MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467
DOI: 10.1046/J.1364-3703.2003.00191.X
Blackwell Publishing Ltd.
Agrobacterium tumefaciens mediated transformation of the
oomycete plant pathogen Phytophthora infestans
I R M A VI J N † A N D FRA N C I N E G O V E R S *
Laboratory of Phytopathology, Wageningen University, and Graduate School Experimental Plant Sciences, Binnenhaven 5, NL-6709 PD Wageningen, the Netherlands
SUMMARY
Agrobacterium tumefaciens is widely used for plant DNA transformation and, more recently, has also been used to transform
yeast and filamentous fungi. Here we present a protocol for
Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late blight. Binary
T-DNA vectors containing neomycin phosphotransferase ( npt )
and β-glucuronidase (gus) fused to oomycete transcriptional regulatory sequences were constructed. Seven days of co-cultivation
followed by transfer to a selective medium containing cefotaxim
to kill Agrobacterium and geneticin to select for transformants,
resulted in geneticin resistant colonies. Under optimal conditions
with Agrobacterium supplemented with a ternary plasmid carrying a constitutive virG gene and in the presence of acetosyringone as inducer, up to 30 transformants per 10 7 zoospores could
be obtained. The majority of these transformants contained a single T-DNA copy randomly integrated at a chromosomal locus.
Using a similar protocol, geneticin resistant transformants of two
other oomycetes species were obtained, Phytophthora palmivora
and Pythium ultimum.
I N T RO D U C T I O N
Phytophthora infestans causes late blight, a devastating disease
posing a world-wide threat to potato production (Agrios, 1997).
The pathogen belongs to the oomycetes, a class of fungus-like
organisms that was traditionally placed within the kingdom
‘fungi’. In the last few decades, however, molecular phylogenetic
analyses have clearly demonstrated that oomycetes evolved
completely independent from fungi and as a consequence their
ability to infect plants evolved independently in the two different
groups of pathogens. Besides P. infestans there are over 60 more
Phytophthora species, all of which are plant pathogens causing
a wide range of diseases on a large variety of plants (Erwin and
*Correspondence: E-mail: [email protected]
†Present address: CatchMabs BV, PO Box 134, NL-6700 AC Wageningen, the Netherlands.
© 2003 BLACKWELL PUBLISHING LTD
Ribeiro, 1996). In addition Pythium spp., that cause seedling dampingoff and root rot, and the destructive downy mildews (Peronosporaceae) belong to the oomycetes (Agrios, 1997). The mechanisms
that oomycetes exploit for infecting and colonizing plants are
largely unknown. In recent years numerous Phytophthora genes
have been cloned and sequenced but due to the diploid nature of
oomycetes, a functional gene analysis of these organisms is still
a major challenge (Kamoun, 2003; Latijnhouwers and Govers,
2003). Here we describe a novel and efficient transformation
method that makes use of Agrobacterium tumefaciens as vector
and that could greatly facilitate studies on the molecular mechanisms underlying pathogenicity in oomycetes.
Traditionally, the soil bacterium Agrobacterium tumefaciens is
widely used for plant DNA transformation (Hansen and Chilton,
1999). Naturally occurring A. tumefaciens strains cause crown
gall tumours by incorporating part of its Ti-plasmid, known as TDNA, into the host cell DNA. The T-DNA, which carries genes
involved in auxin and/or cytokinin biosynthesis, is flanked by
short imperfect direct repeats and integrates into the plant
nuclear genome at random positions. This process is dependent
on the expression of a set of virulence ( vir ) genes that are induced
by compounds secreted from wounded plant cells such as acetosyringone (AS) (Winans, 1992). This naturally occurring DNA
transfer system has been extensively modified, making Agrobacterium mediated DNA transformation the most suitable method
for the genetic modification of plants. In recent years it has been
shown that A. tumefaciens can transfer its T-DNA to a much
broader spectrum of host cells. Different yeast and filamentous
fungi, and even human cells can be modified by the integration
of T-DNA using A. tumeciens as a vehicle (Bundock et al., 1995;
Chen et al., 2000; Covert et al., 2001; De Groot et al., 1998;
Kunik et al., 2001; Zwiers and De Waard, 2001). For some filamentous fungi Agrobacterium mediated transformation appears
to be more efficient than traditionally used methods based on
DNA electroporation or DNA uptake in protoplasts mediated by
polyethylene glycol (PEG) (Hynes, 1996).
Foreign DNA can be transformed into Phytophthora using the
polyethylene glycol (PEG) mediated transformation protocol
developed by Judelson and Michelmore (1991). This method has
been successfully used for the expression of marker genes such
459
460
I. VIJN AND F. GOVERS
as β-glucuronidase (gus) in P. infestans (Judelson and Michelmore, 1991; Kamoun et al., 1998) and green fluorescent protein
(gfp) in Phytophthora palmivora (Van West et al. 1999a) and Phytophthora parasitica var. nicotianae (Bottin et al., 1999). In addition, the introduction of an endogenous gene, either in the sense
or antisense orientation, can induce gene silencing, enabling gene
function studies (Gaulin et al., 2002; Latijnhouwers and Govers, 2003;
Van West et al., 1999b). However, the PEG-mediated transformation method is tedious, with a low efficiency of 0.1–2 transformants per µg DNA per 108 protoplasts (Judelson and Michelmore,
1991; Van West et al., 1998). Recently, Cvitanich and Judelson
(2003) reported a stable transformation of P. infestans using
microprojectile bombardment. They obtained an average of 14
transformants/shot using 10 6 germinated sporangia and 1 µg of
vector DNA. Most primary transformants, however, were heterokaryons of transformed and wild-type nuclei, hence requiring an
extra step of generating single zoospore cultures. Moreover, electroporation seems to be feasible for the transfer of DNA into the
genome of oomycetes. Latijnhouwers and Govers (2003) electroporated zoospores of P. infestans whereas Weiland (2003) electroporated the protoplasts of Pythium aphanidermatum. In both
cases the transformants were stable and the efficiencies seemed
to be slightly higher than with PEG-mediated transformation.
