) to the growth medium for the final 2 h of germination. With this treatment a dramatic improvement in the efficiency of transformation was achieved: yields averaging 21 stable transformants per jtg DNA, comparable to those reported for other filamentous fungi (1) were observed routinely. Southern blot analysis of restriction endonuclease-digested DNA from a random sample of transformants demonstrated the integration of the plasmid in the genome. Hybridization with 32P-labelled probes showed only the resident qa-2 genes in the untransformed recipient strain (Figure IA, B: lane 1). In contrast, the DNA of transformants revealed ectopically integrated copies of qa-2 + gene (Figure IA: lanes 2-5) as well as vector sequences (Figure IB: lanes 2-5), a majority of the integration events being attributable to non-homologous recombination. Electroporation of (3-glucuronidase-treated germinating conidia *
To whom
correspondence
should be addressed
ACKNOWLEDGEMENTS
REFERENCES 1. 2. 3. 4.
Fincham,J.R.S. (1989) Microbiol. Revs. 53, 148-170. Case,M.E. et al. (1979) Proc. Natl. Acad. Sci. USA 76, 5259-5263. Davis,R.H. and de Serres,F.J. (1970) Methods Enzymol. 17, 79-143. Staben,C. et al. (1989) Fungal Genet. Newslett. 36, 79-81.
A
1 2 3 4 5
B
1
23 45 -_.
..
_
-.
23 1 94
6-E
2.3 2.0
Figure 1. Southern blot hybridization of genomic DNA of N. crassa transformants digested with EcoRI. The blot was first hybridized with a 2.2 kb BamHI fragment encoding the qa-2+ gene (A) and (after removal of the probe) with Bluescript vector DNA (B). In panels A and B, the lanes contain DNA (5 ug) as follows: 1, untransformed recipient strain; 2 to 5, DNA from transformants E-17, E-20, E-21 and E-22, respectively. Numbers on the left show position of size markers in kb.
