Curr Genet (2004) 45: 54–60
DOI 10.1007/s00294-003-0454-8
TECHNICAL NOTE
Susanne Zeilinger
Gene disruption in Trichoderma atroviride via
Agrobacterium -mediated transformation
Received: 10 July 2003 / Revised: 21 September 2003 / Accepted: 24 September 2003 / Published online: 29 October 2003

Springer-Verlag 2003
Abstract A modified Agrobacterium-mediated transfor-
mation method for the efficient disruption of two genes
encoding signaling compounds of the mycoparasite
Trichoderma atroviride is described, using the hph gene
of Escherichia coli as selection marker. The transfor-
mation vectors contained about 1 kb of 5¢ and 3¢ non-
coding regions from the tmk1 (encoding a MAP kinase)
or tga3 (encoding an a-subunit of a heterotrimeric G
protein) target loci flanking a selection marker. Trans-
formation of fungal conidia and selection on hygromy-
cin-containing media applying an overlay-based
procedure, which overcomes the lack of formation of
distinct single colonies by the fungus, led to stable clones
for both disruption constructs. Southern and PCR
analyses proved gene disruption by single-copy homol-
ogous integration with a frequency of approximately
60% for both genes; and the loss of tmk1 and tga3
transcript formation in the disruptants was demon-
strated by RT-PCR.
Keywords Agrobacterium-mediated transformation Æ
Gene disruption Æ Trichoderma atroviride Æ
Mycoparasitism
Introduction
Trichoderma atroviride strains are among the most
extensively studied and applied fungal biocontrol agents
because of their ability to antagonize soil-borne plant
Communicated by J. Heitman
S. Zeilinger
Division of Applied Biochemistry and Gene Technology,
Institute for Chemical Engineering,
Vienna University of Technology,
Getreidemarkt 9,
1060 Vienna, Austria
E-mail: szeiling@mail.zserv.tuwien.ac.at
Tel.: +43-1-5880117256
Fax: +43-1-5816266
pathogens (Harman and Björkman 1998; Kubicek et al.
2001). One problem with understanding the mechanisms
underlying biological control in T. atroviride is that the
process of gene disruption and gene replacement,
essential for a functional analysis of candidate genes, is
difficult to achieve in this fungus. While gene disruption
mutants in T. reesei are quite easily obtained using
protoplasts and the calcium/polyethylene glycol proce-
dure for transformation [e.g. Suominen et al. (1993) re-
ported replacement frequencies of 32–52%],
homologous recombination in T. atroviride has only
been successful in very few cases and with low efficiency.
When using conventional protoplasts or biolistic trans-
formation techniques, only three genes (ech42, seb1,
nag1) of T. atroviride have been disrupted (Carsolio
et al. 1999; Woo et al. 1999; Peterbauer et al. 2002;
Brunner et al. 2003). This problem becomes even more
serious when gene disruption results in pleiotropic ef-
fects, e.g. in a reduction in viability. For example, from
several rounds of transformation of T. atroviride pro-
toplasts with a disruption cassette for seb1 (encoding a
transcription factor involved in the response to high
osmolarity stress), only a single disruptant was isolated
(Peterbauer et al. 2002; C. Peterbauer, personal com-
munication). To circumvent this problem, Rocha-Ra-
mirez et al. (2002) applied the expression of an antisense
tga1 cDNA under the control of a derepressible pro-
moter because, in an attempt to disrupt the tga1 gene
(encoding an a-subunit of a heterotrimeric G protein) of
T. atroviride, it was only possible to obtain transfor-
mants either carrying the construct integrated ectopi-
cally or containing more than one nucleus with at least
one carrying a wild-type copy of the gene.
The development of a reliable transformation system
for efficient gene disruption in T. atroviride is a pre-
requisite for improving the understanding of its genetics
and molecular biology, potentially leading to enhanced
application of this fungus as a biocontrol agent.
Here, a description is given for an alternative tech-
nique for the disruption of two genes involved in signal
transduction in T. atroviride [tmk1 (encoding a MAP

