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Applied and Environmental Microbiology, September 2000, p. 4161-4167, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Plant Genome Complexity May Be a Factor Limiting In Situ the
Transfer of Transgenic Plant Genes to the Phytopathogen
Ralstonia solanacearum
Franck
Bertolla,1,*
Regis
Pepin,1
Eugenie
Passelegue-Robe,2
Eric
Paget,2
Andrew
Simkin,3
Xavier
Nesme,1 and
Pascal
Simonet1
Laboratoire d'Ecologie Microbienne, UMR CNRS
5557, Université Lyon I, 69622 Villeurbanne
Cedex,1 Aventis Cropscience,
Biotechnology Department, 69263 Lyon Cedex 09,2
and Génétique Moléculaire des Plantes,
UMR CNRS 5575, Université J. Fourier, 38041 Grenoble
Cedex,3 France
Received 13 April 2000/Accepted 30 June 2000
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ABSTRACT |
The development of natural competence by bacteria in situ is
considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum
is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We
have attempted to determine whether this bacterium could become the
recipient of plant genes. We initially demonstrated that plant DNA was
released close to the infecting bacteria. We constructed and tested
various combinations of transgenic plants and recipient bacteria to
show that the effectiveness of such transfers was directly related to
the ratio of the complexity of the plant genome to the number of copies
of the transgene.
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TEXT |
Nucleotide sequences in databases
indicate that some genes in genomes of soil bacteria actually came from
plants (12, 37). The most likely mechanism by which such
horizontal gene transfers could occur is natural transformation of
bacteria by extracellular DNA released by plants (5).
However, several physical and biological conditions must be fulfilled
before a plant gene can be expressed by a bacterium, explaining why
such events have remained rare and difficult to detect (20).
These conditions include the release of DNA very close to those few
bacteria that have the relevant molecular mechanisms and which actually
develop the physiological state of competence, in which they are able
to take up DNA (20). The naked DNA must also escape any
rapid chemical or enzymatic degradation and avoid being irreversibly
adsorbed onto soil components (16, 42, 43). Finally,
transformation requires that the transforming DNA become integrated
into the bacterial chromosome by homologous or more or less
illegitimate recombination (21).
According to these various conditions, the soil is an unlikely
environment for transformation-mediated gene transfers (5, 39), while much more favorable conditions could be encountered in
plant tissues in which some symbiotic or pathogenic bacteria multiply
actively. This is particularly the case with plants sensitive to the
plant pathogen Ralstonia solanacearum, because a rare
combination of the following positive factors should be examined for
horizontal gene transfer (5). R. solanacearum is
a naturally competent bacterium which was found to develop competence
in vitro (3) but also in situ during the process of
infection of the host plants (4). It can be hypothesized
that the infection process could lead plant DNA released by decaying
plant cells to be in close contact with these invading and
metabolically active bacterial cells. Finally, even the traditional and
genetic barriers to such interkingdom transfers due to the molecular
mechanisms preventing recombination with foreign DNA in bacteria
(3, 18) could be overcome with transgenic plant DNA because
of the prokaryotic origin of the marker genes (11, 15).
Our objectives in this paper were to confirm experimentally these
hypotheses and to determine the various physical or biological factors
which control the transfer of prokaryotic sequences from plants to bacteria.
Evidence of direct contacts between plant DNA and infecting
bacteria.
Our initial goal was to determine whether infection of
sensitive plants by R. solanacearum resulted in close
physical contact between the invading bacteria and the plant nuclear
DNA. We used tomato plants infected with R. solanacearum
strain GMI1000 (bv. 1), since this strain is virulent towards the
tomato (28), with a multiplication rate in planta compatible
with the development of the competence state (3). Seeds of
the transgenic tomato pKHG3 (24) were sown in potting
compost (Ets Grassot, Brignais, France), and the plantlets were grown
for 2 weeks before being inoculated with a suspension of R. solanacearum bacteria according to the method of Bertolla et al.
(4). Segments of leaves, including a portion of the main
leaf vein, were then removed 5 days after inoculation at 30°C and
prepared for microscopy by the protocol of Pépin and Boumendil
(27). Semithin sections (0.7 to 1 µm thick) were cut for
light microscopy, and ultrathin sections (0.07 µm thick) were cut for
electron microscopy. Semithin sections stained with Richardson's
mixture (30) showed numerous bacteria (Fig.