We decided to develop an Agrobacterium mediated transformation protocol for oomycetes, mainly due to its ease in handling
and the good results obtained in yeast and fungi (Bundock et al.,
1995; Chen et al., 2000; Covert et al., 2001; De Groot et al.,
1998; Hynes, 1996; Zwiers and De Waard, 2001). Moreover, as
with microprojectile bombardment this method does not require
protoplasting, a step which has become problematic since
Novozyme 234 is no longer marketed. A satisfactory replacement
enzyme for protoplasting P. infestans has not yet been reported.
In this paper we show that P. infestans can be stably transformed
by A. tumefaciens, that the transferred T-DNA integrates randomly
into the genome and that most transformants have only one TDNA copy integrated. In addition, we show that A. tumefaciens
is able to transfer its T-DNA to Phytophthora palmivora and
Pythium ultimum.
RESULTS AND DISCUSSION
T-DNA transfer from Agrobacterium tumefaciens to
Phytophthora infestans
To establish whether A. tumefaciens transfers T-DNA to the
oomycete P. infestans, we constructed the binary vectors pNptII
(Fig. 2A) and pNptII-Gus (Fig. 3A). pNptII carries a T-DNA that
contains the neomycin phosphotransferase ( nptII ) gene driven by
the oomycete specific Bremia lactucae hsp70 promoter (Judelson
et al., 1991). pNptII-Gus was constructed by inserting into pNptII
the uidA (gus) gene of Escherichia coli under control of the
Fig. 1 Schematic outline of A. tumefaciens mediated transformation of
Phytophthora infestans and typical appearance of geneticin (G418) resistant
transformants. (A) Filter pieces transferred to selection plates after 7 days of
co-cultivation and (B) appearance of G418 resistant colonies after an
additional 7 days at 18 °C.
oomycete specific B. lactucae Ham34 promoter (Judelson et al.,
1991). Both constructs were introduced in the A. tumefaciens
strain LBA1100. In order to enhance T-DNA transfer we supplemented strain LBA1100 with a compatible plasmid carrying a
mutant virG gene (virGn45D) that acts as a constitutive inducer
of other vir genes (Van der Fits et al., 2000).
Co-cultivation of acetosyringone (AS) induced A. tumefaciens
LBA1100 containing pNptII with P. infestans zoospores that were
allowed to encyst and germinate (according to the scheme in
Fig. 1), resulted in the transfer of T-DNA to P. infestans as evidenced by the formation of geneticin resistant colonies. Cocultivation with non-induced A. tumefaciens did not result in resistant P. infestans colonies (Table 1). The introduction of a plasmid
carrying a constitutive virG gene into LBA1100 containing
pNptII diminished the requirement for AS. Geneticin resistant P.
infestans colonies were obtained, even without AS-induction.
However, induction with AS enhanced the efficiency of T-DNA
MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467 © 2003 BLACKWELL PUBLISHING LTD
A. tumefaciens mediated transformation of Phytophthora
461
Table 1 Genetic transformations of P. infestans strain H30P02 by A. tumefaciens LBA1100. Co-cultivation was performed for 7 days at 22 °C in the dark on Hybond
N+ membranes starting with 107 zoospores
Binary plasmid
Ternary plasmid
Acetosyringone
concentration in µg/mL
Average G-418R
colonies per
transformationa
pNptII
pNptII
pNptII
pNptII
pNptII
pNptII-Gus
pNptII-Gus
pNptII-Gus
—
—
pBBR1MCS.virGN54D
pBBR1MCS.virGN54D
pBBR1MCS.virGN54D
pBBR1MCS.virGN54D
pBBR1MCS.virGN54D
pBBR1MCS.virGN54D
0
200
0
100
200
0
100
200
0 (n = 4)
1.75 (n = 4)
1.75 (n = 4)
16.25 (n = 4)
18.75 (n = 4)
0 (n = 2)
7.5 (n = 2)
20 (n = 2)
Range in number
of transformants
0
0–5
0–5
10–30
10–30
0
0–10
10–30
a
As determined 10–14 days after transfer to medium containing geneticin (G-418) and averaged over the indicated number of transformations ( n).
