MICROBIOLOGY

LETTERS
FEMS Microbiology

Letters 137 (1996) 153- 158

An autonomously replicating plasmid transforms Botrytis cinerea

to phleomycin resistance
Milagrosa Santos a, Inmaculada Vallejo a, Laureana Rebordinos a3* ,
Santiago Gutih-rez b, Isidro G. Collado , Jestis M. Cantoral a
Laboratorio de Genhtica .v Microbiologia, Facultad de Ciencias de1 Mar, Unicersidad de Cridiz, Poligono de1 Rio San Pedro,
Puerto Real 11510, Chdiz, Spain
b Departamento de Microbiologia, Facultad de Ciencias Biolbgicas, Unitiersidad de Ledn, Ledn 24071, Spain
Departamento de Quimica Orgcinica, Facultad de Ciencias, Unicersidad de Chdiz. Poligono de1 Rio San Pedro, Puerto Real 11510,
Chdk, Spain
Received 3 1 October

1995; accepted 30 January

1996

Abstract
A transformation
system has been developed for the pathogen fungus Botrytis cinerea, based on the utilization of the
wide host plasmid pUT737 that contains the Sh ble gene, conferring resistance to phleomycin. Transformed protoplasts were
regenerated at lo-25 pg ml- of phleomycin, at a frequency of 25-40 transformants per kg of DNA, and they were
resistant up to 50 pg ml - . Southern hybridization using undigested and digested total DNA showed the presence of circular
autonomously replicating plasmid pUT737 in the transformants. Reisolated plasmid from transformed fungus transformed E.
coli and rescued plasmid was identified as pUT737. Transformants were grown for four generations under non-selective
conditions and replicative plasmids were still detected. Plasmids present in all transformants at this stage had been modified
from native pUT737 and showed the same size and configuration indicating that selection through stabilizing plasmid forms
has happened.
Keywords: Botyvtis cinerea; Phytopathogenic

fungus; Phleomycin

resistance;

1. Introduction
Borrytis cinerea is an economically
important
pathogen because it is the causal agent of the grey
mould on numerous crops. Development of a feasible and efficient transfomiation
system for this fungus is necessary in order to study the fungus-plant

* Corresponding
author. Tel.: + 34 (56) 470 859; Fax:
(56) 470 8 I I ; E-mail: cantoral@galeon.uca.es
0378-1097/96/$12.00
0 1996 Federation
PII SO378-1097(96)00043-2

of European

f34

Microbiological

Replicative

transformation;

Autonomously

replicating

plasmid

interaction, its pathogenicity and the genetic control

of some interesting characteristics.
The particul,ar constitution of the nuclear system
in Botrytis makes it difficult to obtain mutants [l]
and fungal transformation has to be tried using dominant selectable markers conferring resistance to antibiotics, instead of auxotrophic mutants as receptors
for the foreign DNA.
Low frequencies of 2-10 transformants per ,ug of
DNA have been achieved by Botryfis cinerea transformation with the hygromycin resistance gene after
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chromosomal integration of the plasmid [2]. and in

Bortyotinia fuckelianu after biolistic transformation
of conidia [3]. Nevertheless. transformation
by autonomously
replicating plasmids confers some advantages including reisolation of transforming plasmids and increased frequency of transformation. The
development of vectors for replicative transformation
has been tried by in vitro fusions of replicons as
origins of replication
(ori) from plasmids.
autonomously replicating sequences (0r.y). centromeric
regions (ten> or telomeric portions of chromosomes
[4,5]. However, its potential for transformation
of
filamentous fungi has been limited and hence growing interest is emerging around replicative plasmids
generated through in vivo recombination [6.7].
pUT737 plasmid conferring resistance to phleomycin displays some important characteristics: it can
be used both in E. coli and in fungus species.
important to analyze the transformants. and it can be
used as an expression vector. In this paper we report
a method for the transformation of B. cinerea with
pUT737 plasmid that improves the transformation
frequencies in this fungus and that keeps it as a
stable and autonomously replicating form inside the
fungus at least for four generations without selective
pressure.

