Note: Descriptions are shown in the official language in which they were submitted.
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NEMATODE-rNDUClBLE PLANT GENE PROMOTER
The invention relates to regulatory DNA sequences which can be u~ed
for expressing DNA sequences in plant cells. The invention further
comprises chimeric DNA comprising said regulatory DNA ~equences operably
linked to DNA to be expre~sed in plant cells, as well as plants
cont~;n;~ such chimeric DNA in their cells. The invention further
relates to methods for making plants that are resistant, or at least less
susceptible to plant parasitic nematodes, or their effects, as well as to
cells, plants and parts thereof.
STATE OF THE ART
In International patent application W092/17054, a method is
disclosed for the identification and subsequent isolation of nematode
responsive regulatory DNA sequences from Arabidopsis thaliana.
In WO 92/21757 several regulatory DNA sequences have been isolated
from Lycopersicon esculentum, which are responsive to the root-knot
nematode Meloidogyne incognita. Some of these regulatory ~equences
~LEMMI's, for Lycopersicon esculentum - Meloidogyne incognita) are
stimulated, whereas others appear to be repressed by the nematode. It i9
not known whether any of the inducible regulatory sequences are
stimulated by a broader range of nematodes.
Another regulatory sequence that is inducible by the root-knot
nematode Meloidogyne incognita is disclosed in WO 93/06710. A
disadvantage of this regulatory sequence TobRb7 is that it is not
activated by a number of cyst nematodes, among which the Heterodera and
Globodera species. This makes the TobRB7 sequence unsuitable for use in
chimeric constructs aiming at, for example, cyst nematode resistance in
potato.
It is an object of the invention to provide regulatory DNA
sequences which are inducible by both cyst and root knot nematodes and
which can be used to express heterologous DNA sequences under their
control inside the feeding structure of the nematode, preferably, but not
necessarily in a substantially feeding site speci~ic way.
SU~ RY OF THE INVEN~ION
The invention provides a DNA fragment obtainable from Arabidopsis
thaliana that is capable of promoting root knot and cyst nematode-
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inducible transcription of an a~sociated DNA sequence when re-introduced
into a plant. Preferred according to the invention are sequences
represented by nucleotides 1 to 2361 in SEQIDNO: 4. Also envisaged are
portions or variants of a DNA fragment according to the invention capable
of promoting root knot and cyst nematode-inducible transcription of an
associated DNA sequence when re-introduced into a plant. A still further
preferred aspect of the invention comprises a regulatory DNA fragment
that is substantially nematode feeding site-specific.
Further embodiments of the invention comprise chimeric DNA
sequences comprising in the direction of transcription a regulatory DNA
fragment according to the invention and a DNA sequence to be expressed
under the transcriptional control thereof and which is not naturally
under transcriptional control of said DNA fragment. Preferred among the
chimeric DNA sequences according to the invention are those wherein the
DNA ~equence to be expre~sed causes the production of a plant cell-
disruptive substance, such as barnase. In a different embodiment the
cell-disruptive substance comprises RNA complementary to RNA essential to
cell viability. Yet in another embodiment the DNA sequence to be
expressed causes the production of a substance toxic to the inducing
nematode.
The invention finds further use in a replicon comprising a DNA
fragment or chimeric DNA sequence according to the invention, a
microorganism containing such a replicon, as well as plant cells having
incorporated into their genome a chimeric DNA sequence according to the
invention. Further useful embodiments are a root system of a plant
essentially consisting of cells according to the invention, as well as
full grown plants essentially consisting of cells according to the
invention, preferably a dicotyledonous plant, more preferably a potato
plant. Also envisaged are plants grafted on a root system according to
the invention, as well as plant parts selected from seeds, flowers,
tubers, roots, leaves, fruits, pollen and wood and crops comprising such
plants.
The invention also encompasses the use of a DNA fragment according
to the invention for identifying subfragments capable of promoting
transcription of an associated DNA sequence in a plant. Also envisaged is
the use of a chimeric DNA sequence according to the invention for
transforming plants. The invention further provides the use of a
fragment, portion or variant of a regulatory DNA according to the
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invention for making hybrid regulatory DNA sequences.
The following figures further illustrate the invention.
~S~TPTION OF T~E FIGURES
Figure 1. Schematic plasmid map of Binary vector pMOG23.
- Figure 2. Schematic plasmid map of Binary vector pMOG800.
Figure 3. Schematic plasmid map of Binary vector pMOG553.
Figure 4. Schematic plasmid map of Binary vector pMOG819.
Figure 5. Schematic plasmid map of Binary vector pMOG849.
Figure 6. Expression patterns outside the NFS of several pMOG849
transformed Arabidopsis thaliana line~.
Figure 7. Schematic representation of a NFS disrupter gene and a
neutraliser gene in a two component system for engineering of
nematode resistanct plants
Figure 8. Schematic plasmid map of Binary vector pMOG893.
Some ways of practicing the invention a~ well as the -aning of variouR
phrases are explained in more detail below.
DETAILED DESCRIPTION OF T~E INV~.... ION
The present invention provides regulatory DNA sequences obtainable
from Arabidopsis thaliana, which are inducible by root knot and cyst
nematodes and which show a high preference of expression of any
associated DNA inside the special nematode feeding structures of the
plant root. Such a nematode feeding structure is used by an invading
nematode as source of food, whereby the nematode induces a change in the
plant tissue thereby forming either a giant cell (root-knot nematodes) or
a ~yncytium ~cy~t nematodes). A method of isolating regulatory DNA
sequences has been disclosed and clai -.d in a prior application,
WO92/17054, which is incorporated herein by reference.
In principle the regulatory DNA sequences according to the
invention can be used to express any heterologous DNA in any plant of
choice, by placing said DNA under the control of said regulatory DNA
~equences and transforming plants with the resulting chimeric DNA
sequence using known methods. The heterologous DNA is expressed upon
infection of the roots by various root knot nematodes, such as
~eloidogyne incognita, and cyst nematodes, such as Heterodera schachtii
and Globodera pallida ~a more comprehensive, but by no means limiting,
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list is presented in table 2). Advantageouqly, the heterologous DNA may
consist of a gene coding for a substance that is toxic or inhibitive to a
plant parasitic nematode in order to create plants with reduced
susceptibility to plant parasitic nematodes. There exist numerous
examples of such toxic substances, such as the endotoxins of Bacillus
thuringiensis (e.g. EP O 352 052), lectins, and the like.
A more preferred approach for making plants with reduced
susceptibility to plant parasitic nematodes consists in the disruption of
the specialised feeding structure of the plant roots by expressing a
phytotoxic substance under the control of the regulatory DNA sequences
according to the invention. The general principles of this approach have
been disclosed and claimed in International patent applications
W092/21757, W093tlO251 and W094tlO320, which are hereby incorporated by
reference. For the sake of consistency, the phytotoxic substance shall be
referred to hereinafter as the nematode feedings site (NFS) disruptive
substance.
Although the regulatory DNA sequences according to the invention
are substantially specific for the nematode feeding structure, it may be
that due to expression in non-target (i.e. non-NFS) tissue the NFS
disruptive substances under the control thereof have adverse effects on
plant viability and/or yields. Moreover, it was found that the regulatory
DNA sequences according to the invention are active during the tissue
culture phase in the transformation procedure, necessitating the use of a
neutralising substance during this phase. In order to reduce or eliminate
(potential) adverse effects, it is therefore strongly preferred to use a
chimeric NFS-disruptive construct according to the invention in
conjunction with a neutralising gene construct. The details of such a so-
called two-component approach for the engineering of nematode resistant
plants are set out in W093/10251. According to this approach a NFS-
disrupter compound (coding sequence-A) is placed under the control of a
promoter that is at least active in the NFS, and preferably not or hardly
outside the NFS, whereas the unwanted phytotoxic efects outside the NFS
are neutralised by a neutralising compound (coding sequence-B) that is
expressed at least in those tissues wherein the disruptive substance is
produced except for the NFS.