Table 2 A. tumefaciens mediated transformation of different P. infestans
strainsa
Strain
Average G-418R
colonies/transformation
per 107 zoosporesb
Range in number
of transformants
88069
88133c
98014
98020
98027
H30P02
2 (n = 5)
20 (n = 2)
1 (n = 2)
8.5 (n = 2)
1 (n = 2)
24.4 (n = 5)
0–4
10 – 30
0–2
7 – 10
0–2
10 – 30
a
All experiments were performed using A. tumefaciens strain LBA1100
containing plasmid pNptII and supplemented with the constitutive virG gene,
and induced with 100 µM acetosyringone.
b
As determined 10–14 days after transfer to medium containing geneticin
(G-418) and averaged over the indicated number of transformations (n).
c
Low zoospore yield, about 106 per 15 cm Petri dish.
transfer to P. infestans several fold. For the A. tumefaciens strains
harbouring the pNptII-Gus construct, AS-induction was necessary
for T-DNA transfer and the efficiency even increased with higher
concentrations (Table 1).
The choice of P. infestans recipient strains and A. tumefaciens
strains, and also the type of filters used for co-cultivation were
critical for optimal transformation efficiencies. In this study six
different P. infestans strains were tested. From each strain geneticin
resistant transformants were obtained but the number of transformants differed greatly (Table 2). Two strains clearly gave the
highest transformation frequency per fixed number of zoospores,
88133 and H30P02. However, we preferred to use H30P02
because this strain sporulated much better than 88133 and the
number of zoospores that could be harvested from one 15 cm
Petri-dish was much higher. For co-cultivation two different kind
of filters were tested, Nytran and Hybond N+ membranes.
Although the transformants were obtained using both filters,
Hybond N+ membranes yielded two- to threefold the number of
transformants compared to Nytran filters. We have also tried
nitrocellulose filters, but P. infestans did not grow on these filters.
Two different A. tumefaciens strains were tested, LBA1100
(described above) and EHA105. Co-cultivation with AS-induced
EHA105 harbouring the pNptII construct also resulted in
geneticin resistant P. infestans colonies. The number of geneticin
resistant P. infestans colonies was comparable to the number
obtained by transformation with LBA1100 containing the plasmid carrying the constitutive virG gene (results not shown). Thus
far the effect on transformation efficiency after introduction of
the constitutive virG gene in EHA105 has not been tested.
Routinely, 10–30 transformants per 10 7 zoospores were
obtained when co-cultivation occurred on Hybond N + membranes with P. infestans strain H30P02, as recipient and A. tumefaciens strain LBA1100, containing the constitutive virG gene, to
mediate the T-DNA transfer. Typically, 2.5 × 107 zoospores can be
obtained from a 10-day-old P. infestans culture of strain H30P02
grown in a 15-cm Petri dish. With these conditions, a few hundred
transformants can easily be generated in one transformation
experiment.
A Southern blot analysis of over 15 independent transformants
confirmed that the nptII gene was integrated into the genome of
P. infestans. In Fig. 2B, Southern blot analysis of seven of these
is shown. Among the selected geneticin resistant colonies no
false positives were detected, i.e. there were no P. infestans colonies growing on geneticin that did not harbour the nptII gene.
In many of the transformants a single hybridizing band was
detected. In each transformant the size of the band differed, indicating that these transformants have a single copy of the nptII
gene integrated at random sites in the genome. In one transformant two hybridizing bands were observed (see Fig. 2B, lane 6)
presumably representing the integration of two copies of the
Npt II gene. Thus far, no more than two integrated copies were
© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467
462
I. VIJN AND F. GOVERS
were independent transformants, all harbouring only one T-DNA
copy (for one transformant this is shown in Fig. 2B). Co-cultivation
of AS-induced LBA1100 containing both pNptII and the constitutive virG gene with Py. ultimum resulted in three geneticin
resistant transformants. Southern blot analysis showed that all
three transformants had two integrated T-DNA copies. However,
the hybridization patterns in the three transformants were identical, suggesting that all three were derived from a single transformation event (Fig. 2B).
In summary, these results show that A. tumefaciens can transfer its T-DNA to at least to two other oomycetes, P. palmivora and
Py. ultimum, but the conditions we used for transformation may
still be far from optimal. We made no attempts to optimize the
procedure; for example, we tested only one recipient strain of
each of the two species and we did not test the effect of virG as
extensively as we did for P. infestans. The use of other recipient
strains and more virulent or modified A. tumefaciens strains may
significantly increase the efficiency of transformation.
Transfer of T-DNA during asexual reproduction
Fig. 2 Southern blot analysis of geneticin resistant transformants.
(A) Schematic representation of the T-DNA of plasmid pNptII used for
transformation. RB, right border; LB, left border; Phsp Bremia lactucae hsp70
promoter, Npt II, neomycin phosphotransferase; Tham, Bremia lactucae Ham43
terminator. (B) Genomic DNA of seven independent P. infestans transformants,
one P. palmivora transformant, one Py. ultimum transformant and the
untransformed wild-type strains was digested with EcoRI. The T-DNA contains
only one EcoRI restriction site (see Fig. 2A). All blots were probed with a
fragment containing the hsp70 promoter which detects the presence of the
geneticin resistance gene. Sizes at right are in kb.
ever detected among the transformants. Furthermore, no additional vector DNA has been detected in the transformants, either
by Southern blot analysis or by PCR (results not shown), indicating that only the T-DNA was transferred.
