FEMS Microbiology Letters 156 (1997) 95^99
High frequency gene transfer by microprojectile bombardment of intact conidia from the entomopathogenic fungus Paecilomyces fumosoroseus
Cristine Chaves Barreto a, Luciano Cardoso Alves a, Francisco Joseè Lima Aragaìo b, El|èbio Rech b, Augusto Schrank c, Marilene Henning Vainstein a * ;
Departamento de Biologia Celular, Universidade de Bras|èlia, 70910-900 Bras|èlia, DF, Brazil b Cenargen/Embrapa, 70849-970 Bras|èlia, DF, Brazil Centro de Biotecnologia, Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 90540-000 Porto Alegre, RS, Brazil a
c
Received 2 September 1997; accepted 3 September 1997
Abstract
Two different methods, (i) PEG and (ii) biolistic, were employed to transform protoplasts and conidia of Paecilomyces using hygromycin resistance as selectable marker. Transformation frequencies varied from 1.9 to 2.5 transformants Wg31 of DNA by the PEG method, and from 33 to 153 transformants Wg31 of DNA by the biolistic procedure. fumosoroseus
Keywords: Paecilomyces fumosoroseus ;
Fungal transformation; Biolistic; Hygromycin B resistance; Microprojectile bombardment
1. Introduction Paecilomyces fumosoroseus is known mainly as an insect parasite with a wide host range and a worldwide distribution. Together with Metarhizium anisopliae and Beauveria species, P. fumosoroseus has been used in the biological control of insects. The development of e¤cient transformation systems for entomopathogenic fungi is essential to study the mechanisms of virulence and host speci¢city and to construct
* Corresponding author. Centro de Biotecnologia, Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 90540-000 Porto Alegre, RS, Brazil. Tel.: +55 (51) 316-6071; Fax: +55 (51) 319-1079; E-mail:
[email protected]
geneallytic manipulated strains with improved capabilities of biological control [1]. Vector-mediated transformation has been reported for many ¢lamentous fungi using either dominant selectable markers or complementation of auxotrophic mutants with the speci¢c wild-type gene [2,3]. Hygromycin B (HmB) is an aminoglycoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes by interfering with translocation and by causing misreading [4]. An HmB resistance gene, encoding an HmB phosphotransferase which inactivates the antibiotic by phosphorylation, has been isolated from Escherichia coli [5]. The use of HmB resistance as a dominant selection marker has the advantage that, in contrast to dominant vectors based on benomyl resistance or
0378-1097 / 97 / $17.00 ß 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 0 8 - 4
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C. Chaves Barreto et al. / FEMS Microbiology Letters 156 (1997) 95^99
Table 1 E¡ect of the osmotic stabiliser and di¡erent lytic enzymes on the production and regeneration of protoplasts from
P. fumosoroseus
Osmotic stabiliser
Enzyme
Number of protoplasts per mg of mycelium ( 104 )
Protoplast regeneration (%)
KCl 0.7 M KCl 0.7 M KCl 0.8 M KCl 0.8 M Mannitol 1.2 M Mannitol 1.2 M Sorbitol 1.2 M Sorbitol 1.2 M
LETa Novozym/cellulaseb LET Novozym/cellulase LET Novozym/cellulase LET Novozym/cellulase
9.82 6.17 5.26 1.30 3.77 1.96 7.87 1.42
5.6 19.2 2.46 12.2 2.20 9.5 16.23 32.30
a b
U
Lysing enzyme from Trichoderma harzianum (Sigma). Both enzymes at 1 mg ml31 . The results are means of at least three determinations each corresponding to two independent experiments.
acetamide metabolism, it does not interfere with other cellular or metabolic functions of the transformants. The most common method for fungal transformation requires the production of protoplasts, its mixing with a plasmid that usually contains the gene of interest and an appropriate promoter sequence, and the fusing of protoplasts. While sometimes e¤cient transformation is achieved with protoplasts, their preparation demands a careful monitoring of the individual steps, along with the optimisation of conditions for each batch of cell wall-degrading enzymes. Other procedures have also been used: treatment with alkali metal ions [6] or with glass beads [7] may permit direct uptake of DNA without the requirement of protoplasts. Recently, biolistic transformation has become available, and there are some reports of its use for ¢lamentous fungi [8^12]. In this paper we report transformation of P. fumosoroseus by biolistic bombardment of conidia as compared with a protoplast-mediated procedure. We demonstrate that either method results in integrative transformation of recipient strains; however, biolistic protocols produced a higher transformation e¤ciency.