According to the two-component approach a suitable promoter-A is
defined as a promoter that drives expression of a downstream coding
sequence inside the NFS, at levels sufficient to be detrimental to the
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s
metabolism and/or functioning and/or viability of the NFS, while this
promoter should preferably, but not necessarily, be inactive in tissues
outside the NFS; it should at least never be active outside NFS at such
levels that the activity of the di~ruptive ~ubstance, encoded by coding
sequence-A, can not be neutralized sufficiently by product~ from coding
sequence-B.
The properties of the regulatory DNA ~equences according to the
invention, in particular the 4, 2.1 and 1.5 kBp fragments of #1164, make
them highly useful in the two-component approach, as is illustrated by
way of Examples herein. Obviously, numerous mutations such as deletions,
additions and changes in nucleotide sequence and/or combinations of those
are possible in the regulatory DNA sequences according to the invention
which do not alter the properties of these sequences in a way crucial to
their intended use. Such mutations do, therefore, not depart from the
present invention.
Moreover, as is well known to those of skill in the art, regulatory
regions of plant genes consist of disctinct subregions with interesting
properties in terms of gene expression. Examples of subregions as meant
here, are enhancers but also silencers of transcription. These elements
may work in a general (constitutive) way, or in a tissue-specific manner.
As is illustrated in the examples, several deletions may be made in the
regulatory DNA sequences according to the invention, and the subfragments
may be tested for expression patterns of the associated DNA. Variou~
subfragments so obtained, or even combinations thereof, may be useful in
methods of engineering nematode resistance, or other applications
involving the expression of heterologous DNA in plants. The use of DNA
sequences according to the invention to identify functional subregions,
and the subsequent use thereof to promote or suppress gene expression in
plants is also encompa~sed by the present invention.
Within the context of this invention, the terms NFS disruptive
substance and neutralizing substance embraces a series of selected
compounds that are encoded by DNA whose gene products (either protein or
RNA or antisense-RNA) are detrimental to the metabolism and/or
functioning and/or viability of NFS or organelles therein and for which
neutralizing substances are known that are able, when expres~ed
simultaneously in the same cell as the disruptive substance, to repress
the activity of the disrupting substance. Preferred combinations of
disrupting and neutralizing substances are e.g. barnase / barstar from
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Bacillus amyloliquefaciens (Hartley, 1988, J. Mol. Biol. 2Q2, gl3-915),
restriction endonuclea~es / corresponding methyla~es ~uch a~ E~QRI from
E.coli (Green et al., 1981, J. Biol. Chem. 2~, 2143-2153) and EcoRI
methylase or similar combination~ as described in the review for type II
restriction modification systems ~Wilson, 1991, Nucl. Acid Res. 12,
2539-2566), bacteriocins and corresponding immunity proteins, e.g.
colicin E3 / immunity protein from E. coli tLau et al. 1985, Nucl. Acid
Res. 12, 8733-8745) or any disruptive substance coding gene which may be
neutralized by simultaneou~ production of antisense RNA under control of
promoter-B, such as DNA sequences encoding Diptheria Toxin Chain A (Czako
& An, 1991, Plant Phy~iol. 95, 687-692), RNAses such as RNAse Tl,
ribonucleaseq or proteases and ribozymes against mRNA that code for
phytotoxic proteins.
According to another aspect of the invention combination~ of
disrupting and neutralizing substances comprise respectively gene4
inhibitory to an endogenous gene that encodes a protein or polypeptide
product that is essential for cell viability and, as a neutralizing gene,
a gene that encodes a protein or polypeptide product capable of
~ubstituting the function of the endogenous protein or polypeptide
product. Such disruptive genes may be selected from the group consisting
of (a) genes encoding ribozymes against an endogenous RNA transcript, (b)
gene~ which when transcribed produce RNA transcripts that are
complementary or at least partially complementary to RNA transcripts of
endogenous genes that are essential for cell viability, a method known as
antisense inhibition of gene expression (disclosed in EP-A 240 208), or
(c) genes that when transcribed produce RNA transcripts that are
identical or at least very similar to transcripts of endogenous genes
that are essential for cell viability, an as yet unknown way of
inhibition of gene expression referred to as co-suppression (disclosed by
Napoli C. et al., 1990, The Plant Cell 2, 279-289).
According to a preferred embodiment of the invention use is made of
antisense genes to inhibit expression of endogenous genes essential for
cell viability, which genes are expressed in the nematode feeding
structures by virtue of regulatory DNA sequences according to the
invention fused upstream to the said antisense gene.
The disruptive effect brought about by the antisense gene
inhibitory to the vital endogenous gene is neutralized by the expression
of a neutralizing compound-B, which expression is under the control of a
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promoter-B as defined, said compound-B being a protein or polypeptide
product which is identical or similar to the protein or polypeptide
encoded by the endogenous vital gene and capable of substituting the
function of the endogenous gene product in the host plant. It is
preferred that the nucleotide sequence of the RNA transcript encoded by
the neutralizing gene is divergent from the endogenous vital gene RNA
transcript to avoid a possible co-suppre~sive effect. Hence, it i~
preferred that the neutralizing gene encodes a protein or polypeptide
with essentially the same function as the endogenous vital gene, but
through an RNA transcript intermediate that is divergent; neutralizing
genes which fit this description can be suitably obtained by screening a
database for genes obtainable from a different plant species, or even a
different non-plant species, such as yeasts, animal eukaryotes or
prokaryotes. Preferably, the nucleotide sequence identity of the
transcripts encoded by the disruptive antisense transgene and the
neutralizing sense transgene is less than 90%, preferably les~ than 80%,
yet more preferably said neutralizing sense transgene encodes a protein
or polypeptide gene product that is not identical in amino acid sequence
to the disrupted gene product and wherein the nucleotide sequence
identity of the transcripts encoded by the neutralizing transgene is less
than 75%.
Target genes for anti~ense disrupter genes are selected from those
coding for enzymes that are essential for cell viability, also called
housekeeping enzymes, and should be nuclear encoded, preferably as single
copy genes, although a small size gene family would also be suitable for
the purpose of the invention. Furthermore, the effect of antisense
expression of said genes must not be nullified by diffusion or
tranQlocation from other cells or organelles of enzyme products normally
qynthesized by such enzymes. Preferably, genes coding for
membrane-translocating enzymes are chosen as these are involved in
establish;ng chemical gradients across organellar membranes. Inhibition
of such proteins by antisense expres~ion can not, by definition, be
cancelled by diffusion of substrates across the membrane in which these
proteins reside. The translocated compound is not limited to organic
molecules but can be of inorganic nature; e.g. P, H, OH or electrons.
Preferably, the membrane-translocating enzymes should be present in
organelles that increase in numbers during parasitism, thereby
illustrating the essential role that such organelles have in cells
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comprising the NFS. Specific examples for such organelles are
mitochondria, endoplasmic reticulum and plasmodesmata (Hussey et ~1. 1992
Protoplasma 167; 55-65, Magnusson & Golinowski 1991 Can. J. Botany 5~;
44-52). A list of target enzymes is given in Table 1 by way of example
but the invention is not limited to the enzymes mentioned in this table.
More detailed listings can be assembled from series as Biochemistry of
Plants (Eds. Stumpf & Conn, 1988-1991, Vols. 1-16 Academic Pre~s) or
Encyclopedia of Plant Physiology (New Series, 1976, Springer-Verlag,
Berlin).
Although only in some cases, the gene coding for these enzymes have been
isolated and, therefore, the number of gene copies are not known, the
criteria that have to be met are described in this invention.
15 ~XAMPT.~.~ OF TAR~.FT FNZyMF~ FOR ANTISENSE ExPRF~SION IN NFS ~n SF.N.