1A) in mature and
differentiating xylem tissue both inside and outside
the primary cell walls of the vessels under the light microscope. They
were often in the sheath of cells associated with vessels, some of them
having intact nuclei. Ultrathin sections were floated onto copper grids
and contrasted with uranyl acetate and lead citrate (29).
Bacteria were also seen inside cortical parenchyma cells, which
contained abundant cytoplasmic and nuclear remnants (Fig. 1B, C, D, and
E). Most of the infected cells were adjacent to intact collapsed cells
that clearly contained nuclei and chloroplasts. Infection due to the
intensive spread of bacteria involved bacterial lytic enzymes, such as
endoglucanases, polygalacturonases, and pectin methylesterases
(31, 34, 35, 38). Although these enzymes are not absolutely
required for wilting, they permit bacteria to spread from cell to cell
by degrading plant cell walls and tissues, thus releasing host DNA.
Direct observations indicate that physical contact between the plant
DNA and invading bacteria cells is possible. In view of results
obtained by members of our group on the development of competence in
planta (4), these data confirm the potential of an R. solanacearum-based model to fulfill most of the criteria required
for natural gene transfer in the environment. These include the release
of plant DNA in close contact with bacteria that are physiologically
capable of being genetically transformed. We next provided the plants
and bacteria with nucleotide sequences favoring the integration of the
plant DNA into the recipient genome by homologous recombination and the
expression of the marker genes.
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FIG. 1.
(A) Light microscope observations of a
longitudinal section of tomato xylem tissues infected by R. solanacearum. Bacteria were mainly inside the tracheids and
associated parenchyma cells, some of which still contained a cytoplasm
and a nucleus. (B, C, D and E) Transmission electron microscope views
of tomato parenchyma cells infected by R. solanacearum. (B)
R. solanacearum bacteria in a parenchyma cell, the contents
of which were necrotic but which still had a recognizable nucleus, are
shown. (C) R. solanacearum bacteria are shown in
differentiating vascular tissues; an organelle could be identified as a
degraded nucleus. (D) Heavy infection of a parenchyma cell, which still
contained an organelle that could be a nucleus, is shown. (E) R. solanacearum bacteria are shown in the extraplasmatic space of a
plasmolysed parenchyma cell containing living organelles, such as a
chloroplast, mitochondria, and a nucleus. Abbreviations: b, bacteria;
c, chloroplast; ep, extraplasmatic space; m, mitochondria; n, nucleus;
pm, plasma membrane; pn, putative nucleus. Bars indicate lengths of 10 µm (A), 5 µm (B, C, and D), and 1 µm (E).
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Construction of sequence-compatible transgenic plants and R. solanacearum strains.
The first strategy was to construct
transgenic plants in which the transgene included sequences of the
R. solanacearum strain flanking the marker plant genes, to
provide homologous sequences between the bacterial and plant genomes.
This required construction of the binary plasmid pZpop1, with
spectinomycin and gentamycin resistance genes flanked by the
popA gene sequences from R. solanacearum GMI1000
(Fig. 2). The plasmids used in the plant
transformations were transferred into Agrobacterium
tumefaciens EHA105 by electroporation (22). Disks of
PBD6 tobacco leaves and tomato plants were transformed with A. tumefaciens according to the protocols from Rogers et al.
(32) and Fillatti et al. (14), respectively.
Calli were cultured on selective medium containing 100 mg of
kanamycin/liter for tobacco or 50 mg of kanamycin/liter for tomato for
about 5 weeks. The resulting resistant green plantlets were placed in soil, and their DNA was checked. Plant DNA was extracted from tobacco
and tomato leaves by the cetyltrimethylammonium bromide method
(13), and Southern hybridizations (33) indicated
that one to four copies of transgenic DNA (T-DNA) had been integrated into the plant nuclear genomes (Table 1).
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FIG. 2.
Physical map of the T-DNA of plasmid pZpop1 used to
transform plants. The gentamycin resistance gene aacC3-IV
that was extracted from pUC1813AM/Gm (9) after a
SalI digest was cloned into the XhoI site of the
pFB2 plasmid to give pFB21. A SacII cassette containing the
antibiotic resistance genes flanked by 719 and 907 bp of the
popA gene from the R. solanacearum GMI1000 strain
was cloned into the SmaI site of the binary vector pPZP212
(17). LB and RB are the left and right borders of the
T-DNA.