T-DNA transfer to other Pythiaceae
To determine if Agrobacterium-mediated transformation was
applicable to a broader range of oomycetes we tested T-DNA
transfer to two other plant pathogenic Pythiaceae, Phytophthora
palmivora and Pythium ultimum. The protocol was slightly modified. In particular the number of zoospores used as a starting
material for the co-cultivation was lower and the co-cultivation
period was shorter. For further details see the Experimental procedures. For P. palmivora, five geneticin resistant colonies were
obtained. A Southern blot analysis showed that these transformants
A. tumefaciens mediated transformation of pNptII-Gus to P.
infestans H30P02 resulted in gus expressing, geneticin resistant
P. infestans colonies. Figure 3C shows the GUS staining of one of
these transformants (marked with ‘P’). To demonstrate that the TDNA was stably integrated and transferred to the next generation
via asexual reproduction, single zoospore cultures of three gus
transformants were generated and analysed for gus expression
and T-DNA integration by Southern blot analysis. When plating
the zoospores on a medium without antibiotics, a few hundred
colonies were easily obtained. The transfer of these colonies to a
medium containing geneticin resulted in growth of all single
zoospore cultures. Southern blot analysis of three of these
zoospore cultures, its parent and a wt control is shown in Fig. 3B.
With probe I a 2 kb EcoRI fragment was detected in the parent,
showing that the complete nptII gene was integrated into the
genome. The same probe was used for a blot containing BamHI
digested genomic DNA of a wild-type strain, the parent and the
zoospore progeny. This resulted in the hybridization of a 9 kb
fragment harbouring the hsp-promoter driving the nptII gene and
the chromosomal sequences linked to the T-DNA left border in the
parent as well as in the zoospore progeny. With DNA of the wildtype strain, no hybridization was detected. GUS staining of the
cultures resulted in blue coloured colonies, from both the
parental line and the singles zoospore cultures, while the wildtype culture stayed white (Fig. 3C). Re-hybridization of the blot
containing the BamHI digested genomic DNA with probe II
showed a band that was slightly larger than 9 kb. The hybridizing
fragment harbours the ham-promoter driving the gus gene and
the chromosomal sequences linked to the T-DNA right border, and
is present in both the parent and the zoospore progeny. As can be
MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467 © 2003 BLACKWELL PUBLISHING LTD
A. tumefaciens mediated transformation of Phytophthora
463
that a single transformation event has led to the formation of the
primary transformant and that this transformant developed from
a single cell. Analyses of the zoospore progeny of the other two
gus expressing transformants showed identical results, except
that not all single zoospore cultures showed gus expression (two
out of five). Nevertheless, the cultures did grow in geneticin and
a Southern blot analysis showed that the gus gene was present
in all single zoospore progeny. Why these cultures no longer
express the gus gene is not known, but it might be due to a phenomenon called transcriptional gene silencing. Loss of gus
expression has been described before by Judelson and Whittaker
(1995) who obtained gus transformants of P. infestans by the
PEG-mediated transformation of protoplasts.
Transformation efficiency and copy number
Fig. 3 Asexual inheritance of integrated T-DNA in P. infestans. (A) Schematic
representation of the T-DNA of plasmid pNptII-Gus used for transformation.
Pham, Bremia lactucae Ham34 promoter, Gus, uidA gene of Escherichia coli.
For other abbreviations see legend of Fig. 2A. (B) Southern blot analysis of a
gus expressing transformant and its single zoospore progeny. The genomic
DNA of an untransformed wild-type strain (Wt) and the geneticin resistant and
gus expressing parent (P) transformant was digested with EcoRI. EcoRI cuts
twice in the T-DNA, releasing a 2 kb fragment containing the nptII gene under
control of the hsp promoter and ham terminator, which can be detected with
probe I. The genomic DNA of the same untransformed wild-type strain, the
parent and the single zoospore progeny (Z1 to Z3) was digested with BamHI.
BamHI cuts only once in the T-DNA. In this case probe I detects the presence
of the Npt II gene, whereas probe II detects the presence of the gus gene.
(C) Histochemical staining of GUS activity. Wt, untransformed wild-type; P, Gus
expressing parent transformant; Z1–Z4, single zoospore F1 progeny of parent
P. In this black and white picture GUS activity (in blue) is seen as a dark staining.
seen in Fig. 3B, the fragment detected with probe II was not
identical in size to the fragment detected with probe I. Since deprobing of probe I was not complete, a faint signal of the first
hybridization was still visible. Taken together, these results show
An advantage of A. tumefaciens mediated transformation compared to the PEG-mediated transformation, the method that has
been mostly used up to now, is that it is less labour intensive and
at first sight more efficient: a few hundred transformants can easily be obtained in a single transformation experiment. However,
for A. tumefaciens mediated transformation the DNA to be transformed has to be cloned into a binary vector, while for the PEGmediated transformation, cloned genomic fragments carrying the
gene of interest can be used immediately for co-transformation
with a plasmid containing the selectable marker. To compare the
real efficiencies between the two transformation methods is
difficult. For the PEG-mediated transformation the number of
transformants obtained per microgram of DNA determines the
efficiency, while in the A. tumefaciens mediated transformation
the amount of input DNA is not know. A difference we did find
was the copy number of DNA integrations. Transformants
obtained with PEG-mediated transformation often carry many
transgene copies, while the A. tumefaciens mediated transformation usually resulted in the integration of only one or two copies.
The same difference in number of transgene copies has been
observed in filamentous fungi (Covert et al., 2001; de Groot
et al., 1998).