EXPRF~SION ouTsIn~ NFS
~nzym~ pathway/or~anelle
20 ATP synthase mitochondrion
adenine nucleotide translocator mitochondrion
phosphate translocator mitochondrion
tricarboxylate translocator mitochondrion
dicarboxylate translocator mitochondrion
25 2-oxo-glutarate translocator mitochondrion
cytochrome C mitochondrion
pyruvate kinase glycolysis
glyceraldehyde-3P-dehydrogena~e glycolysis
NADPH-cytochrome P450 reductase lipid metabolism
fatty acid synthase complex lipid metabolism
glycerol-3P-acyltransferase lipid metabolism~5 hydroxymethyl-glutaryl CoA reductase mevalonic acid pathway
aminoacyl transferase nucleic acid metabolism
transcription factors nucleic acid metabolism
elongation factors nucleic acid metabolism
A suitable promoter-B is defined as a promoter that drives
expression in substantially all cells wherein coding sequence-A is
expressed, with the proviso that it does not drive expression inside a
nematode feeding structure, or not effectively. (With 'substantially all
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cells' iq meant at least those cells that should be viable in order to
get normal plant growth and or development required for commercial
exploitation of such plants). As an illustration of plants in which the
disruptive effect is not neutralized in exactly all cells of the host
plant and which are nevertheless viable and suitable for commercial
exploitation, are those which express a disrupter gene according to this
invention in stamen cells; this may yield male-sterile plants, which i9
even regarded as a commercially attractive trait in some crops. Suitable
examples of the promoter-B type can be obtained from plants or plant
viruses, or may be chemically synthesized. The regulatory sequences may
also include enhancer sequences, such as found in the 35S promoter of
CaMV (Ray et al., 1987, Science 2~, 1299-1302), and mRNA stabilizing
sequences quch as the leader sequence of Alfalfa Mosaic Virus RNA4
~Brederode et al., 1980, Nucl. Acids Res. ~, 2213-2223) or any other
sequences functioning in a like manner.
Alternatively, to provide for expression in all or effectively all
plant tissues, a promoter-B/coding-Sequence-B can be complemented with a
qecond promoter-B'/coding-sequence-B having an expression pattern which
is partly overlapping or entirely complementary to promoter-B/coding-
sequence-B, with the proviso that neither promoter-B nor promoter-B'
drives expression in the NFS. Also hybrid promoters, comprising (parts
of) different promoters combined as to provide for the required
expression pattern as defined herein, fall within the scope of the
preqent invention.
Preferebly, promoter-B is the Cauliflower Mosaic Virus 35S promoter
or derivatives thereof, which is generally considered to be a strong
constitutive promoter in plant tissues ~Odell et al. 1985 Nature ~
810-812). Another preferred example for promoter-B is the strong root
promoter ~QlD (Leach & Aoyagi 1991 Plant Sci. 79; 69-76) from plasmid
pRiA4 of Agrobacterium rhizogenes; the 5' flanking region of ORF15
(Slightom et al. 1986, J. Biol. Chem. 261, 108-121). The suitability of
other constitutive promoters such as the nopaline synthase promoter
(Bevan, 1984, Nucl. Acids Res. 12, 8711-8721) or figwort mosaic virus
promoter (EP-A 426 641) for use as promoter-B can be tested through
fuqion to marker genes such as GUS (Jefferson, 1987, Plant Mol. Biol.
Reporter 5, 387-405), transfer of these constructs to plants and
histochemical analysis of such transgenic plants after infection with
PPN.
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Other regulatory sequences such as t~ ;n~tor sequences and
polyadenylation signals include any such sequence functioning as such in
plants, the choice of which is within the level of skill of the average
skille~ person in the art. An example of such sequences is the 3'
fl~nk; ng region of the nopaline synthase ~nos) gene of Agrobacterium
tumefaciens ~Bevan, 1984, Nucl. Acids Res. 12, 8711-~721).
Further details of the two component approach can be found in
WO93~10251 (herein incorporated by reference).
The choice of the plant species is primarily determined by the
amount of damage through PPN infections estimated to occur in agriculture
and the amenability of the plant species to transformation. Plant genera
which are damaged during agricultural practice by PPN and which can be
made significantly less susceptible to PPN by ways of the present
invention include but are not limited to the genera mentioned in Table 2.
Nematode species as defined in the context of the present invention
include all plant-parasitic nematodes that modify host cells into
specially adapted feeding structures which range from migratory
ectoparasites (e.g. Xiphlnema spp.) to the more evolved sedentary
endoparasites (e.g. Heteroderidae, Meloidogynae or Rotylenchulinae). A
list of parasitic nematodes are given in Table 2, but the invention is
not limited to the species mentioned in this table. More detailed
listings are presented in Zuckerman et al. (eds., in: Plant Parasitic
Nematodes, Vol. I 1971, New York, pp. 139-162).
T~RL~ 2
F.~AMPT.F..S OF PLANT-PAR~SITIC NFM~TODES ANn THFIR
PRTNCIP~T HOST PL~NTS
Nematode Species Principal Host Plants
MF~loi~ Jy~e
M. hapla wide range
M. incognita wide range
M. exigua coffee, tea, Capsicum, Citrullus
M. indica Citrus
35 M. javanica wide range
M. africana coffee
M. graminis cereals, grasses
M. graminicola rice
M. arenaria wide range
Heterodera L Globo~ra
H. mexicana Lycopersicon esculentum, Solanum spp.
H. punctata cereals, grasses
G. rostochiensis Solanum tuberosum, Solanum spp, Lycopersicon esculentum
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G. pall~A Solanum tuberosum
G. tabacum Nicotiana tabacum, Nicotiana 8pp.
H. cajani Cajanus cajan, Vigna sinenqis
H. glycinea Glycine max, Glycine qpp.
5 H. oryzae Oryza sativa
H. schachtii Beta qpp, Braaqica qpp,
H. trifolii Trifolium spp.
H. avenae cerealq, grasses
H. carotae Daucuq carota
10 H. cruciferae Cruciferae
H. goettingiana Pisum sativum, Vicia 8pp.
Within the context of this invention, a plant i~ said to show
reduced qusceptibility to plant paraqitic nematodes tPPN) if a
statistically significant decrea~e in the number of mature females
developing at the qurface of plant rootq can be observed as compared to
control plants. Suqceptible / reaistance classification according to the
number of maturing females is ctandard practice both for cyst- and
root-knot nematodeq (e.g. T~Mon~;a, 1991, Plant Disease 75, 453-454;
20 Omwega et al., 1990, Phytopathol. 80, 745-748).
A nematode feeding qtructure according to the preqent
invention qhall include an initial feeding cell, which shall mean the
cell or a very limited number of cells destined to become a nematode
feeding ~tructure, upon induction of the invading nematode.
A NFS disruptive effect according to the invention iq not
limited to adverse effects on the NFS only; also disruptive effects are
contemplated that, in addition, have an adverse effect on nematode
development by way of direct interaction.
Several techniques are available for the introduction of
rec~ ';nAnt DNA contsining the DNA sequences as described in the present
invention into plant hosts. Such techniques include but are not limited
to transformation of protoplastq using the calcium/polyethylene glycol
method, electroporation and microinjection or (coated) particle
bombardment tPotrykus, 1990, Bio/Technol. 8, 535-542).
In addition to these so-called direct DNA transformation
methods, transformation systems involving vectors are widely available,
such aq viral vectors ~e.g. from the Cauliflower Mosaic Virus tcaMv) and
bacterial vectors te.g. from the genus Agrobacterium) tPotrykus, 1990,
Bio/Technol. ~, 535-542). After qelection and/or screening, the
protoplasts, cell~ or plant parts that have been transformed can be
regenerated into whole plants, using methods known in the art ~Horsch et
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al., 1985, Science 225, 1229-1231). The choice of the transformation
and~or regeneration techniques is not critical for this invention.