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We also modified the
R. solanacearum strain with sequences
from the transgenic plant. Because some of the transgenic plants
contained the
nptII marker gene, conferring resistance to
kanamycin,
we cloned a part of this
nptII gene in
R. solanacearum to provide
the recipient bacterial strain with
sequences in which homologous
recombination could occur. We constructed
the plasmid pFB3 containing
the deleted
nptII gene from the
plasmid pBin19 flanked with sequences
of the
popA gene from
the
R. solanacearum strain GMI1000 (Fig.
3). pFB3 was transferred to strain
GMI1000 by natural transformation
according to the method of Bertolla
et al. (
3). Other transformations
of
R. solanacearum were also performed by this method. The
popA sequences permitted the integration of the deleted
nptII gene
into the
R. solanacearum genome by
homologous recombination. The
recipient strain GMI1000FB3 was checked
by PCR using primers FGPnptII1544
and FGPnptII2347' (
4).
Moreover, the sensitivity of the gentamycin-resistant
clones to
ampicillin confirmed the absence of replicative plasmids
in the strain
GMI1000FB3.
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FIG. 3.
Construction of the pFB3 plasmid containing a defective
T-DNA. The BglII-BglII T-DNA fragment from pBin19
(6) was ligated into the BamHI linearized
pBluescript vector to create plasmid pT-DNA. This plasmid was digested
with NcoI and BalI to generate a deletion of 354 bp within the nptII gene before being ligated to the
gentamycin resistance cassette resulting from the digestion of pMGm
with NcoI-SmaI to give plasmid p T-DNA. The
SacII-SacII popA gene that was
recovered from pKSpopA (1) was ligated into a
SacII linearized pBluescript whose BamHI
restriction site was deleted previously to give plasmid pFB1. pFB1 was
modified by the ligation of the BamHI-BglII
aad gene (40) into the unique BamHI
site of the popA gene, creating pFB2 in order to provide
suitable conditions for the integration of this defective T-DNA.
Plasmid pFB3 was constructed by cloning the 5.5-kb
SpeI-XhoI fragment of defective T-DNA from
pT-DNA into the SpeI-XhoI sites of the vector
pFB2.
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Plasmids pKHG3 and pBin19, which contained the
nptII marker
gene in the T-DNA region, were used to transform tobacco and tomato
plants and so to provide the corresponding transgenic plants (Table
1).
Virulence of the recipient R. solanacearum
strains.
The symptoms of infection of the transgenic tomato plants
Lycopersicon esculentum pKHG3 and ZPop1, inoculated by
the strain GMI1000FB3 and the wild-type strain GMI1000, respectively,
were similar, including severe necrosis around the infection sites and
the pith of wilted plants, which appeared to be water soaked, brown and
hollow. Infected plants wilted within 6 days after the first appearance
of symptoms (at 2 days). The infected stems were crushed and
homogenized in sterile distilled water with an Ultra-Turrax T25
homogenizer at 25,000 rpm (Janke and Kunkel, Staufen, Germany) to
determine the bacterial population dynamics. The plant tissue suspensions were then plated on Boucher gelose (BG) media
(8), supplemented with 12 µg of gentamycin/ml for strain
GMI1000FB3. The population kinetics in planta of the GMI1000 and
GMI1000FB3 strains were similar and reached 4.5 × 109 ± 1.7 × 109 and 6.6 × 109 ± 1 × 109 CFU g of fresh
material
1 (n = 3) after 5 days, respectively.
In vitro transformation of the recipient R. solanacearum strains with plasmid and transgenic plant DNA.
Two conditions were tested to validate the various donor plant
DNA-recipient bacterial strain combinations. We first transformed the
recipient R. solanacearum strains in vitro with the various plasmids used to transform the plants. R. solanacearum
GMI1000 was transformed with 0.1 µg of the binary recombinant plasmid pZpop1. Clones resistant to gentamycin (12 µg ml1) and
spectinomycin (40 µg ml1) were detected at frequencies
reaching 3.76 × 107 ± 2.14 × 108 (n = 3). Similar results were
obtained with strain GMI1000FB3 transformed by plasmids pKHG3 and
pBin19 on BG media supplemented with 25 µg of kanamycin/ml (Table
2). Natural transformation of this
recipient strain by plasmids restored a functional copy of the
nptII kanamycin resistance gene. The presence of the marker genes in transformants was checked by PCR with primers complementary to
part of the aacC3-IV gene (FGPaac1,
5'-TCCTTCTGAAGGCTCTTCTC-3', and FGPaac2,
5'-GCAATACGAATGGCGAAAAG-3') and with the set of primers targeting the nptII gene (4). The expected PCR
products were detected, including 601- and 803-bp-long DNA fragments
for the aacC3-IV gene in strain GMI1000 and the restored
nptII marker gene in GMI1000FB3, respectively (data not
shown). Neither the back trap method (41) nor the plasmid
extraction kit from Qiagen (Courtaboeuf, France) detected any plasmid,
indicating that the marker gene was integrated into the recipient
genome (results not shown). This set of experiments indicated that the
targeted sequences could be integrated and expressed in R. solanacearum, whatever the endogenous (popA gene) or
exogenous (nptII gene) origin of the sequences involved in
the homologous recombination mechanism.