2. Materials and methods
2.1. Plasmid, Paecilomyces strains and growth, and DNA manipulation P. fumosoroseus, isolated in Brazil and provided by the Fungal Collection of the Microbiology Laboratory at Brasilia University, was the recipient in transformation experiments. DNA from plasmid pAN7-1, bearing an E. coli HmB resistance gene (hph) £anked upstream by the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and downstream by the A. nidulans trpC transcription termination signals [13], was used for transformation. Malt yeast glucose medium (MYG) was used to grow P. fumosoroseus. To select transformants, 150 Wg ml31 of HmB was added to the medium. 2.2. Transformation of P. fumosoroseus For protoplast preparation, spore suspensions were treated with a solution of 0.1 M phosphate bu¡er pH 7.5 and Tween 80 (0.01%) for 3 h at 28³C, followed by incubation in 0.1 M phosphate
Table 2 Activity of chitinase, L-glucanase and protease of the lytic enzymes used for protoplast production Lytic enzyme LETa Novozym/cellulase (1:1)
L-Glucanase (U mg protein31 ) 1.41 1032 2.17 1032
U U
Chitinase (U mg protein31 )
Protease (U mg protein31 )
432.36 133.46
5.40 11.58
Lysing enzyme from Trichoderma harzianum (Sigma). The results are means of at least three determinations each corresponding to two independent experiments.
a
C. Chaves Barreto et al. / FEMS Microbiology Letters 156 (1997) 95^99
97
Fig. 1. Hybridisation patterns of P. fumosoroseus wild-type and some transformants probed with 32 P-labelled pAN7.1. Molecular mass in kb is indicated on the left. A: Southern blot of HindIII- and EcoRI-digested pAN7.1 (lanes 1 and 2) and of EcoRV-, HindIII- and EcoRI-digested DNA from wild-type strain (lanes 3, 4 and 5, respectively) and from biolistic transformants TB1 (lanes 6, 7 and 8), TB2 (lanes 9, 10 and 11) and TB3 (lanes 12 and 13). B: Southern blot of HindIII- and EcoRI-digested DNA from PEG transformants TP4 (lanes 1 and 2) and TP5 (lanes 3 and 4).
bu¡er pH 7.5 for a further 3 h at 28³C. This prior spore treatment ensures a complete spore germination when inoculated in liquid medium to produce mycelium. Protoplasts were prepared from 7.5 mg of mycelia in 1 ml of the appropriate osmotic stabiliser, either KCl (0.7 or 0.8 M), mannitol (1.2 M) or sorbitol (1.2 M), and 1 mg of a mixture of Novozym (NovoZym 234, Novo BioLabs) and cellulase (Boehringer Mannheim GmbH) (1:1), and incubated at 30³C for 3 h with gentle shaking. For transformation by the polyethylene glycol (PEG) method, protoplasts were resuspended in 0.7 M KCl/50 mM CaCl2 and incubated at room temperature for 20 min. The protoplasts were mixed with uncut plasmid DNA (10 Wg) and kept on ice for 20 min. 1 ml of PEG solution (PEG 6000 50%/50 mM glycine/50 mM CaCl2 /1.2 M sorbitol) was added and the incubation proceeded for a further 20 min. Regeneration medium was MYG osmotically stabilised with 1.2 M sorbitol. Plates were incubated at 28³C and HmB-resistant
Table 3 E¡ect of the method on transformation e¤ciency Transformation method
Number of spores ( 107 )
E¤ciency (transf. per Wg DNA)
PEG Biolistic Biolistic Biolistic Biolistic
^ 0.5 1.0 2.0 3.0
1.9^2.5 33^45 64^70 120^134 140^153
U
The results are means of at least three determinations each corresponding to two independent experiments.