According to a preferred embodiment of the present invention
use is made of so-called binary vector 9y9tem (diqclosed in EP-A 120 516)
in which Agrobacterium strains are used which contain a helper pla~mid
with the virulence genes and a compatible pla~;A, the binary vector,
conta;n;ng the gene construct to be transferred. This vector can
replicate in both E. coli and in Agrobacterium; the one used here is
derived from the binary vector Binl9 ~Bevan, 198g, Nucl. Acids Res. 12,
8711-8721). The binary vectors as used in thiq example contain between
the left- and right-border sequences of the T-DNA, an identical
NPTII-gene coding for k~ cin resistance (Bevan, 1984, Nucl. Acids Res.
12, 8711-8721) and a multiple cloning 9ite to clone in the required gene
constructs.
Recent scientific progress shows that in principle monocots
are amenable to transformation and that fertile transgenic plants can be
regenerated from transformed cells. The development of reproducible
tiqsue culture systems for these crops, together with the powerful
methods for introduction of genetic material into plant cell~ has
facilitated transformation. Presently, preferred methods for
transformation of monocots are microprojectile bombardment of explants or
suqpension cells, and direct DNA uptake or electroporation (Sh;m~m~to, et
al., 1989, Nature 338, 274-276). Transgenic maize plants have been
obtained by introducing the Streptomyces hygroscopicus bar gene, which
encodes phosphinothricin acetyltransferase (an enzyme which inactivates
the herbicide phosphinothricin), into embryogenic cells of a maize
suspension culture by microparticle bombardment (Gordon-Kamm, 1990, Plant
Cell, 2, 603-618). The introduction of genetic material into aleurone
protopla~ts of other monocot crops such as wheat and barley has been
reported (Lee, 1989, Plant Mol. Biol. 13, 21-30). Wheat plants have been
regenerated from embryogenic suspension culture by selection only the
aged compact and nodular embryogenic callus tissues for the establishment
of the embryogenic suspension cultures (Vasil, 1990 Bio/Technol. ~,
429-434). Also an Agrobacterium-using method for the transformation of
rice has been disclosed recently (WO 95tl6031). The combination with
transformation systems for these crops enables the application of the
present invention to monocots. These methods may also be applied for the
transformation and regeneration of dicots.
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The following examples are given only for purposeq of
illustration and do not intend to limit the scope of the invention.
EXPERIMENTAL PART
DNA procedures
All DNA procedures were carried out according to qtandard
methods described in Maniatis ~Molecular Cloning, A laboratory Manual 2nd
Edition, Cold Spring Harbor Laboratory, 1990).
Trans~ormat~on o~ Ar~b i dop~ ~ ~
Transformation wa~ carried out using co-cultivation of
Arabidopsis thaliana (ecotype C24) root segments with Agrobacterium
strain MOG101 containing a suitable binary vector as described by
Valvekenq et al. (1988, Proc. Nat. Acad. Sci. USA ~, 5536-5540) which i8
as follows:
Arabidopsis seeds were vernalized for 7 days at 4~C before
germination. Seeds were surface-~terilized for 2 min in 70% EtOH,
transferred to 5% NaOCl/0.5~ NaDodSO4 for 15 min rinsed five times with
sterile distilled water, and placed on 150 x 25 mm Petri disheA
cont~;n;ng germination medium ~GM) (Table 3) to germinate. Petri disheq
were sealed with gas-permeable medical tape (Urgopore, Chenove France).
Plants were grown at 22~C in a 16-hr light/8-hr dark cycle. The same
growth-room conditions were used for tissue culture procedures.
All plant media were buffered with 2-(N-morpholino)ethanesulfonic acid at
0.5g/liter (pH 5.7: adjusted with 1 M KOH), solidified with 0.8~ Difco
Bacto agar, and autoclaved at 121~C for 15 min. Hormone9 and antibiotics
were dissolved in dimethyl Aulfoxide and water, respectively, and were
added to the medium after autoclaving and cooling to 65~C.
Intact roots were incubated for 3 days on solidified 0.5/0.05
medium (Table 3). Roots were then cut into small pieces of about 0.5 cm
(herein referred to as "root explants") and transferred to 10 ml of
liquid 0.5/0.05 medium; 0.5-1.0 ml of an overnight Agrobacterium culture
- was added. The root explants and bacteria were mixed by gentle shaking for about 2 min.
Subsequently, the root explanta were blotted on sterile filter paper to
remove most of the liquid medium and cocultivated for 48 hr on 0.5/0.05
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agar. The explants were then rinsed in liquid 0.5/0.05 medium containing
1000 mg of vancomycin (Sigma) per liter. The pieces were blotted and then
incubated on 0.15/5 agar (Table 3) supplemented with 750 mg of vancomycin
and 50 mg of Xm per liter. Three weeks after infection with agrobacteria
containing a chimeric neo gene, green Km-resistant (KmR) calli were
formed in a background of yellowish root explants. At this point the root
explants were transferred to fresh 0.15t5 agar containing only 500 mg of
vanc: ~in and 50 mg of Km per liter. Three weeks later most green call
had formed shoots. Transformed shoots were transferred to 150 x 25 mm
Petri dishes containing GM to form roots or seeds or both. In these Petri
dishes, many regenerants formed seeds without rooting. Rooted plants
could also be transferred to soil to set seed. The following modification
was made to obtain the initial root material 6 sterilized Arabidopsis
~halian~ C24 seeds were germinated in 50 ml GM (250 ml Erlenmeyer) on a
rotary shaker (100 rpm) in a growth room for 9 days under low light
conditions. Transgenic plants were regenerated from shoots grown on
selection medium (50 mg/l kanamycin), rooted and transferred to
germination medium or soil.
20 TART~ 3
PT,~T ~F'.nTA
CIM SIM
GM R3~ PG1* 0.5/0.05 0.05/7* 0.15/5*
Salts + ~itamins MS MS B5 B5 MS B5
Sucrose, g/L 10 30 -- -- 30 --
Glucose, g/L -- -- 20 20 ~~ 20
30 IAA, mg/L -- 5 -- -- 0.05 0.15
2,4-D, mg/L -- 0.5 2 0.5 -- --
2ipAde, mg/L -- -- -- -- 7 5
Xin, mg/L -- 0.3 0.05 0.05 -- --
L, liter; IAA, indole-3-acetic acid; Kin, kinetin; 2ipAde, N6-(2-
isopentenyl)adenine; CIM, callus-inducing medium; SIM, shoot-inducing
medium; MS, Murashige & Skoog medium ; B5, Gam~org B5 medium
Transformation o~ potato
For the transformation of Solanum tuberosum var. Kardal a protocol as
described in Hoekema et al. 1989 Bio/Technology 7, 273-278 was used with
several modifications.
Peeled surface-sterilized potato tubers were cut in 2 mm thick slices.
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These were used to cut out disks of 1 cm in diameter around the periphery
of the slice. The disks were collected in WM ~Murashige & Skoog medium,
containing 1 mg/l ~hi; ;ne HCl, 0.5 mg/l pyridoxine Hcl, 0.5 mg~l
~ nicotinic acid, 100 mg/l myo-inositol, 30 g/l sucrose, 0.5 g/l MES pH
5.8). Inoculation with Agrobacterium tumefaciens 9train EHA105 ~Hood et
al. 1993 TransgeniC Research 2, 20a-218) was done by replacing the WX
with 100 ml fresh WM containing the resuspended pellet of 10 ml
Agrobacterium culture grown freshly in LB + appropriate antibiotic to an
OD600 of 0.5-0.7. After incubating the tuber disks for 20 min in the
bacterium suspension they were transferred to solidified CM ~WM
supplemented with 8 g/l agar, 3.5 mg/l zeatin riboside, 0.03 mg/l indole
acetic acid) at a density of 20 explants/petridish. After two days the
disks were transferred to PM (CM supplemented with 200 mg/l cefotaxime,
100 mg/l vancomycin) to select against the Agrobacteria. Three days later
the disks were transferred to SIM plates (CM supplemented with 250 mg/l
carbenicillin, 100 mg/l kanamycin) at a density of 10 explants/petridish
to select for the regeneration of transformed shoots. After 2 weeks the
tissue disks were transferred to fresh SIM, and after another 3 weeks
they were transferred to SEM ~SIM with 10 x lower concentration of
hormones). About 8-9 weeks after co-cultivation the shoots were large
enough to cut them from the callus tissue and tran~fer them to glass
tubes ~Sigma, Cat.nr. C5916) containing 10 ml of RM (wM containing 0.5 x
MS salts, 0.5 x vitamins, 10 g/l sucrose, 100 mg/l cefotaxime, 50 mg/l
vancomycin and 50 mg/l kanamycin) for rooting maintenance in vitro and
vegetative propagation.