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TABLE 2.
Transformation frequencies of R. solanacearum
recipient strains with transgenic plant DNA and plasmids
containing T-DNA
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As a second step, recipient strains GMI1000 and GMI1000FB3 were
transformed in vitro with DNA extracted from the transgenic
tobacco and
tomato plants Zpop1, pKHG3, and pBin19 (Table
1).
Concentrations of DNA
from 0.1 to 1,000 µg ml
1 were tested. We also used
plant DNA that had been treated with
restriction endonucleases that did
not affect the transgene region
to mimic the partial degradation of DNA
that could occur in the
plant. Transformants remained undetectable at
all the DNA concentrations
tested and with long and restricted DNA
fragments, indicating
that transformation, if any, occurred at
frequencies below 1.36
× 10
9 (Table
2). The
transformation efficiency was not increased when
the
R. solanacearum recipient strains were transformed by electroporation
(
10), indicating that the lack of transformation was not due
to the natural process of DNA
uptake.
In planta tests to track plant-bacterium gene transfers.
Transgenic tomato plants (pKHG3) were inoculated with R. solanacearum strain GMI1000FB3 containing the deleted
nptII gene under conditions that permitted the bacterium to
become competent. Infected stems were removed and crushed 5 days later
and were plated on BG media supplemented with 25 µg of kanamycin/ml.
Kanamycin-resistant bacteria were detected at about 102 CFU
g of fresh plant material1. However, hybridization of the
genomic DNA of these clones with a nptII probe remained
negative, indicating that kanamycin resistance was not encoded by
nptII (data not shown). Moreover, their amplified and
restricted 16S ribosomal gene, with the FGPS5 and FGP1509' primers
(25), showed patterns that did not correspond to those for
R. solanacearum (data not shown). These clones did not
belong to R. solanacearum but were part of the indigenous,
epiphytic, potentially opportunistic but unidentified microflora that
naturally contained the gene for kanamycin resistance but which did not demonstrate that horizontal gene transfer had occurred.
We also tested strain GMI1000 as a potential recipient of the
R. solanacearum-indigenous
popA sequences, specifically
cloned
in the transgenic tomato plant Zpop1 (Fig.
2). The crushed
infected
plant tissues did not produce any spectinomycin- and
gentamycin-resistant
colonies when plated on BG medium supplemented
with 50 µg of spectinomycin/ml
and 12 µg of gentamycin/ml,
indicating that no gene transfer was
detected.
It can be argued that a transfer event would not have any positive
effect on the fitness of the strain which would have permitted
the
specific multiplication of the transformants and favored their
detection. We therefore tried to provide the natural medium with
selection pressure by inoculating the transgenic plant tissues
on days
2 and 5 after infection with 0.1 ml of antibiotic solution
(50 µg of
kanamycin/ml for plants pKHG3; 24 µg of gentamycin/ml
and 100 µg of
spectinomycin/ml for plants Zpop1), according to
the protocol described
by Bertolla et al. (
4). The plants were
then crushed on day
7. In spite of these numerous assays to boost
the growth of potential
recombinant clones, transformants remained
undetectable. These negative
results confirmed those in which
R. solanacearum strains
were transformed in vitro with plant DNA
and indicated that the
frequency of horizontal gene transfer in
planta must be below about
4.27 × 10
9 ± 8.8 × 10
10
transformants per recipient cell (
n = 20).
Factors limiting transformation.
Some of the numerous factors
which may limit gene exchanges between plants and bacteria could not be
used to explain these negative results. The successful transformation
of R. solanacearum with plasmid DNA issued from
Escherichia coli (3) and A. tumefaciens (19) could exclude any influence of
restriction and modification mechanisms (7). This could mean
that R. solanacearum belongs to the group of competent
bacteria in which DNA is translocated into the cytoplasm as
single-stranded molecules, so escaping the degradation mechanisms
specific to double-stranded DNA.