2. Material and methods

2. I. Struins, media and plasmids
B. cinereu UCA 992 is a wild-type strain obtained
from Domecq (Jerez de la Frontera, Cgdiz, Spain). It
was used as a recipient in the transformation experiments and is deposited in the Universidad de Cjdiz.
Facultad de Ciencias Mycological Herbarium Collection (UCA) [8]. The fungus was grown on malt-agar
medium and mycelial cultures were obtained by
growing the fungi in shaken Czapeck-Dox
liquid
medium. Czapeck-Dox solid medium was used to
regenerate B. cinereu protoplasts. Plasmid pUT737
(Cayla, France) contains the sh ble gene conferring
phleomycin
resistance.
E. coli DH5a strain was
used for the maintenance
and propagation of the
plasmid. The plasmid was isolated from liquid culture by standard procedures [9].

2.2. Prrpurution
fiwmutioti

ofprotol,lcr.Fts urld B. cinereu trutw

Botrytis protoplasts were prepared according to

Cantoral et al. [IO] and adjusted to a final concentration of I-S X IO protoplast ml- as determined
microscopically with a hemocytometer. For B. cirwrpu transformation
SO ~1 of protoplast suspension
was mixed with 0.5-5 pg of plasmid DNA in 2-10
~1 of TE buffer and 12 ~1 PCM solution (25% PEG
8000, 50 mM CaCl,. 10 mM MOPS. pH 5.8). The
mixture was kept on ice for 30 min. 500 ~1 of the
same PCM solution was added, mixed gently and
incubated for 20 min at room temperature. Finally.
500 ~1 of I M sorbitol was added. Aliquots of 300
~1 from the reaction were added to 4 ml of molten
Czapeck-Dox medium pH 7.0 containing
I M sorbitol. 1.48 Difco agar and different amounts of
and immediately
overlaid onto solid
phleomycin
Czapeck-Dox medium with the same composition to
select transformants. Plates were incubated at 23C.

The standard
lowed [I I].

transformation

procedure

was fol-

2.4. B. cirlereu DNA isolution and DNA munipulation

The method followed for DNA isolation was described by Cantoral et al. [IO]. DNA manipulation
was according to standard techniques [9] using nylon
membranes for DNA hybridization. The probe consisted of the WI-DraIII
fragment of 395 pb from
pUT7.37 labelled by nick translation with Pl.
2.5. Mitotic stabili~
Conidiospores
from Botqiis
cinereu transformants were harvested from solid Czapeck medium
containing phleomycin.
They were resuspended in
0.9% NaCl and then plated on malt-agar medium to
allow sporulation.
Four consecutive
non-selective
platings were carried out before challenge
with
phleomycin
containing
medium,
to determine
whether or not they maintained the antibiotic resistance, and before analysis by hybridization.

M. Sanros et al. / FEMS Microbiology Letters 137 (19961 153-158

3. Results and discussion

3.1.

Transformation

phleomycin

of

Botrytis

cinerea

and

selection

The sensitivity of wild-type strain UCA 992 to

phleomycin was tested by hyphal transfer onto maltagar plates containing a range of phleomycin concentrations. 10 pg ml- of antibiotic was the minimal
antibiotic concentration that completely inhibited
growth of the fungal mycelium. Conidia were germinated on the same medium and appeared more resistant to phleomycin than mycelium so that at 10 pg
ml-r a low amount of conidia still germinated and
15 pg ml- was necessary for a complete inhibition
of the germination.
Protoplasts were plated onto solid Czapeck-Dox
medium containing 1.4% Difco agar, which produced both the best regeneration results and the
highest frequency of transformation (compared to 1.8
and 2% agar). High concentrations of agar have been
described to increase the number of abortive colonies
[2]. 40-50% of protoplasts regenerated after 2-3
days of plating medium free of antibiotics, however
regeneration was completely inhibited by 5 pg ml-
of phleomycin.
B. cinerea
protoplasts were transformed with
pUT737 plasmid and plated on Czapeck-Dox medium
containing 1 M sorbitol and lo-25 pg ml- of
phleomycin. We first used KC1 as an osmotic stabilizer but it interfered with phleomycin, in contrast
sorbitol did not show any interaction and from the
two concentrations tested, 1 and 1.2 M, the first
produced a higher number of regenerative transformants. Positive controls were non-transformed protoplasts plated on antibiotic free medium that regenerated at frequencies around 30% after PEG treatment,
while for negative controls, protoplasts were plated
on phleomycin containing medium and none regenerated.
Two kinds of transformed colonies appeared after
4-5 days: (i) large colonies that developed normally
on selective medium producing conidia and sclerotia.
These colonies represented 80% of the total colonies
and were the true transformants that grew up to 50
pg ml- of phleomycin. Conidia collected from
them were also resistant to 50 pg ml-. (ii> 20% of
transformants were false transformants and fall into