~anAl ~n~ of nematode~, growth and infection of plant roots
Arabidopsis seeds were surface sterilized and sown in petri dishes ~0: 9
cm) on B5 medium containing 20 g/l glucose and 20 mg~l kanamycin. After 3
days at 4~C the plates were incubated for 2 weeks in a growth chamber at
22~C with 16-hr light/8 hr-dark cycle. Kanamycin-resistant plants were
then transferred to soil-filled translucent plastic tubes ~30x15x120 mm,
Kelder plastibox b.v., The Netherlands). The tube9 were placed tilted at
an angle of 60 degrees to the vertical axis causing the roots to grow on
the lower side of the tubes. This allows to monitor the infection process
by eye and facilitates removal of the root system from the soil for GUS
analysis. Infection was done after two more weeks by injecting a
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auspension containing 500 second stage larvae of Heterodera schachtii ~in
3 ml H2O) per root system or 300 second stage larvae of Meloidogyne
incorJn;ta per root system into the 30il.
S~milarly, potato shoots which had rooted on k~- yCin~COntaining RM
medium were transferred to qoil-filled tran~lucent plastic tubeq
(30x15x120 mm, Kelder plactibox b.v., The Netherland~) and grown tilted
for another 2 week~ at 22~C with 16 h light/8 h dark cycle. Infection was
done by injecting a suspension conta;ning 500 ~econd stage larvae of
Globodera pallida (in 3 ml H2O) per root sy~tem into the soil.
GUS aaaay
GUS activity was determined at various times during the infection proce~
by thoroughly washing the root sy~tems to remove most of the adhering
soil and incubating them in X-Gluc solution (1 mg/ml X-Gluc, 50mM NaPO4
(pH7), lmM K4Fe(CN)6, lmM K K3Fe(CN)6, 10mM EDTA, 0.1% Triton X100) at
37~C over night. After removal of the chlorophyll from the ti~que by
incubation with 70% ethanol for aeveral hour~ GUS staining was monitored
under the microscope.
E~ample
ConJtruction of binary vector pMOG800
The binary vector pMOG800 i~ a derivative of pMOG23 (Fig. 1,
depo~ited at the Centraal Bureau voor sch; -lcultures, Oosterstraat 1,
Baarn, The Netherlands on January 29, 1990 under number CRS 102.90) in
which an additional KpnI restriction site was introduced into the
polylinker between EcoRI and SmaI. This plasmid contains between the left
and right borders of T-DNA a kanamycin resistance gene for selection of
transgenic plant cell~ (Fig. 2). A sample of E. Coli DH5 alpha,
harbouring pMOG800, was deposited at the Centraal Bureau voor
Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on Augu~t 12,
1993 under number CBS 414.93.
E~ample 2
Conatruction of promoterleaa GUS con~truct pMOG553
Construction of this vector is described in Goddijn et al. 1993
Plant J 4, 863-873. In this reference an error occurs; the construct
contains a CaMV 35S RNA terminator behind the ~-glucuronidase gene
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inqtead of the indicated nos terminator. The sequence between the T-DNA
borders of this binary vector is available from the EMBL databaqe under
acceqqion number: X84105.pMOG553 carries the HygR marker for plant
transformation (Fig. 3).
Esample 3
Identification and isolation of a trapped NFS-prefQrential
promoter fragment in Arabidop~i~ thaliana
The binary vector pMOG553 waq mobilized by triparental mating to
Agro~acterium tumefaciens strain MOG101. The resulting strain was used
for Arabidopsis root transformation. More than 1100 transgenic
Arabidopsis plant lines were obtained in this way. Tranqgenic plants were
grown to maturity, allowed to self-fertilize and the resulting qeeds (S1)
were harvested and vernalized. Subqequently S1 qeedq were germinated on
nutrient solution (Goddijn et al. 1993 Plant J 4, 863-873) solidified
with 0.6% agar, 10 mg/l hyy-olllycin and stored at 4~C for a 4 day
imbibition period. At day 5 the plateq were transferred to room
temperature and moderate light (1000 lux, 16 h L / B h D) for
germination. Fourteen days old see~l;ngs were transferred to potting soil
in tilted translucent plastic tubes (30x15x120 mm) for further growth at
5000 lux (20~C). Growing the plants in this way causes most of the root
system to grow on the lower side of the tubes in the interphase between
80il and tube. After two weeks the roots were infected with nematodes as
described in the Experimental part. At qeveral time points after
inoculation (ranging from 2 -14 days), the root systems were analyzed for
GUS activity as described in the Experimental part. Line pMOG553#1164 was
identified as a line which showed rather strong GUS expression inside
qyncytia and giant cells induced by Heterodera schachtii and Meloidogyne
incognita, respectively. In un-infected control plants (as well as in the
infected plants) of this line very weak GUS expression was detected in a
few cells at the base of young lateral roots and in some green parts of
the plant.
In line 1164 this phenotype was found to segregate at a 1:3 ratio,
indicating that the GUS construct is present at one locus per genome. The
presence of only one T-DNA copy was confirmed by Southern analyqis.
A 1.5 kb fragment of the trapped promoter sequence adjacent to the GUS
open reading frame was isolated by inverted PCR. Genomic DNA of this line
was cleaved with the restriction enzyme MscI, which cleaves once in the
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GUS coding region, and religated. By subsequent digestion of the circular
DNA with the enzyme SnaBI a linear fragment waq obtained with known GUS
sequences at the ends and the fl~nk;n~ plant sequence in between. This
fragment was amplified using the primer set GUSinvS (5' CTT TCC CAC CAA
CGC TGA TC 3' SEQIDNO: 1) and GUS7 (5' GTA ATG CTC TAC ACC ACG CCG 3'
SEQIDNO: 2), cloned in a multi-copy vector and sequenced (see below).
To clone this amplified fragment back in front of GUS the plant sequence
was re-amplified from Arabidopsis genomic DNA using the primers GUSinv5
and 1164XBM (5' TCT AGA GGA TCC TGG CCA TAC AAA TCA ACG TTT AC 3'
SEQIDNO: 3). A pfu DNA polymerase carrying a proofreading activity was
used to reduce the error rate. Primer 1164XBM introduces a ~amHI site at
the 5 end of the promoter, which allowed to clone the 1480 bp BamHI
promoter fragment back in front of GUS in construct pMOG819 without
changing the sequence between the GUS open reading frame and the plant
promoter.
Erample 4
Construction of promoterleQ3 GUS construct pMOG819
This vector was constructed by cloning the GUSintron coding region
(V~nc~nneyt et al. 1990, Mol. Gen. Genet. 220; 245-250) of pMOG553 as a
BamHI-Eco~I fragment in the polylinker of pMOG800. The binary vector
pMOG819 (Fig. 4) qerves to introduce the cloned promoter fragments for
further expression analysis after transformation of plantq.