kinase) and tga3 (encoding an a-subunit of a heterotri-
meric G protein)], based on Agrobacterium tumefaciens
T-DNA transfer.
Agrobacterium-mediated transformation was origi-
nally developed for plants, but has been applied suc-
cessfully to several fungi (De Groot et al. 1998; Covert
et al. 2001; Malonek and Meinhardt 2001; Mullins et al.
2001). T-DNA integrates into the fungal genome at a
random position by illegitimate recombination and has a
100- to 1,000-fold higher efficiency than conventional
transformation methods (De Groot et al. 1998). As
A. tumefaciens can introduce DNA into intact fungal
cells, such as hyphae and spores (De Groot et al. 1998),
it is not necessary to use protoplasts as recipients. Using
intact cells reduces both the number of manipulations
required and the equipment needed. Integration of
T-DNA via homologous recombination has been de-
scribed for some filamentous fungi, e.g. Aspergillus
awamori (Gouka et al. 1999), Mycosphaerella gramini-
cola (Zwiers and De Waard 2001), and Glarea lozoyensis
(Zhang et al. 2003).
The Agrobacterium-mediated transformation method
described here offers an efficient and easy-to-use tool for
generating disruptant strains by homologous recombi-
nation in the economically important biocontrol fungus
T. atroviride P1.
Materials and methods
Strains and culture conditions
T. atroviride strain P1 (formerly T. harzianum, ATCC 74058;
Kullnig et al. 2001) was grown on potato-dextrose agar (PDA) at
28 C until sporulation. A conidial suspension was prepared by
harvesting the conidia from the plate, suspending them in 0.9 M
NaCl, and separating them from mycelial carryover by filtration
through a column filled with glasswool.
Agrobacterium tumefaciens strain LBA1100, carrying the viru-
lence (vir) genes essential for T-DNA transfer, and binary vector
pTAS5 were kindly provided by Paul Hooykaas (Leiden Univer-
sity, The Netherlands). LBA1100 was routinely grown on LB agar
containing spectinomycin (250 lg ml)1).
For plasmid constructions, Escherichia coli strain JM109 was
used and grown on LB agar supplemented with ampicillin (100 lg
ml)1) or kanamycin (50 lg ml)1) as appropriate (Sambrook et al
1989).
Construction of disruption plasmids
For the construction of pTSZ-Dtmk1, a ca. 1.1-kb HindIII/XbaI
fragment containing the 5¢ flanking sequences of the T. atroviride
MAP kinase gene (tmk1; AF452096) was obtained by PCR-ampli-
fication, using genomic DNA of T. atroviride as template and primers
ptmk1(F)/HindIII (5¢-ATAAGCTTGGTTTC GTTTCAACAAA
AGA-3¢) and ptmk1(R)/XbaI (5¢-ATTCTAG AGACTGCCGTT
GTGGTT-3¢). After digestion with the appropriate restriction en-
zymes, the fragment was inserted into HindIII/XbaI-cut pAN 7-1
(Punt et al. 1987). Subsequently, the resulting plasmid was digested
with BglII/StuI and a ca. 1-kb fragment containing the 3¢ flanking
sequences of the tmk1 gene was obtained by PCR amplification
with primers ttmk1(F)/StuI (5¢-TAAGGCCTTACCAAGA GAT
TATGCGGTA-3¢) and ttmk1(R)/BglII (5¢-TAAGATCTACTAC
GATTGGATGAAAACAGC-3¢) and inserted. The whole
55
disruption cassette was then excised from the resulting plasmid by
digestion with HindIII/BglII. The BglII site was blunted and the
cassette was ligated with the 7,990-bp backbone from pTAS5, after
HindIII/KpnI digestion and blunting of the KpnI site, resulting in the
binary disruption vector pTSZ-Dtmk1. For the construction of
pTSZ-Dtga3, a ca. 1.9-kb HindIII/XbaI fragment containing the 5¢