Similar as has been described for plants (van der Fits et al.,
2000), the introduction of the ternary plasmid carrying the
constitutive virG gene improved the transformation efficiency
of P. infestans considerably, especially after induction of the A.
tumefaciens strains with acetosyringone. For some constructs,
induction with AS was necessary, even in the presence of the
constitutive VirG gene. This indicates that a very high virulence of
A. tumefaciens is needed for a successful transformation of P.
infestans. Since it was possible to obtain a comparable amount
of transformants when using the A. tumefaciens strain EHA105
as when using LBA1100 harbouring the constitutive VirG gene,
the transformation efficiency might benefit from the introduction
of the constitutive VirG gene into EHA105.
© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467
464
I. VIJN AND F. GOVERS
Concluding remarks
In this paper we demonstrate that oomycetes can be genetically
modified by A. tumefaciens and we describe a protocol that is
applicable to Phytophthora infestans. Now that it has been
shown that the Agrobacterium-mediated transformation method
can be used for the expression of heterologous genes, its use
should be extended towards the over-expression or regulated
expression of endogenous genes and targeted knock-down
mutagenesis. In filamentous fungi, targeted mutagenesis can be
achieved by gene disruption via homologous recombination
(Bundock et al., 1995; Gouka et al., 1996). However, due to its
diploid nature the use of homologous recombination, if at all
possible, will be a laborious procedure in P. infestans. Van West et al.
(1999b) demonstrated that with PEG-mediated transformation of
P. infestans it is possible to silence the expression of a target
gene by the introduction of either sense or antisense gene copies.
In that study they found silencing of the inf1 elicitin gene in up
to 20% of the transformants with no correlation between copy
number of the transgene and the efficiency of gene silencing;
even in single copy transformants the target gene was silenced.
Latijnhouwers and Govers (2003) used two transformation methods in order to silence the same target gene, i.e. the G-protein β
subunit gene gpb1. With the PEG-mediated transformation they
obtained a higher efficiency of gene silencing than with zoospore
electroporation. It was also remarkable that the number of plasmid
integrations was higher in the PEG-mediated transformants,
but to what extent this determined the efficiency of gene silencing was not clear. One has be aware that the efficiency of silencing can also be highly dependent on the target gene, e.g.
expression levels or stage of expression may be important. We
expect that gene silencing can also be induced by the introduction of extra gene copies via A. tumefaciens mediated transformation. Thus far, however, we have not found gene silencing in
up to 100 transformants that were transformed with the elicitin
gene inf1, suggesting that the silencing efficiency is lower than
with PEG mediated transformation (van West et al., 1999b). We
are currently studying the effects on silencing by introducing
inverted repeat constructs. In many organisms, inverted repeats
increase the silencing frequencies enormously. Combining an
efficient DNA transformation system, like that described here,
with a highly efficient silencing strategy will greatly facilitate the
possibilities of performing gene-function analysis in P. infestans.
E X P E R I M E N T A L P RO C E D U RE S
Strains and plasmids
The P. infestans, P. palmivora and Py. ultimum strains that were
used in this study as recipient strains and the A. tumefaciens
strains used, are listed in Table 3. Table 4 gives an overview of the
plasmids.
Table 3 Strains used in this study
Strain
Relevant traitsa
Reference
Origin or provider
Van der Lee et al. (1997)
This laboratoryb
This laboratory
This laboratory
This laboratory
This laboratory
This laboratory
Mchau and Coffey, 1994
Van West et al. (1999a)
N.A.R. Gow, University
of Aberdeen, UK
Pythium ultimum var. sporangiiferum
CBS 219.65
www.cbs.knaw.nl
J. Raaijmakers, Wageningen
University, the Netherlands
Agrobacterium tumefaciens
LBA1100
Beijersbergen et al. (1992)
P.J.J. Hooykaas, Leiden
Hood et al. (1993)
University, the Netherlands
H. van Attikum, Leiden
University, the Netherlands
Phytophthora infestans
88069
88133
98014
98020
98027
H30P02
Phytophthora palmivora
P6390
EHA105
Dutch field isolate, A1
Dutch field isolate, A2
Dutch field isolate, A1
Dutch field isolate, A2
Dutch field isolate, A2
F1 progeny of cross
80029 × 88133, A1
Field isolate from South
Sulawesi, Indonesia, A2
a
A1 = A1 mating type, A2 = A2 mating mating type.
Phytophthora culture collection, Laboratory of Phytopathology, Wageningen University.
b
MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467 © 2003 BLACKWELL PUBLISHING LTD
A. tumefaciens mediated transformation of Phytophthora
465
Table 4 Plasmids used in this study
Plasmid
Characteristics
Reference
Origin or provider
pTH209
pHAMT35G
pMOG800
pNptII
pNptII-Gus
pBBR1MCS.virGN54D
Npt II gene
gus gene
Binary plasmid
Binary plasmid
Binary plasmid
Constitutive VirG gene,
ternary plasmid
Judelson et al. (1991)
Judelson and Michelmore (1991)
Honée et al. (1998)
This study
This study
Van der Fits et al. (2000)
H.S. Judelson, UC Riverside, USA
H.S. Judelson, UC Riverside, USA
Constructs
pNptII was constructed by the insertion of a 2.1 kb BamHI/EcoRI
fragment from pTH209 containing the hsp70 promoter of Bremia
lactucae fused to the neomycin phosphotransferase ( nptII ) gene
and the B. lactucae ham34 terminator (Judelson et al., 1991) into
the binary vector pMOG800 (Honée et al., 1998). pNptII-Gus was
constructed by subcloning the 3.3 kb HindIII/EcoRI fragment from
pHAMT35G containing the uidA (gus) gene of Escherichia coli
fused to the B. lactucae Ham34 promoter and terminator (Judelson
et al., 1991) into pBluescript KS + (Stratagene). Subsequently
the fragment was excised with Hin dIII and Sst I and cloned into
the Hin dIII and Sst I sites of pNptII. E. coli strain DH5α was used
as the recipient strain for all plasmids. pNptII and pNptII-Gus
were electroporated to A. tumefaciens strains LBA1100 and
EHA105. All DNA manipulations were performed using standard
procedures (Sambrook et al., 1989).