25Esampl~ 5
Analysi~ of promoter fragments after re-introduction into
Arabidop~is
The PCR product from tag 553#1164 was cloned back in front of a
GUS gene on the binary vector pMOG819 to make pMOG849 (Fig. 5). A sample
of E. coli DH5a harbouring pMOG849 has been deposited at the Centraal
Bureau voor schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on
May 4, 1995 under number CBS 308.95. To determine the tissue-specific
activity of the cloned promoter fragment the resulting clone pMOG849 waq
mobilised to Agrobacterium tumefaciens and the corresponding strain was
used to transform wildtype Arabidopsis thaliana plants. Per construct 24-
30 transformants were produced. Seeds from the primary transformants were
harvested and grown up for infection asqays with 8eterodera schachtii as
deqcribed in the Experimental part. GUS analysis after nematode infection
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showed that 79% of the lines transformed with pMOG849 expressed the
reporter gene in syncytia. Some weak expression was also found in the
area of lateral root branching, in the vascular tissue of roots and
leaves, in the centre of the rozette and in some flower tissues. GUS
expre~sion outside the syncytium showed strong variation from line to
line (see Fig. 6). Presumably, this variation is a result of genome
position effects on the introduced requlatory sequences. NevertheleRs, in
most lines, an expression pattern was found that was very similar to the
originally tagged line 553#1164.
~ven though the activity of the promoter fragment in the various
pMOG849 lines waq generally much weaker than the GUS-activity inside
syncytia, none of the syncytium-positive lines was entirely specific for
the feeding sites.
GUS-expression was also found in giant cells induced by infection
with ~eloidogyne incognita in the same lines which expressed GUS in
syncytia induced by Heterodera schachtii. This shows that the #1164
fragment can be used as a nearly feeding site specific promoter to
engineer plants having reduced susceptibility to Meloidogyne incognita
and Heterodera schachtii.
During the tissue culture phase, it was observed that the #1164
regulatory sequence was also active as a promoter, thus prompting the
need to use a neutralizing gene if the #1164 promoter fragment is
transferred to Arabidopsis with a plant cell disruptive gene under its
control, such as barnase (see Example 8 and 9).
The 553~1164-based PCR fragment was used as a probe to isolate the
corresponding genomic clone. A genomic fragment of 2.1 Rb (see SEQIDNO:
4) was then used in a similar approach as described above (pMOG889
contains genomic 553~1164 fused to GUSintron). Again, nematode-induced
GUS expression could be observed in syncytia and giant cells after
nematode infection of Arabidopsis roots with H. Schachtii and M.
incognita respectively.
Example 6
Sequence determination of promoter tag pMOG553#1164
The sequence of the genomic clone of #1164 was determined by the
primer walking strategy on CsCl purified DNA, using the automatic
sequencer ALF of Pharmacia. Fluor dATP was used in combination with the
AutoRead sequencing kit. The procedure is described in Voss et al. (1992)
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Mol Cell Biol 3, 153-155. The sequence is depicted in SEQIDNO: 4.
Example 7
Cloning of promoter ~ub~ragment (8)
Five subfragments of promoter #1164 were made by PCR using the
primers as shown in table 4. The primer numbering is the same as that
used in the Sequence Listing. For all amplifications the proofreading DNA
polymerase pfu was used and pMOG849 served as target DNA. All 5' end
primers contain an XhoI site. Thus, all PCR generated deletion fragments
10 of the 1164 promoter could be reintroduced in pMOG819 using this XhoI
site and the BamHI site, which is located in the multiple cloning site of
pMOG553 and was retained in the tagged linell64 between the GUS coding
region and the tagged plant sequence. The numbers refer to the constructs
resulting from the subfragments cloned in pMOG819; the primers 6044-1 to
15 6044-6 correspond with SEQIDNO's 6 to 11, respectively.
TARTF 4
pMOG 5' end primer 3'end primer
958 6044-1 6044-6
959 6044-2 6044-6
960 6044-3 6044-6
961 6044-4 6044-6
962 6044-1 6044-5
After reintroduction of these gene cassettès into plants expression
patterns, timing and the like can be determined as described for the 1.5
Kb #1164 fragment in Example 3. Fragments found to have useful patterns
and/or timing may subsequently be used to drive expression of other
heterologous DNA sequences (both sense/coding and antisense) and/or used
to make hybrid promoter constructs. Furthermore, further analysis yields
insight in several regulatory elements such as silencers, enhancers and
the like, and creates the possibility of willfully influencing expression
patterns and/or timing. To illustrate how the promoter fragments
according to invention can be used to impart reduced susceptibility to
nematodes this is now illustrated for the genomic 2.1 Kb #1164 fragment,
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cloned in front of Barnase, as an example of a NFS-disrupter gene.
EYample 8
Cloning of #1164 in front of barnase
A 2.1 Kb genomic DNA fragment containing the S' tagged
sequence from line 1164 was cloned in front of barna~e, a Bacillu~
amyloli4uefaciens derived RNase gene, to engineer plants resistant to
sedentary plant nematodes. The genomic fragment was obtained by screening
400000 clones of a genomic library of Arabidopsis ecotype C24 with the
10 #1164 iPCR product (see Example 3). From one of the hybridizing cloneq a
4 kb EcoRI fragment was isolated and subcloned in the multicopy pla~mid
pKS (Stratagene). Sequence analysis revealed that this clone contained
2.1 kb of sequence 5' to the T-DNA insertion in line 1164 and 1.9 kb of
3' sequence.
To restore the exact sequence context in front of the GUS
coding region a 546 bp SnaBI fragment from pMOG849 Apanning the promoter-
GUS fusion was inserted at the SnaBI site of the genomic clone. A 2325 bp
HindIII fragment was isolated from the resulting clone, containing the
entire 5' tagged sequence from the genomic EcoRI subclone. This fragment
was cloned in front of the barnase gene in construct pFL8 ~described
below), resulting in clone pFL15.
A fragment containing the barnase coding region was PCR
amplified on pMT416 DNA (Hartley, sub) using primer~ 5'
CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 12) and 5'
C~GClCGAGCCTAGGCACAGGTTATCAACACGTTTG 3' (SEQIDNO: 13). These primers
introduce flanking Bam~I and XhoI restriction sites to facilitate cloning
of the fragment. The fragment was cloned in the multiple cloning site in
a vector containing the barstar gene under control of a Taq promoter
(necessary to overcome toxicity of barnase in bacteria). To eliminate
toxicity of barnase expression in subsequent cloning steps a ST-LSl
intron was inserted in the StyI site of barnase. An NcoI site was created
at the barnase translation initiation codon by recombinant PCR using the
primers 5' CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 14) and 5'
CTTACTCGAGCCATGGTAA~lllClGC 3' (SEQIDNO: 15), resulting in pOG16.1. The
5' untranslated sequence of barnase was further modified to resemble the
corresponding sequence in the original line pMOG553#1164 by annealing the
following oligonucleotides 5'
GATCTAGACTCr,A~.AA~CTTGGATCCCCGGGTAGGTCAGTCCCC 3' (SEQIDNO: 16) and 5'
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CATGGGGGACTGACCTACCCGGGGATCCAAGCTTCTCGAGTCTA 3' tSEQIDNO: 17) and
ligating the resulting adapter between the BglII site and the NcoI site
of pOG16.1, resulting in clone pFL8. The adapter introduced a HindIII
site 5' to the barnase coding region which was used to insert the 1164
promoter yielding pFL15. In addition, by this procedure a fragment
containing the Tag promoter and the barstar gene were exchanged with this
adapter.