flanking sequences of the T. atroviride a subunit of a heterotrimeric G
protein gene (tga3; AF452097) was obtained by PCR-amplification,
using genomic DNA of T. atroviride as template and primers
ptga3(F)/HindIII (5¢-ATAAGCTTGTGCCTAGTCGCTAGG-3¢)
and ptga3(R)/XbaI (5¢-TATCTAGACTGTCGGCGTTCTTT
GAGG-3¢). It was then inserted into pAN 7-1, as described above.
The resulting plasmid was digested with BglII/StuI and a ca. 1.2-kb
fragment containing the 3¢ flanking sequences of the tga3 gene
obtained by PCR-amplification with primers ttga3(F)/StuI (5¢-AT-
AGGCCTAAAGCGCGTTATGACC-3¢) and ttga3(R)/BglII
(5¢-ATAGATCTAGTATATTAATATAAATGACCGC-3¢) was
inserted. The whole disruption cassette was then excised as described
above and fused with the 7,990-bp backbone of pTAS5 by HindIII/
blunt-end ligation, resulting in the binary disruption vector pTSZ-
Dtga3. Figure 1 outlines the design of the constructs used for targeted
mutations of the tmk1 and tga3 genes.
Finally, both constructed disruption vectors were transformed
into A. tumefaciens LBA1100 by electroporation (Mozo and
Hooykaas 1991) and transformants were selected on LB agar with
spectinomycin (250 lg ml)1) and kanamycin (100 lg ml)1). Agro-
bacterium strains containing the respective binary vectors were
identified by PCR, applying primers ptmk1(F)/HindIII and
ttmk1(R)/BglII for identification of pTSZ-Dtmk1 and primers
ptga3(F)/HindIII and ttga3(R)/BglII for identification of pTSZ-
Dtga3.
A. tumefaciens-mediated transformation
The transformation procedure is based on previous protocols
(Bundock et al. 1995; De Groot et al. 1998; Zwiers and De Waard
2001), with some essential modifications described in the following.
LBA1100-derived strains transformed with pTSZ-Dtmk1 or pTSZ-
Dtga3 were grown as described by Zwiers and De Waard (2001).
For co-cultivation, 100 ll of A. tumefaciens culture with an optical
density at 660 nm (OD660) of 0.3, grown in both the presence and
absence of acetosyringone (AS; 200 lM), was mixed with 100 ll of
a conidial suspension (106, 107, or 108 ml)1) from T. atroviride P1
and the mixture was plated onto sterile cellophane discs placed on
induction medium (Zwiers and De Waard 2001) with or without
200 lM AS and then incubated at 25 C. After growth on induc-
tion medium, the cellophane discs were transferred to selection
medium [PDA plates supplemented with 200 lg hygromycin B
(HygB) ml)1 (with 200 lM cefotaxime to inhibit growth of
A. tumefaciens)] and covered with 5 ml of an overlay consisting of
PDA with 200 lg HygB ml)1. Colonies appearing after incubation
at 28 C were transferred to PDA with 200 lg HygB ml)1 and
afterwards purified to mitotic stability by two rounds of single-
spore isolation.
DNA and RNA manipulations
Genomic DNA was isolated as described by Gruber et al. (1990); and
RNA was isolated as described by Chomczynski and Sacchi
(1987). Southern hybridizations were carried out as described by
Sambrook et al. (1989). RT-PCR analysis was performed using primer
pairs tmk1strF222–242 (5¢-GGTTGCCATCAAGAAGATCAC-3¢)
and tmk1strR608–628 (5¢-GATCTCGGTGCAGAACATTGG-3¢),
or tga3strF63–84 (5¢-CGACAAAGAGTTGGAC CAGGAC-3¢) and
tga3strR468–488 (5¢-GCTCCATATGGCCTGCACAGC-3¢), respec-
tively.
For proving the intactness of the loci 5¢ and 3¢ of the integrated
disruption constructs, PCR analysis was performed with one pri-
mer positioned in the hph marker gene and the second in the 5¢
56