colonies were searched for over a period of 8^25 days. For the biolistic method, intact conidia were used. 5^30 Wl of a conidial suspension (109 ml31 ) were placed in the centre of a Petri dish for bombardment. Microparticles were prepared essentially as previously described [9]. Immediately after bombardment, the suspension of conidia was transferred to 5 ml of MYG and incubated at 28³C for 18 h. The conidia were collected by centrifugation and resuspended in 2 ml of MYG. Aliquots (200 Wl) were plated on selective medium and re-incubated at 28³C. 2.3. Isolation and manipulation of DNA
DNA was extracted from mycelia by the Raeder and Broda method [14]. Standard procedures for restriction endonuclease digestion, agarose gel electrophoresis and Southern blotting were carried out as described by Sambrook et al. [15]. 3. Results and discussion
To transform P. fumosoroseus, both the standard PEG protocol and a biolistic procedure were employed. To produce protoplasts from this fungus, 0.7 M KCl was the best osmotic stabiliser and LET (lysing enzymes from Trichoderma harzianum, Sigma) the best cell wall-degrading enzyme. However, for protoplast regeneration, 1.2 M sorbitol and a mixture of Novozym and cellulase were far more e¡ective (Table 1). To understand the better
C. Chaves Barreto et al. / FEMS Microbiology Letters 156 (1997) 95^99
98
lytic capacity of LET in comparison to Novozym
L-glucanase
and cellulase, their chitinase,
coincided with high molecular mass DNA and no
and pro-
evidence of fast migrating plasmid DNA homolo-
tease activities were assayed. As shown in Table 2,
gous to the probe was found. To explore the struc-
the chitinase-speci¢c activity of LET is much higher
ture of integrated plasmid DNA in more detail, ge-
HindIII,
than that found for the Novozym/cellulase mixture.
nomic DNA was digested with
Since chitin is the most abundant polysaccharide of
the pAN7-1 vector once. Analysis of randomly se-
the fungal cell wall, it may explain LET's better lys-
lected
ing capacity and the lower protoplast regeneration
patterns of integration. Most transformants exhib-
achieved
ited
the
during
the
transformation
same by
incubation
the
PEG
period.
method,
For
0.7
M
a
transformants
more
integration
plasts and 1.2 M sorbitol was used for protoplast
vector.
Even
though
optimal
pattern,
or
complex
independent
of
the
sites
of
tandem
arrays
of
the
pAN7-1
conditions
A major advantage of microprojectile bombard-
were given for production and regeneration of pro-
ment is that protoplast formation is not necessary,
toplasts,
thus providing a fast and easy scale-up method for
transformation
the
complex
simple
method used for transformation, suggesting multiple
KCl/Novozym/cellulase was used to produce proto-
regeneration.
revealed
which cuts
e¤ciencies
by
the
PEG
method were very low, varying from 1.9 to 2.5 transformants per formation
Wg
frequencies
hamper
the
application
of
such molecular methods as gene isolation and gene disruption.
As
transformation of intact conidia.
DNA (Table 3). These low trans-
an
alternative
method
to
Acknowledgments
increase
transformation frequencies and to avoid the proto-
This
work
received
¢nancial
support
from
plast preparation step, the biolistic method has been
PADCT, CNPq and FAPDF. C.C.B. is the recipient
used to transform ¢lamentous fungi [8^11]. By using
of a fellowship from CNPq and L.C.A. from PIBIC-
this method, we demonstrated that conidia of
UnB.
lomyces
Paeci-
can be e¤ciently transformed. Transform-
ants were recovered at a frequency of 33^153 DNA,
depending
on
the
number
of
spores
Wg31
bom-
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