Example 9
Construction rolD-B~
Construct pFL11 contains a chimeric barstar gene in a binary
vector. This construct was cloned in the following way. The barstar
coding region resides on a HindIII/BamHI fragment in construct pMT316
(Hartley (1988) J Mol Biol 202, 913-915). The HindIII site was changed
into a BamHI site by ligating in this site the self-annealing adapter 5'
AGCTCGGATCCG '3 (SEQIDNO: 18). Subsequently, the resulting BamHI fragment
was cloned between a double enhanced CaMV 35S promoter and a nos
tenminator in the expres~ion cassette pMOG180, described in WO93/10251,
re~ulting in pOG30. Using the adapter 5' GGCTGCTCGAGC 3' (SEQIDNO: 19)
the HindIII site at the 3' end of the nos terminator was changed into an
XhoI site and the EcoRI site at the 5' end of the promoter was changed
into a HindIII site using the adapter 5' AATTGACGAAGCTTCGTC 3' (SEQIDNO:
20). Then the 35S promoter was replaced by the promoter from the
Agrobacterium rhizogenes RolD gene. This promoter was excised as a
HindIII/BamHI fragment from construct pDO2, obtained from F. Leach (Leach
and Aoyagi (1991) Plant Sci 79, 69-76). From the resulting clone, pOG38,
the barstar gene including promoter and terminator was excised by
digestion with HindIII and XhoI and inserted in the respective sites of
the polylinker in pMOG800, resulting in pFL11.
Finally, the chimeric #1164 promoter-barnase gene was cleaved out
of pFL15 as an EcoRI fragment and inserted in the unique EcoRI site of
pFL11 between barstar and the NptII marker gene in a tandem orientation,
resulting in pMOG893.
Exa~ple 10
~ransformation of potato plants with pMOG893 and testing for
increased resistance against Globodera pallida
The binary vector pMOG~93 was mobilised to Agrobacterium tumefaciens and
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the resulting strain was used for transformation of tuber discs from the
potato cultivar Kardal as described in the Experimental part. A total of
98 transgenic lines were obtained. These lines were propagated
vegetatively by cutting shoots in segments containing at least one node
S and rooting them in vitro. Per line 15 plants are tested for increased
resistance to Globodera pallida as described in the Experimental part. It
is expected that potato plants transformed with the pMOG893 contained
Barna~e/Barstar construct show reduced susceptibility to Globodera
pallida due to the nematode-induced expression of Barnase inside the
(developing) nematode feeding structure.
The above examples merely serve to illustrate the invention and are
not meant to indicate it~ limits. Numerous modifications will readily
occur to the person skilled in the art which are within the scope of the
invention.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: MOGEN International N.V.
(B) STREET: Einsteinweg 97
(C) CITY: Leiden
(D) STATE: Zuid-Holland
(E) COUNTRY: The Netherlands
(F) POSTAL CODE (ZIP): NL-2333 CB
(G) TELEPHONE: 31-71258282
(H) TELEFAX: 31-71-221471
(ii) TITLE OF INVENTION: REGULATORY DNA SEQUENCES
(iii) NUMBER OF SEQUENCES: 20
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /note= "primer that anneals to uidA
gene (Beta-glucuronidase) at position 224-205 from
the tagging construct pMOG553.(X83420)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CTTTCCCACC AACGCTGATC 20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
10 GTAATGCTCT ACACCACGCC G 21
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA ~genomic)
(iii) HYPOTHETICAL: YES
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "5'overhang with a XbaI and
a BamHI site"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 13..35
(D) OTHER INFORMATION: /note= "this part of the primer
anneals to sequence 6044-0 at position 646 to 668"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TCTAGAGGAT CCTGGCCATA CAAATCAACG TTTAC 35
(2) INFORMATION FOR SEQ ID NO: 4:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2163 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana
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(B) STRAIN: C24
(ix) FEATURE:
(A) NAME/KEY: CDS
~B) LOCATION: 2161... 2163
(D) OTHER INFORMATION: /codon_start= 2161
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 2128... 2163
(D) OTHER INFORMATION: /note= "Sequence of pMOG553
upstream (5') of the uid A translation initiation
codon up to the RB/plant genome transition."
15 (ix) FEATURE:
(A) NAME/KEY: promoter
(B) LOCATION: 1..2127
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 787..804
(D) OTHER INFORMATION: /label= primer6044-1
/note= "annealing of primer 6044-1 (table 4) to
amplify subfragment"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 1147..1169
(D) OTHER INFORMATION: /label= primer6044-2
/note= "annealing of primer 6044-2 (table 4) to
amplify subfragments"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
35 (B) LOCATION: 1853.. 1880
(D) OTHER INFORMATION: /label= primer6044-3
/note= "annealing of primer 6044-3 (table 4) to
amplify subfragments"
40 (ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 1918..1940
(D) OTHER INFORMATION: /label= primer6044-4
/note= "annealing of primer 6044-4 (table 4) to
amplify subfragments"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 1897..1917
(D) OTHER INFORMATION: /label= primer6044-5
/note= "annealing of primer 6044-5 (table 4) to
amplify subfragments (opposite strand)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
SUBSTITUTE SHEET (i-lULE 26~
I
CA 022~7l49 l998-l2-02
W 097/46692 PCTAEP96102437
27
GAATTCCATC AAATATTAAC TTTTAATATC ACTACATCAT CACCAGATAT GGATGAATAT 60
TTATATAATA TTCACTGCCA ATTAGTTCTT TTAACATATA TATGCTGCTT GTACATATAG 120
GTCATCCAAA TTTTTAGGGT TCAAAACAAA ACrAAAAr~AA ACA~AAAAGA TCTGATAAAA 180
AGTCTTCATT TTAGAr~.A~.G GATCAAACTT ATCTAGTGCA TCTGAATGAA AAAAAATGAT 240
CTTAACACTG CAGGTGAAGG CTGGCTCAAT CTTTGACAAT ATATTGATCT GCGATGACCC 300
GGCATATGCA AGAAGCATCG TGGATGACTA TTTTGCCCAA CACAGGGAGG TAGATTTCCA 360
AG'lC~ 7lA TATC.l-l-G ~llCll-lIG GATAAAATCA AAGAAGTTTT TTGATCTTGC 420
AAGTGTGTAG TAATTGCAAA TGGATTTTCT GCATGCTATT ATATACGAAA ATGTCTTATT 480
AGTGAATTTG ATATGCTATA TACTTGGCCA TATGCACCAG TCTGAGAAGG AGTTATTTGC 540
G~AAG~A~AG AAArAAAGAA AAGCTAGAGA AGATGAGGTT TGTAGTTCAC AAAAAAGTTC 600
1.~-~ ~.~ TTCAAGTCTT CTCTGTATAT CCTAGTTAAC GAGCATGGCC ATACAAATCA 660
ACGTTTACAG GAAGCTCGGA TAGCACGGGA AGAAGGTGAA CGCAGGCGGA AAGAAAGGGA 720
CrACCGGTAT Gr.A~ArAG~A GGAGGCGTGA CAAACGGGTA AGTACTTATT TGAGTCCAAA 780
TGAATTATAA CCTTCTCAAC TCTGTTTTAT CTGGAAACCA AGTGAGTGAA TATTGTTGGA 840
AA-GC71llGG .~ L~ ~11~1 11 lGl 11 ~GC AGCCGAATCC ACGTGATTAT ATGGATGATT 900
ACCATGTAAG TC~lC~--~- ATCTCAACCA CTTTAAAAAG AATGGTTTAT GCATTTTAGT 960
ACTGAATCAT CTTAACTGTT CTAAAAATGT AA~7~ 7llA TGATTCTGAA ~llCGI~lAG 1020
35 GACGAGCTAT GAGGCGCAGA GTGGTGGGCA TTTGGCAAAG CATTGGGGGA AATTATCTAT 1080
ATTTTGCCTT TGAATGTGTA C~l~7lll~7lA ATTTCATAAT TTGTAACCTT TTGTATTCAT 1140
ATTCTTATAA TGTATTTTGG CATGAAAACT TGACTTGTTA illIlCCCTT CCAATACAAA 1200
AATTCTAAAA TTGGCAAGAA CGACTTACTA CCATGCAGTG ATTTGTGAAG TTTGATAGTG 1260
GTGGTAATTT TAAllGl--'C ACr.ArAGAAA AIII~lClAT ATCCTGAAGA AGATAGCTGA 1320
45 GTTGAACTGA GAGGTTGGCG TTTCTTAGTG AAAATAr.AAA AAATAGAAAT CTTTAGCTAG 1380
AAAGTGTGGT GTGGACCCGA CTGATGGTAA CCATGTTCAT TTGGAGGAAC TAATGTGAAT 1440
ATTAGCTAAA AGCATATTGT TGAGTGTTGA CAAAATGACA ACAGATAAAT CGTCAAATAC 1500
TACTCCACCT AGCTAATATT lli~ii-AAC TAATGTTAGA AAGCCACCTA TTTGCATCCG 1560
TAATGATAAA AACTAAAAAA ATATTAGATT ATTAGAGTGA TACATTTTGT GTGAAAAcGT 1620
55 AAAC~AAAGT rAAAAGAAAG AAAAACGAAA GAAATTTAAA TGCGGTTTAT GGTGGGCACA 1680
SUBSTITUTE SHEET (RULE 26)
... .. . . . ...... . .