The successful transformation of another naturally competent bacterium,
such as
Acinetobacter calcoaceticus, by DNA from transgenic
plants (
11,
15) confirms that plant DNA can be internalized
by bacteria. In our study we hypothesized that specific proteins
or
histones bound to plant DNA which did not inhibit transformation
in
A. calcoaceticus would not affect specifically
R. solanacearum.
The fact that these two bacteria differ in their
efficiency of
transformation, the frequency of which is routinely
10
2 in
A. calcoaceticus (
26) and
less than 10
6 in
R. solanacearum, could
explain the different rates of transformation
of these two bacteria by
plant
DNA.
Another key factor to be considered is the characteristics of the donor
DNA: its number of copies of target sequences, the
size of the plant
genome, and the ratio between the transgene
and the whole plant genome.
An increase of the complexity of the
donor DNA should reduce the
frequency of transformation for a
given gene due to increased
competition with nontarget
sequences.
In order to check the influence of donor DNA complexity on the
frequency of transformation,
R. solanacearum was transformed
in vitro with various amounts of pZpop1 plasmid DNA (100, 10,
1, 0.1 and 0.01 ng), alone or diluted in 5 µg of nontransgenic
plant DNA
(from
L. esculentum, var. Ailsa Craig). The minimal
amount
of pZpop1 plasmid providing detectable transformants was
0.01 ng when
the pure plasmid solution was used (Fig.
4), corresponding
to 6.7 × 10
5 copies of transforming sequences. This value was
actually lower
than the actual number of transforming sequences in 5 µg of transgenic
tomato or tobacco plant DNA, indicating that the
absolute copy
number of selectable sequences in transgenic plant DNA
would be
high enough to provide transformants at detectable
frequencies.
Moreover, plasmid DNA mixed with plant DNA background
always provided
a 10-fold-lower transformation frequency than an
identical concentration
of pure plasmid DNA solution (Fig.
4). The
minimal amount of plasmid
pZpop1 (1 ng) in mixed DNA that provided
detectable transformants
corresponded to 6.7 × 10
7
transforming molecules, 15 times higher than the actual copy
number in
the genome of transgenic tomato plants (genome, about
1 × 10
9 bp) and 67 times more than that in tobacco plants
(genome, about
4.6 × 10
9 bp). These data confirm that
the efficiency of transformation
of
R. solanacearum is
directly related to the complexity of the
donor DNA. The efficiency of
transformation of a marker gene present
as only a few copies in
transgenic plant DNA must be at least
100-fold less than that for
plasmid solutions. Transfers could
thus occur at frequencies not lower
than 10
11, which is 6 orders of magnitude higher than
those expected for
Erwinia chrysanthemi, another bacterial
plant pathogen (
36).
Moreover, the fact that
R. solanacearum develops competence in
planta and acts directly to
release plant DNA indicates that such
transfer events could occur in
the environment at quite relevant
frequencies. These data confirm the
usefulness of models based
on direct symbiotic but mainly pathogenic
plant-bacterium relationships
for investigating horizontal gene
transfers in situ.
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FIG. 4.
Influence of the concentration of pZpop1 on the
transformation frequencies of R. solanacearum GMI1000.
R. solanacearum GMI1000 cells were transformed with 100, 10, 1, 0.1, and 0.01 ng of a pure pZpop1 solution (open squares).
Transformations were also conducted with the same amounts of plasmid
diluted in 5 µg of wild-type tomato DNA (solid circles). The open
circles indicate transformation frequencies below the detection limit
(dotted line). Error bars show standard deviations of triplicate
experiments. The mean value symbols from three replicate experiments
occasionally obscure the smaller standard error bars.
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ACKNOWLEDGMENTS |
We thank Stephane Peyrard and the Electron Microscopy Center
(CMEABG) of the Claude Bernard University in Lyon for technical assistance.
This work was supported by the Biotechnology program of the
Ministère Français de l'Enseignement Supérieur et de
la Recherche (MENRT). F.B. and A.S. were funded by a grant from the
MENESER and by the European Agriculture and Fisheries Program (contract number FAIR-98-5002), respectively.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire
d'Ecologie Microbienne du Sol, UMR CNRS 5557, Bâtiment 741, Université Lyon I, 43 bd. du 11 Novembre 1918, F-69622
Villeurbanne Cedex, France. Phone: 33 4 72 44 82 89. Fax: 33 4 72 43 12 23. E-mail: bertolla{at}biomserv.univ-lyon1.fr.
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Applied and Environmental Microbiology, September 2000, p. 4161-4167, Vol. 66, No. 9
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