or 3¢ non-coding regions of the tmk1 or tga3 genes outside the

sequences used for construction of the respective disruption
vectors: hphF (5¢-CGACGTCTGTCGAGAAGTTTCTG-3¢) and
hphR (5¢-GCCAGTGATACACATGGGGATC-3¢), Ptmk1(5¢-CC
GTTCCCAAGGCCAGCTG-3¢) and Ttmk1 (5¢-GTCGACCTG
CAGGTCAACGGATC-3¢), or Ptga3 (5¢-CA CAGGACGGGA
CAGCGATTC-3¢) and Ttga3 (5¢-CAACAACACAACAGCAG-
CAC-3¢). As control, PCR fragments from the native tmk1 or tga3
gene loci were amplified using DNA from wild-type strain P1 as
template and primer pairs Ptmk1 and tmk1R (5¢-GGTGCTTGA
GGGCATCCTC-3¢) or Ttmk1 and tmk1F (5¢-GA GGATGCC
CTCAAGCACC-3¢) for tmk1 and using Ptga3 and tga3R
(5¢-GTGGCCTGGGTAAGACTGTGATG-3¢) or Ttga3 and
tga3F (5¢-CATCACAGTCTTACCCAGGCCA-3¢) for tga3.

Results and discussion

Construction of disruption vectors

To create A. tumefaciens vectors suitable for gene dis-

ruption in T. atroviride, the respective disruption cas-
settes were constructed in plasmid pAN7-1 (Punt et al.
1987), containing the E. coli hph (HygB phosphotrans-
ferase-encoding) gene under control of the A. nidulans
gpdA promoter and trpC terminator. Subsequently, the
whole disruption construct consisting of the
pgpdA::hph::ttrpC marker cassette flanked by at least
1 kb of 5¢ and 3¢ non-coding sequences of the tmk1 and
tga3 genes, respectively, was inserted between the left
and right border repeats of the binary vector pTAS5
(Fig. 1). The resulting plasmids, pTSZ-Dtmk1 and
pTSZ-Dtga3, were subsequently transformed into
A. tumefaciens strain LB1100 and positive Agrobacte-
rium transformants identified by PCR, as described in
the Materials and methods section.

Optimization of conditions for Agrobacterium-mediated

transformation of T. atroviride
To test the Agrobacterium-mediated transformation of
T. atroviride, fungal conidia (106, 107, or 108 ml)1,
respectively) were co-cultivated with the generated
A. tumefaciens strains containing the respective disrup-
tion constructs. Initial transformation experiments were
carried out by plating the co-cultivations onto sterile
nitrocellulose membranes placed on induction medium
with or without AS (an inducer of the A. tumefaciens vir
genes) and incubating at 25 C for 2–3 days as described
for other filamentous fungi (De Groot et al. 1998;
Gouka et al. 1999; Covert et al. 2001; Mullins et al.
2001). After 24 h (when 107 conidia were applied in the
co-cultivation) or 48 h (in the case of 105 and 106 coni-
dia) the membranes were completely covered with a
mycelial lawn, even after transferring the discs to selec-
tive medium, making it impossible to distinguish real
transformants from the background. For this reason,
the hitherto-described procedure had to be modified and
adapted to the fast growth of T. atroviride on Agro-
bacterium induction medium. First, to facilitate the
Fig. 1 Physical maps of the disruption cassettes of pTSZ-Dtmk1
(A) and pTSZ-Dtga3 (B) and schematic representations of the
respective wild-type and disrupted loci after the homologous
integration events by double cross-over. A ApaI, Ac ClaI, E
EcoRI, H HindIII, N NheI, S StuI, Sm SmaI, X XbaI. LB Left
border, RB right border. P 5¢ non-coding region, T 3¢ non-coding
region of the respective gene. Grey arrows indicate the primers used
for characterization of the disruptants (a Ptmk1, b tmk1R, b¢
tmk1F, c Ttmk1, d hphR, e hphF, f Ptga3, g tga3R, g¢ tga3F, h
Ttga3)
detection of fungal colonies, sterile transparent cello-
phane discs were used instead of the white nitrocellulose
membranes and second, after co-cultivation on induc-
tion medium, the cellophane discs were transferred to
selection medium and immediately covered with 5 ml of
an overlay consisting of PDA with 200 lg ml)1 HygB.
After 57 days, small clearly visible colonies appeared on
top of those plates originating from co-cultivation in the
presence of AS, whereas no HygB-resistant colonies
could be detected from co-cultivation experiments in the
absence of AS or from controls of T. atroviride conidia
alone. In two independent experiments and with both