Culture conditions of Phytophthora and Pythium
species and preparation of zoospores
Sporulating cultures of P. infestans were obtained by growth of
the strains for 10–12 days at 18 °C in the dark on rye medium
amended with 2% w/v sucrose (Caten and Jinks, 1968). Zoospores
were obtained by flooding the cultures with 25 mL water per
15 cm Petri dish followed by incubation at 4 °C for 2.5 h, during
which zoospores are released from the sporangia. Subsequently,
the water, containing mainly zoospores, was filtered through a
50 µm mesh to remove mycelial pieces. In general, 106 zoospores/
mL were released in the water.
P. palmivora zoospores were obtained by flooding sporulating
cultures, which were grown for 5–7 days at 25 °C in the light on
0.2 × V8 medium supplemented with 5 g/L CaCO3 (Erwin and
Ribeiro, 1996) with 25 mL water per 15 cm Petri dish. Sporangia
containing the zoospores were harvested by rubbing the cultures
carefully with a glass rod. The sporangia suspension was poured
through a 50 µm mesh to remove mycelial pieces and incubated
at −20 °C for 15 min to release zoospores. Approximately 105−
106 zoospores/mL were released in the water.
J. Memelink, Leiden
University, the Netherlands
Sporulating Py. ultimum cultures were obtained by growing
the strain on 0.2 × V8 agar medium supplemented with 10 g/L
CaCO3 for 2–3 days at 18 °C in the dark in 9 cm Petri dishes. The
agar containing the growing mycelium was cut in four pieces and
each piece was transferred to a Petri dish containing 20 mL sterile
de-mineralized (demi) water. The water was refreshed after 1 h. The
flooded agar-plugs were incubated for another 4 days at 18 °C in
the dark. For release of zoospores the water was refreshed with
20 mL precooled 10 °C demi-water. After an additional 2 h at
18 °C the released zoospores were harvested by decanting the
water. In general 104 zoospores/mL were released in the water.
Culture conditions of Agrobacterium tumefaciens
A. tumefaciens cells were grown overnight at 28 °C in minimal
medium (MM) (Hooykaas et al., 1979) containing 50 µg/mL kanamycin, 250 µg/mL spectinomycin and 25 µg/mL chloramphenicol. Subsequently 1 mL of the culture was washed twice with
1 mL induction medium (IM; MM salts and 40 m M 2-( N morpholino)ethanesulphonic acid (MES), pH 5.3, 10 mM glucose,
0.5% (w/v) glycerol) supplemented with 100 µM acetosyringone
(AS), 10× diluted in fresh IM + AS and grown for another 5 h at
28 °C. The final OD600 of the cultures should be ≈ 0.25. Before
co-cultivation the cells were washed twice with an equal volume
of sterile demi water.
Co-cultivation and selection of transformants
For transfer of the T-DNA from A. tumefaciens to P. infestans, 1 mL
of bacteria was added to 50 mL of water containing zoospores
(approximately 5 × 107 zoospores). After 30 min, encystment of
the zoospores was induced by manual shaking for 2 min, and the
cysts were allowed to germinate for 2 h at room temperature. The
mixture of germinated cysts and Agrobacterium cells was harvested by centrifugation at 260 g for 5 min and plated on
either Hybond N+ membranes (Amersham International, Little
Chalfont, Buckinghamshire, UK), or Nytran membranes (Schleicher and Schuell, Keene, NH) and transferred to IM agar plates
containing 5 mM glucose and 100 µM AS. For each co-cultivation
© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467
466
I. VIJN AND F. GOVERS
a total of four 9 cm membranes was used. After co-cultivation for
7 days at 22 °C in the dark the filters containing both P. infestans
and A. tumefaciens were cut into 1 cm2 pieces and transferred
upside-down to Plich agar plates (Van der Lee et al., 1997) with
5 g/L glucose containing 200 µg/mL cefotaxim (Duchefa Biochemie BV, Haarlem, the Netherlands) to kill the A. tumefaciens and
4 µg/mL geneticin (G-418, Gibco BRL, Gaithesburg, MD, USA) to
select for P. infestans transformants. After about 10 days geneticin
resistant colonies appeared and were transferred to rye medium
amended with 2% w/v sucrose and with 200 µg/mL cefotaxim
and 5 µg/mL G-418.