CA 022~7l49 l998-l2-02
W097/46692 PCT~EP96/02437
28
AAl~l.vLGA CCTGGTGTGT CCClllCCCA CTTAAATGTA CGGCTGATAA TCACATCAGT 1740
GGCGACTTTA GGAAATAGAA AATTCGCACA ATTGACTCGA TACGCATTAA AGTCGTAATC 1B00
ACTAGACATT TTTGTTATCT ~lCCll,AGT G~l-C~1~lA ATCTGGAACG TCCTTATAAT 1860
AACATAAGAT AAATATTTAC TTAATTAGCT ACGGAACTAC ATTAGTATTC AATTGATATA 1920
ACTAATGGTA ATTACTAATT AATTGCGGAA AGCC~AGA~A GGTGATGGTG CACG~-lGCAT 1980
GT~.AA~AGCT TTTGATACGT AAGTGGAGCA CTCATGATAA GCGAAGTTGT CTATTTATAA 2040
AGTTTAATTT ACTGTGCTTT TTATAATGTG ACACACTATT GGAATCCAAT GACTGCATTA 2100
TTTATTTATA TGTAAAAAAA AAAGTCTCAA AGCTTGGATC CCCGGGTAGG TCAGTCCCTT 2160
ATG 2163
Metl
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 am1no acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS
(A) LSNGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
~iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
~ix) FEATURE:
(A) NAME/KEY: misc_feature
~B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "5' overhang containing the
XhoI and the EcoRI sites"
SIJBSTITUTE SHEET (RULE 26)
CA 022~7l49 l998-l2-02
W 097/46692 PCT/EP96tO2437
29
(ix) FEATURE:
(A) NAME/REY: primer_bind
~B) LOCATION: 13..30
~D) OTHER INFORMATION: /note= "this part of the primer
anneals the sequence of 6044-0 ~SEQIDNO: 4) at
position 787-804"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CTCGAGAATT CTATAACCTT CTCAACTCTG 30
~2) INFORMATION FOR SEQ ID NO: 7:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 35 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA ~genomic)
~vi) ORIGINAL SOURCE:
~A) ORGANISM: Arabidopsis thaliana
~B) STRAIN: C24
~ix) FEATURE:
~A) NAME/KEY: misc_feature
~B) LOCATION: 1..12
~D) OTHER INFORMATION: /note= "5' overhang containing the
XhoI and EcoRI site"
~ix) FEATURE:
~A) NAME/REY: primer_bind
35 ~B) LOCATION: 13.. 35
~D) OTHER INFORMATION: /note= "this part of the primer
anneals to the sequence of 6044-0 (SEQIDNO: 4) at
position 1147-1169"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
cTcGAr~AATT CTATAATGTA TTTTGGCATG AAAAC 35
~2) INFORMATION FOR SEQ ID NO: 8:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 37 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA ~genomic)
~iii) HYPOTHETICAL: YES
SUBSTITUTE SHEET (RULE 26)
CA 022~7149 1998-12-02
W 097l46692 PCT~P96/02437
~ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "5' overhang containing a
XhoI and a EcoRI site"
(ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 13..37
(D) OTHER INFORMATION: /note= "this part of the primer
anneals to the sequence of 6044-0 (SEQIDNO: 4) at
position 1853-1880"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CTCGAGAATT CTATAATAAC ATAAGATAAA TATTTAC 37
(2) INFORMATION FOR SEQ ID NO: 9:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 35 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA ~genomic)
~iii) HYPOTHETICAL: YES
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "5' overhang containing the
XhoI and EcoRI site"
~ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 13..35
(D) OTHER INFORMATION: /note= "part of primer
annealing to the sequence of 6044-0 (SEQIDNO: 4) at
position 1918-1940"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
45 CTcGA~AATT CTATAACTAA TGGTAATTAC TAATT 35
~2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA (genomic)
SUBSTITUTE SHEET (RULE 26)
CA 022~7149 1998-12-02
W 097/46692 PCT~EP96tO2437
31
~iii) HYPOTHETICAL: YES
~ix) FEATURE:
IA) NAME/KEY: misc_feature
~B) LOCATION: 1..14
tD) OTHER INFORMATION: /note= "part of primer
restores sequence of 6044-0 tSEQIDNO: 4) from
position 2128 to 2142 while causing a deletion of
10 fragment 1909 to 2127
(ix) FEATURE:
(A) NAME/KEY: primer_bind
~B) LOCATION: 15..35
(D) OTHER INFORMATION: /note= "this part of the primer
anneals to the sequence of 6044-0 (SEQIDNO: 4) at
position 1897-1917"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGATCCAAGC TTTGATCAAT TGAATACTAA TGTAG 35
~2) INFORMATION FOR SEQ ID NO: 11:
~i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 20 base pair~
tB) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA tgenomic)
tiii) HYPOTHETICAL: YES
~ix) FEATURE:
(A) NAME/KEY: primer_bind
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /notes "primer that anneals to uidA
gene (Beta-glucuronidase) at position 224-205 from
the tagging construct pMOG553.(X83420)"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CTTTCCCACC AACGCTGATC 20
~2) INFORMATION FOR SEQ ID NO: 12:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
tC) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA (genomic)
SUBSTITUTE SHEET (Rl ILE 26)
.. .. . . . . ...
CA 022~7l49 l998-l2-02
W097/46692 PCT~P96/02437
32
~iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
5 CGGACTCTGG ATCCGGAAAG TG 22
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 ba~e pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GC~CGAGC CTAGGCACAG GTTATCAACA CGTTTG 36
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CGGACTCTGG ATCCGGAAAG TG 22
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTTACTCGAG CCATGGTAAG ~ GC 27
(2) INFORMATION FOR SEQ ID NO: 16:
SUBSTITUTE SHEET ~ULE 26~
CA 022~7l49 l998-l2-02
W 097/46692 PCTAEP96102437
33
(i) SEQUSNCE CHARACTERISTICS:
~A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GATCTAGACT CGAGAAGCTT GGATCCCCGG GTAGGTCAGT CCCC 44
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
30 CATGGGGGAC TGACCTACCC GGGGATCCAA GCTTCTCGAG TCTA 44
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AGCTCGGATC CG 12
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SU~STITUTE SHEET (RULE 26)
.. . , .. ~, ..... ~ .. ...... . . .. . .. .
CA 022~7l49 l998-l2-02
W O 97/46692 PCT/EP96/02437
34
(ii) MOLECULE TYPE: DNA (genomic)
~iii) HYPOTHETICAL: YES
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GGCTGCTCGA GC 12
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA ~genomic)
~iii) HYPOTHETICAL: YES
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
AATTGACGAA GCTTCGTC 18
SUBSTITUTE SHEET (RULE 26)
T- .