155

two categories. The first type were small colonies

apparently resistant to phleomycin but not able to
develop into mature transformants and to replicate
after subculturing in selective medium, these are
called abortive colonies. The other type of false
transformants grew normally but failed to produce
conidia and did not grow after subculturing. Although this phenotypic variability has been attributed
to the use of mycelium as the material for transformation and has been widely described in transformation of filamentous fungi [12], it has also been
described in fungi transformed using intact conidia
131.
The transformation frequency achieved was 25-40
transformants per pg of plasmid DNA, obtained
from several independent transformation experiments. This transformation frequency is higher than
that described for Botrytis cinerea using hygromicyn
resistance as a selectable marker [2].
3.2. Molecular analysis of transformants
The location of plasmid pUT737 DNA after transformation was studied by hybridization using the
32P-labelled SalI-DraIII fragment of 395 pb as a
probe. DNAs extracted from phleomycin resistant
Botrytis cinerea transformants, (i) intact DNA and
(ii) DNA digested with Hind111 or EcoRI, which
cleave pUT737 once to yield linear plasmid
monomers of 5.4 kb, were run on an agarose gel. No
hybridization was detected with total DNA from
untransformed Botrytis cinerea. However, clear hybridization bands were obtained with total DNA
from five transformants either without digestion or
digested with Hind111or EcoRI. While no hybridization signals were obtained on chromosomal DNA
two bands were seen corresponding in size to supercoiled pUT737 monomer and dimer running faster
than the chromosomal DNA. Long exposures never
revealed labelled chromosomal DNA. DNA digested
by Hind111 or EcoRI showed a single band corresponding in size to linear 5.4 kb of pUT737
monomer. Moreover no bands were seen which might
correspond to junction fragments, which would have
been expected if pUT737 were integrated into the
chromosome. Transformants 1, 3, 4 and 5 showed
different resistance levels although a relation between copy number and phleomycin resistance could

123456769

I
~,,
*nw?

were digested with HirzdIII or EcoRl and the fragment of 5.4 kb was produced (Fig. 21. Double digestions with these enzymes liberated two fragments of
4.3 and I. I kb as expected (not shown).

23
9.4

6.6
-5.4

3.4. Stubilig of phleomycin resistclnce und presence

of replicating ,free plasmid in B. cinerea trunsformun ts

2.3
2.0

Fig. I. Southern hybridization analysis of DNA

lrom H. CIIICIYCI

transformants.

l-5.

DNA

and the parental strain. Lane5

from transformants I-5.

7 and 9, partially

undigested

Lane 6. control 8. ciwrrcl.

digested DNA

from tranhformants

Lane\

I and 3,

respectively. Lane 8, total digestion from transformant 4. 5 kg 01

DNA

was loaded on each lane of 0.8% agarose gel. Molecular

size markers on the right are in kb and were obtained from

Hind111 digested ADNA.