vectors, pTSZ-Dtmk1 and pTSZ-Dtga3, most transfor-
mants were obtained using 107 conidia, even though the
co-cultivation for this condition was carried out for 24 h
only. This is somewhat different to the report for
Agrobacterium-mediated transformation of Fusarium
oxysporum, where Mullins et al. (2001) showed that the
co-cultivation period correlated positively with the effi-
ciency of transformation. No significant difference ex-
isted in the number of transformants between the two
constructs. For both pTSZ-Dtmk1 and pTSZ-Dtga3, the
transformation frequency was in the range 30–
50 transformants in 107 conidia, while 1–5 transfor-
mants could be detected on the plates originating from
106 conidia and no colonies appeared on the plates with
105 conidia. De Groot et al. (1998) reported a trans-
formation frequency of 240 transformants in 107 conidia
for T. reesei when the fungus was transformed by
Agrobacterium-mediated transformation with a plasmid
bearing the hph resistance gene under control of the
gpdA promoter and trpC terminator sequences from
Fig. 2A–D Southern blot analysis. Genomic DNA was isolated
from randomly chosen hygromycin B-resistant strains originating
from Agrobacterium-mediated transformation with plasmids pTSZ-
Dtmk1 (A) or pTSZ-Dtga3 (B), and the T. atroviride wild-type strain
P1. The DNA was digested with EcoRI (A, C), ApaI/NheI (B) or
SmaI/ClaI (D) and probed with the respective 32P-labeled disrup-
tion cassettes given in Fig. 1. Arrows in A indicate the 3.2-kb and
4.2-kb bands originating from a double cross-over event at the
tmk1 locus. Arrows in C indicate the 4.1-kb and 5.7-kb bands
originating from a double cross-over at the tga3 locus. The
positions of molecular DNA size markers are shown on the right
57
Aspergillus nidulans but without any sequences homol-
ogous to the Trichoderma genome.
Highly efficient disruption of tmk1 and tga3 by targeted
integration through Agrobacterium-mediated
transformation
About 50 T. atroviride transformants from each con-
struct were recovered and purified to mitotic stability by
repeated transfer to selective medium and two rounds of
single spore isolation. Southern blot analyses of 12
randomly chosen HygB-resistant strains originating
from transformation with the pTSZ-Dtmk1 disruption
construct and hybridization with the whole tmk1 dis-
ruption cassette as probe (Fig. 1A) showed that the
disruption cassette had integrated into the T. atroviride
genome in all transformants tested. Seven transformants
(58%) showed strong indications for single-copy inte-
gration at the homologous tmk1 gene locus character-
ized by the appearance of bands at 3.2 kb and 4.2 kb
and the absence of the 5.1-kb EcoRI band observed in
untransformed T. atroviride P1 (Fig. 2A). Integration at
the tmk1 gene locus was further verified exemplarily by
ApaI/NheI digestion of genomic DNA isolated from two
putative Dtmk1 disruptant strains. In both cases, the
internal 2.9-kb fragment obtained with DNA from the
wild type was replaced by bands at 4.2 kb and 1.1 kb
(Fig. 2B). In the case of pTSZ-Dtga3, eight transfor-
mants were chosen and analyzed by Southern blot, using
the whole tga3 disruption cassette as probe (Fig. 1B).
All of them had the construct integrated into the
58