Co-cultivation of A. tumefaciens and P. palmivora was started
as described above for P. infestans. The only difference was that
co-cultivation was performed at 25 °C and the period of cocultivation was shortened to 5 days. P. palmivora transformants
were selected on Plich medium with 5 g/L glucose and containing
200 µg/mL cefotaxim and 2.5 µg/mL G-418. P. palmivora transformants were transferred to V8 medium supplemented with 5 g/ L
CaCO3 containing 200 µg/mL cefotaxim and 20 µg/mL G-418.
In addition, co-cultivation of A. tumefaciens and Py. ultimum
was performed as described above. Pythium transformants were
obtained after 3 days of co-cultivation at 18 °C and selected on
Plich medium with 5 g/L glucose and containing 200 µg/mL cefotaxim and 75 µg/mL G-418.
Single zoospore cultures
Single zoospore cultures of P. infestans transformants were
obtained by harvesting zoospores from sporulating cultures as
described above and by plating dilutions of the zoospore
suspension on rye medium amended with 2% w/v sucrose
without antibiotics. The dilutions of the suspensions ranged from
104 to 106, resulting in approximately 1 to 1000 zoospores per
plate. To check for antibiotic resistance and gus expression,
single colonies were transferred to rye medium supplemented
with 5 µg / mL G418. GUS staining was performed as described
below.
DNA isolation and Southern blot analysis
For DNA isolation all transformants were grown in liquid Plich
medium with 25 g/L glucose containing 200 µg/mL cefotaxim
and the appropriate concentration of G-418. For P. infestans and
P. palmivora the mycelium was harvested after 10–14 days,
while for Pythium the mycelium was harvested after three days.
Genomic DNA was isolated from mycelium as described by
Pieterse et al. (1991). The DNA was digested (10 µg genomic DNA
per sample), separated on a 1% agarose gel and transferred by
vacuum blotting (1.5 h, 700 mPA) in 0.4 M NaOH to Hybond N+
membranes (Amersham International, Little Chalfont, Buckinghamshire, UK). Hybridization with 32P-labelled probes was per-
formed at 65 °C in 0.5 M sodium phosphate buffer and 7% SDS
(pH 7.2). The filters were washed at 65 °C in 0.5 × SSC (75 mM
NaCl and 7.5 mM sodium citrate) and 0.1% SDS and exposed to
Kodak X-OMAT-AR films (Eastman Kodak Co., Rochester, NY).
GUS staining
Mycelium was submerged in β-glucuronidase (GUS) staining
solution (0.5 mg/mL 5-bromo-4-chloro-3-indoxyl-β-D-glucuronide
(X-gluc, Biosynth AG, Staad, Switzerland) in 100 m M sodium
phosphate (pH 7.0), 1% Triton X-100, 1% DMSO and 10 mM
EDTA) and vacuum infiltrated for 10 min, followed by overnight
incubation at 37 °C or until the blue colour appeared.
ACKNOWLEDGEMENTS
We are very grateful to all colleagues mentioned in Tables 3 and
4, who kindly provided plasmids and Phytophthora, Pythium or
A. tumefaciens strains. The work was financially supported by
the Syngenta Phytophthora Research Consortium coordinated by
Syngenta (Research Triangle Park, NC, USA).
R E F E RE N C E S
Agrios, G.N. (1997) Plant Pathology. San Diego, CA: Academic Press.
Beijersbergen, A., de Dulk-Ras, A., Schilperoort, R.A. and Hooykaas, P.J.J.
(1992) Conjugative transfer by the virulence system of Agrobacterium
tumefaciens. Science. 256, 1324–1327.
Bottin, A., Larche, L., Villalba, F., Gaulin, E., Esquerre-Tugaye, M.T.
and Rickauer, M. (1999) Green fluorescent protein (GFP) as gene
expression reporter and vital marker for studying development and
microbe – plant interaction in the tobacco pathogen Phythophthora parasitica var. nicotianae. FEMS Microbiol. Lett. 176, 51–56.
Bundock, P., den Dulk-Ras, A., Beijersbergen, A. and Hooykaas, P.J.J.
(1995) Trans.-kingdom T-DNA transfer from Agrobacterium tumefaciens
to Saccharomyces cerevisiae. EMBO J. 14, 3206 –3214.
Caten, C.E. and Jinks, J.L. (1968) Spontaneous variability of single isolates
of Phytophthora infestans. I. Cultural variation. Can. J. Bot. 46, 329–347.
Chen, X., Stone, M., Schlagnhaufer, C. and Romaine, C.P. (2000) A
fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl. Environ. Microbiol. 66, 4510–
4513.
Covert, S.F., Kapoor, P., Lee, M. and Nairn, C.J. (2001) Agrobacterium
tumefaciens-mediated transformation of Fusarium circinatum. Mycol.
Res. 105, 259 – 264.
Cvitanich, C. and Judelson, H.S. (2003) Stable transformation of the
oomycete, Phytophthora infestans, using microprojectile bombardment.
Curr. Genet. 42, 228 – 235.
De Groot, M.J.A., Bundock, P., Hooykaas, P.J.J. and Beijersbergen, A.G.M.
(1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol. 16, 839 – 842.
Erwin, D.C. and Ribeiro, O.K. (1996) Phytophthora Diseases Worldwide.
St Paul, MN: APS Press.