The plasmid size iz, indicated hy the

arrow.

not be established. The lowest resistance was for

transformant 4 (to 20 pg ml ); 1, 3 and 5 resisted
40 pg ml- and interestingly transformant number
2, which showed an extra band, had the highest
resistance, to 50 pg ml . This band could correspond to the fastest one present in transformants
after four non-selective
generations (Fig. 11. Long
exposures of the autoradiograms
also showed the
extra band in transformants
I. 3. 4 and 5.
3.3. Trun,$wmation
lated plasmids

of Escherichiu

coli with reiso-

If free plasmids were responsible for fungal transformation it ought to be easy to reisolate by retransforming E. coli with transformant
DNA. E. coli
DHSa was transformed with total DNA isolated
from four Botrytis transformants that had shown the
presence of free plasmid by Southern blot analysis of
DNA. Colonies were selected on LB solid medium
containing
100 pg ml- ampicillin.
Controls selected on ampicillin supplemented medium did not
grow and competent cells transformed with pUT737
produced E. coli transformants
to a frequency of
106-10 transformants per pg of DNA.
Transformed
E. coli with fungal DNA was obtained at a low frequency of 100 colonies per pg of
DNA. Uncut plasmid DNA was extracted from transformants and compared to pure pUT737. Plasmids

Although meiotic instability of Neurosporu transformants, formerly attributed to autonomously replicating plasmids, was later explained as a result of
destruction of transformation-generated
repeats by
the RIP (repeat-induced point mutation) process [ 131,
it has been argued that transformation
with a free
plasmid could result in reduced stability of the transformed sequence, against the general mitotic stability
described in transformed fungi through chromosomal
integration [ 141. Botqtis transformants were subcultured in non-selective medium for four generations,
then transferred to antibiotic containing medium and
analyzed. All of them were resistant to phleomycin
and the size and/or colony morphology were indistinguishable from those of the wild-type strain and
the only difference was that growth rate was slower
than the wild-type. Southern blot hybridizations
of
uncut total DNA produced no signal corresponding
to chromosomal DNA, instead three bands migrating
above chromosomal DNA were detected.
The plasmid size was the same in the four analyzed transformants and bigger than the plasmid size
seen in Botrytis transformants from the first culture,
indicating that a kind of plasmid rearrangement,
likely through in vivo recombination,
and further
selection towards stabilization has occurred. Recombination apparently increased both copy number (Fig.

6.6 _
5.4 4.4 -

Fig. 2. Hybridization

of plasmid DNA

transformed with total DNA from

Bottyis

resistance to phleomycin. Lane I. pUT737

co/i transformants derived from
respectively.

extracted from
cinrrea

control. Lanes 2-3.

B. cinerea

4;

transformed by

transformants

E.
l-3,

M. Santos et al./ FEMS Microbiologp Letters 137 (1996) 153-158

A
-0

_23 CHR

1.57

ing a variety of phenomena such as DNA replication

and recombination
as well as for some applications
in gene cloning and in gene expression.

Acknowledgements

We thank Domecq (Jerez de la Frontera, Cadiz,

Spain) for the donation of the Botrytis cinerea strain
UCA 992. This work was supported by grants from
UCA (PRE- 1994) and CICYT (PB92- 110 1).

-0
_ CHR
- 23
-

9.4

6.6

4.4

Fig. 3. Southern blot hybridization of undigested DNA from B.

cinerea transformants
of first culture generation and after four
non-selective generations. 5 pg of DNA was loaded on each lane
of 0.7% agarose gel. 0 is the gel origin and CHR the position of
chromosomal DNA on the gel. (A) Lanes l-3, first order transformants 2, 3 and 4. Lane 4, untransformed
Botqvis cinerea. Lanes
5-7, transformants 2, 3 and 4 from antibiotic containing medium
on fifth growing generation, after four culture generations without
selective pressure. (B) Overexposed films. Lanes I-4, first culture
transformants.
Lanes 5-7, fifth order transformants
after four
culture generations.

3) and resistance level, so that after four generations

without selective pressure all transformants grew on
100 pg ml- of phleomycin
and contained some
modified plasmid forms indicating the importance of
recombination
towards effective replication and partition of plasmids.
Several strategies developed in vivo for replication of foreign extrachromosomal
DNA in fungi
have been reported including formation of concatemers of linearized plasmids in Ustilago maydis [ 151
and Phytophthora in&tans [ 161 and plasmid-plasmid recombination
to form a replicating dimer in
Aspergillus nidulans [ 171.
The mapping of the modified plasmid is in
progress and its analysis should be useful for study-

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Letters 137

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