genome, five of them (62%) with a single copy at the

homologous tga3 gene locus, as indicated by the
appearance of bands at 4.1 kb and 5.7 kb and the ab-
sence of the 7.6-kb band observed in untransformed
strain P1 after digestion of the genomic DNAs with
EcoRI (Fig. 2C). This result was exemplarily confirmed
for two putative Dtga3 disruptant strains by SmaI/ClaI
digestion, resulting in replacement of the internal 2.6-kb
fragment obtained when using wild-type DNA by a 4.8-
kb band (Fig. 2D). To test whether integration of the
disruption constructs occurred by a straight double-
crossover event without any deletions or recombina-
tions, the intactness of the 5¢ and 3¢ non-coding regions
of the respective genes was analyzed by PCR. To this
end, primers were chosen in the DNA regions upstream
and downstream of those used for the construction of
the disruption vectors (Fig. 1). PCR analysis with one
primer positioned in the hph reporter gene and the sec-
ond lying outside the disruption construct and DNA
from two of the Dtmk1 disruptant strains as template
yielded a 2.9-kb amplification product for the 5¢ region
and a 3.3-kb fragment for the 3¢ region, which can only
be obtained in those strains where the disruption con-
struct has integrated at the tmk1 gene locus, leaving
the 5¢ and 3¢ flanking regions intact (Fig. 3A). Two
randomly chosen positive Dtga3 disruptants were ana-
lyzed in the same way, yielding a 3.7-kb fragment using
primers hphF and Ptga3 for amplification of the 5¢ re-
gion and a 3.5-kb product when applying primers hphR
and Ttga3 for amplification of the 3¢
region (Fig. 3B).
Fig. 3 PCR analyses proving the knockout of the tmk1 (A) or tga3
In all cases, about 300 bp from the obtained DNA (B) gene by homologous recombination and the intactness of the
fragments containing the junctions between the inte- respective 5¢ and 3¢ regions outside the disruption constructs in two
grated construct and the respective native locus were disruptants. As controls, amplifications of the native gene loci were
sequenced, showing that no deletions and/or recombi- performed, using the genomic DNA of the wild-type strain as
template. The primer combinations used for PCR amplification are
nations had occurred during the crossover event. given above the agarose gel
These results show that the high efficiency of targeted
integration is not gene-specific, as Agrobacterium-medi-
Agrobacterium-mediated transformation, which is simi-
ated disruption of both the tmk1 and the tga3 gene oc-
lar to what is reported for polyethylene glycol-mediated
curred with about the same frequency. It should be
transformation of T. reesei protoplasts (Suominen et al.
noted that disruption of both genes led to pleiotropic
1993).
effects, which was previously shown to strongly impair
The absence of the tmk1 and tga3 transcripts in the
gene-knockout in T. atroviride (Peterbauer et al. 2002;
respective disruptants was confirmed by RT-PCR anal-
Rocha-Ramirez et al. 2002). Knockout of the tmk1 gene,
ysis, using primer pairs tmk1strF222–242 with tmk1str
encoding a MAP kinase, led to a slightly (about 15–
R608–628 and tga3strF63–84 with tga3strR468–488,
20%) reduced growth rate on PDA plates and an
aberrant sporulation pattern, similar to what is de-
scribed for T. virens (Mukherjee et al. 2003). Knockout
of tga3, encoding a Ga subunit, resulted in a heavily
changed colony morphology (e.g. hyperbranched hy-
phae), hypersporulation with dark green pigmented
conidia, and a reduced (about 55%) growth rate on
PDA (Fig. 4).
Generally, the frequency of homologous recombina-
tion leading to gene disruption in filamentous fungi is
influenced by the size of the homologous DNA flanking
the selectable marker and varies from one locus to an-
other (Bird and Bradshaw 1997). This report shows that, Fig. 4 Colony morphology of Dtmk1 and Dtga3 disruptant strains
for T. atroviride, about 1 kb at each side of the selection in comparison with the wild-type strain P1 when grown on potato-
marker is sufficient for homologous integration using dextrose agar for 7 days

Fig. 5A, B RT-PCR analysis of tmk1 and tga3 transcripts in wild-
type (gDNA) and disruptant (cDNA) strains. A Disruptant Dtmk1
(strains Dtmk1-1, 2, 4, 11, 12) and B disruptant Dtga3 (strains
Dtga3-1, 3, 5, 6) are shown. As controls, the 407-bp tmk1 and 426-
bp tga3 PCR products obtained with genomic DNA from the wild-
type strain T. atroviride P1 are shown (gDNA)
respectively. As shown in Fig. 5, a 281-bp band of the
tmk1 gene and a 321-bp band of the tga3 gene was
produced in the wild-type strain P1. In the Dtmk1 dis-
ruptants, the 281-bp tmk1 band is absent, whereas the
tga3 transcript is produced, proving the intactness of the
RNA preparation; and, in the Dtga3 strains, the 321-bp
tga3 band is missing, whereas the tmk1 gene is tran-
scribed normally.
In summary, the generation of T. atroviride disrup-
tants by homologous recombination using Agrobacte-
rium-mediated transformation is highly efficient and the
vast majority of the transformants originate from the
integration of a single copy of the disruption cassette.
These results make Agrobacterium-mediated transfor-
mation a very feasible alternative for gene disruption in
this economically important fungus.
Acknowledgements P.J.J. Hooykaas is appreciated for providing
A. tumefaciens strain LBA1100 and plasmid pTAS5. C.P. Kubicek
and R.L. Mach are acknowledged for providing the infrastructural
facilities and for critically reviewing the manuscript. This work was
supported by grants from the Austrian Programme for Advanced
Research and Technology of the Österreichische Akademie der
Wissenschaften and from Fonds zur Förderung Wissenschaftlicher
Forschung (P15483).
59
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