Gaulin, E., Jauneau, A., Villalba, F., Rickauer, M., Esquerré-Tugayé, M.
and Bottin, A. (2002) The CBEL glycoprotein of Phytophthora parasitica
MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467 © 2003 BLACKWELL PUBLISHING LTD
A. tumefaciens mediated transformation of Phytophthora
var. nicotianae is involved in cell wall deposition and adhesion to cellulosic
substrates. J. Cell Sci. 115, 4565 – 4575.
Gouka, R.J., Punt, P.J., Hessing, J.G.M. and van den Hondel, C.A.M.J.J.
(1996) Analysis of heterologous protein production in defined recombinant
Aspergillus awamori strains. Appl. Environ. Microbiol. 62, 951–1957.
Hansen, G. and Chilton, M.D. (1999) Lessons in gene transfer to plants
by a gifted microbe. In: Plant Biotechnology: New Products and Applications (Hammond, J., McGarvey, P. and Yusibov, V., eds), pp. 21–57. Berlin:
Springer-Verlag.
Honée, G., Buitink, J., Jabs, T., De Kloe, J., Sijbolts, F., Apotheker, M.,
Weide, R., Sijen, T., Stuiver, M. and de Wit, P.J.G.M. (1998) Induction
of defense-related responses in Cf9 tomato cells by the AVR9 elicitor peptide of Cladosporium fulvum is developmentally regulated. Plant Physiol.
117, 809–820.
Hood, E.E., Gelvin, S.B., Melchers, L.S. and Hoekema, A. (1993) New
Agrobacterium helper plasmids for gene transfer to plants. Transgenic
Res. 2, 208–218.
Hooykaas, P.J.J., Roobol, C. and Schilperoort, R.A. (1979) Regulation of
the transfer of Ti-plasmids of Agrobacterium tumefaciens. J. Gen. Microbiol. 110, 99–109.
Hynes, M.J. (1996) Genetic transformation of filamentous fungi. J. Genet.
75, 297–311.
Judelson, H.S. and Michelmore, R.W. (1991) Transient expression of
genes in the oomycete Phytophthora infestans using Bremia lactucae
regulatory sequences. Curr. Genet. 19, 453–459.
Judelson, H.S., Tyler, B.M. and Michelmore, R.W. (1991) Transformation
of the oomycete pathogen Phytophthora infestans. Mol. Plant-Microbe
Interact. 6, 602–607.
Judelson, H.S. and Whittaker, S.L. (1995) Inactivation of transgenes in
Phytophthora infestans is not associated with their deletion, methylation, or mutation. Curr. Genet. 28, 571–579.
Kamoun, S. (2003) Molecular genetics of pathogenic oomycetes. Eukaryotic Cell, 2, 191–199.
Kamoun, S., van West, P. and Govers, F. (1998) Quantification of late
blight resistance of potato using transgenic Phytophthora infestans
expressing β-glucuronidase. Eur. J. Plant Pathol. 104, 521–525.
Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C. and Citovsky, V.
467
(2001) Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl
Acad. Sci. USA, 98, 1871–1876.
Latijnhouwers, M. and Govers, F. (2003) A Phytophthora infestans Gprotein β subunit is involved in sporangia formation. Eukaryotic Cell,
2(5), in press.
Mchau, G.R.A. and Coffey, M.D. (1994) Isozyme diversity in Phytophthora palmivora: evidence for a southeast Asian Centre of origin. Mycol.
Res. 98, 1035–1043.
Pieterse, C.M.J., Risseeuw, E.P. and Davidse, L.C. (1991) An in planta
induced gene of Phytophthora infestans codes for ubiquitin. Plant Mol.
Biol. 17, 799–811.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory Press.
Van der Fits, L., Deakin, E.A., Hoge, J.H.C. and Memelink, J. (2000) The
ternary transformation system: constitutive virG on a compatible plasmid
dramatically increases Agrobacterium-mediated plant transformation.
Plant Mol. Biol. 43, 495–502.
Van der Lee, T., de Witte, I., Drenth, A., Alfonso, C. and Govers, F.
(1997) AFLP linkage map of the oomycete Phytophthora infestans.
Fungal Genet. Biol. 21, 278–291.
Van West, P., de Jong, A.J., Judelson, H.S., Emons, A.M. and Govers, F.
(1998) The ipiO gene of Phytophthora infestans is highly expressed in
invading hyphae during infection. Fungal Genet. Biol. 23, 126–138.
Van West, P., Kamoun, S., Van’t Klooster, J.W. and Govers, F. (1999b)
Internuclear gene silencing in Phytophthora infestans. Mol. Cell. 3, 339–348.
Van West, P., Reid, B., Campbell, T.A., Sandrock, R.W., Fry, W.E.,
Kamoun, S. and Gow, N.A.R. (1999a) Green fluorescent protein (GFP)
as a reporter gene for the plant pathogenic oomycete Phytophthora
palmivora. FEMS Microbiol. Lett. 178, 71–80.
Weiland, J.J. (2003) Transformation of Pythium aphanidermatum to
geneticin resistance. Curr. Genet. 42, 344–352.
Winans, S.C. (1992) Two-way chemical signalling in Agrobacterium– plant
interactions. Microbiol. Rev. 56, 2 – 31.
Zwiers, L.H. and de Waard, M.A. (2001) Efficient Agrobacterium tumefaciensmediated gene disruption in the phytopathogen Mycosphaerella graminicola.
Curr. Genet. 39, 388 –393.
© 2003 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2003) 4(6), 459–467